Comparison of speed of onset of fazadinium, pancuronium, tubocurarine and suxamethonium. Under light general anaesthesia the speed of onset of action of fazadinium, pancuronium, tubocurarine and suxamethonium has been assessed by measuring the decrease in the twitch height of the adductor pollicis muscle following administration of the drugs. Suxamethonium produced 95% depression of the twitch height significantly faster than any of the other drugs, while the onset of fazadinium was significantly more rapid than that of the other non-depolarizing neuromuscular blocking drugs.


A comparison of four beta-adrenoceptor antagonists in patients with asthma. 1 Cardiovascular and airways response to two non-cardioselective beta-adrenoceptor blocking drugs, propranolol and pindolol (with partial agonist activity) and two cardioselective beta-adrenoceptor blocking drugs, acebutolol (with partial agonist activity) and atenolol, were compared in twelve patients with asthma. 2 All four drugs produced a significant reduction in resting pulse rate and prevented the increase in heart rate following inhaled isoprenaline (1,500 microgram). 3 Seven patients in clinical remission showed no significant bronchoconstrictor response to any of the drugs. In the remaining five patients, bronchoconstriction was greatest following propranolol (mean reduction in FEV1 26.6%) and least following atenolol (mean reduction in FEV1 6.5%). 4 The bronchodilator response to inhaled isoprenaline was blocked by propranolol and pindolol but not by acebutolol and atenolol. 5 Partial agonist activity did not appear to be clinically useful.


Circulatory and alpha-adrenoceptor blocking effects of phentolamine. 1 Intravenously administered phentolamine provoked immediate decreases in diastolic blood pressure but increases in heart rate and cardiac output. 2 These immediate circulatory effects had largely disappeared twenty minutes after administration and at this time phentolamine did not inhibit increases in blood pressure which were provoked during hand immersion in ice-cold water. 3 Log dose-response curves of noradrenaline induced increases in systolic and diastolic pressure 20 min after intravenous phentolamine were shifted to the right in a parallel manner compared with the curves before phentolamine administration. 4 It was concluded that the immediate and short acting effects induced by phentolamine are due to a non-specific vasodilator effect but in addition phentolamine causes a longer acting alpha-adrenoceptor blockade at vascular adrenoceptor sites. However, by producing both pre- and post-synaptic alpha-adrenoceptor blockade this may explain why this drug exerts only a weak antihypertensive effect.


The second Lilly Prize Lecture, University of Newcastle, July 1977. beta-Adrenergic receptor blockade in hypertension, past, present and future. All beta-adrenoceptor blocking drugs that have been described share the common property of being competitive inhibitors. They differ in their associated properties, the presence or absence of cardioselectivity, membrane stabilizing activity, and partial agonist activity. Recently some beta-adrenoceptor blocking drugs have been reported which also possess alpha-adrenoceptor blocking activity. The associated properties have been used as a basis for classifying beta-adrenoceptor blocking drugs (Fitzgerald, 1969, 1972). The presence or absence of cardioselectivity is most useful for dividing beta-adrenoceptor blocking drugs. The non-selective drugs (Division I) can be further divided according to the presence or absence of intrinsic sympathomimetic activity (ISA) and membrane stabilizing activity (Fitzgerald's groups I-IV). Group I possess both membrane activity and ISA, e.g. alprenolol, oxprenolol, group II just membrane action, e.g. propanolol, group III ISA but no membrane action, e.g. pindolol. Fitzgerald placed pindolol in group I but should be placed in group III as it possesses a high degree of beta-adrenoceptor blocking potency in relation to its membrane activity (Prichard, 1974). Finally drugs in group IV have neither ISA nor membrane action, e.g. sotalol, timolol. The cardioselective drugs (Division II) can be similarly sub-divided into groups I-IV according to the presence or absence of ISA or membrane action (Fitzgerald grouped all these together as group V). Lastly there are new beta-adrenergic receptor blocking drugs which in addition have alpha- adrenergic receptor blocking properties (Division III).


Prevention and treatment of premature labour by drugs: review of controlled clinical trials. This review sought evidence from 18 controlled clinical trials about the benefits of drugs (hormones, ethanol or beta-sympathomimetic agents) used to prevent premature birth. Of the 16 trials comparing drug with placebo, 13 were judged to be methodologically adequate. In eight trials, drugs were given prophylactically and in five they were given therapeutically. Two of the therapeutic trials found the drug more effective than placebo in postponing delivery. However, only one therapeutic trial and one prophylactic trial found the drug to affect favourably the outcome for the infant. The side effects of the drugs were not systematically studied. Additional clinical trials are needed to justify the use of drugs to inhibit labour.


Influence of pH on the efficacy of pilocarpine. Pilocarpine 4% solutions at pH 4.1 and 5.8 were compared in a double-blind clinical trial on 24 eyes of patients with primary open-angle glaucoma. Each drug was used over a period of 1 week. No significant difference in the lowering of intraocular pressure was found, and the near-neutral solution of pilocarpine was found to be equally stable when compared to the acid solution over a 6-month period.


Properties of trout hemoglobins reconstituted with unnatural hemes. Native globins isolated from trout hemoglobin compoents I and IV have been reconstituted with proto-, meso-, and deuteroheme, and the spectral and functional properties of the reconstituted hemoglobins have been investigated. Equilibrium and kinetic studies allow the following conclusions. (a) The properties of the proto-reconstituted hemoglobins are very similar, or indistinguishable, from those of the native Hb's I and IV. (B) The CO binding kinetics for both proteins were found to be consistent with the equilibrium data: the overall association rate constant increases (and the autocatalytic character of the reaction decreases) in the order proto, meso, deutero. (c) A marked pH dependence of both ligand affinity and cooperativity is maintained in the reconstituted Hb's IV: at pH 6 the fractional saturation with oxygen in air (Root effect) is lower for proto- than for meso- and deutero-Hb IV. The results obtained, including partial photodissociation experiments at different pH values, can be considered, to a first approximation, consistent with the basic features of a simple two-states model.


Effect of pH on the cross-bridge arrangement in synthetic myosin filaments. Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.


Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site. Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.


Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies. We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism.


Analogous effect of protons and inositol hexaphosphate on the alteration of structure of nitrosyl fetal human hemoglobin. We have determined the low temperature EPR spectra and room temperature ligand dissociation rate constants of human NO-hemoglobins F and A as a function of pH and inositol hexaphosphate levels in order to assess the contribution of a quaternary structural equilibrium in the two proteins to their spectral and functional properties. Our results are consistent with an increased stability of a ligated low affinity structure in the fetal protein; the functional properties of this structure appear to be essentially the same in the two hemoglobins, even though its stability relative to a high affinity conformation is different. The pH dependence of the NO dissociation constant in both adult and fetal hemoglobin can be assigned primarily to the pH-dependent equilibria of high and low affinity forms as monitored by EPR.


Spin-label studies of the sulfhydryl environment in bovine plasma albumin. 1. The N--F transition and acid expansion. The environment of the sulfhydryl group in plasma albumin was previously characterized by employing spin-labels of varying chain lengths (Hull, H. H., Chang, R., & Kaplan, L. J. (1975) Biochim. Biophys. Acta 400, 132). It was established that the sulfhydryl is in a crevice approximately 10 A deep but this crevice was not identified further. We now report the results of titrating albumin through the acidic conformational transitions while monitoring the electron-spin resonance of the bound nitroxide. With short spin-labels a general change is observed as the pH is lowered but the N--F transition is not discernible. However, with a spin label previously shown to project to the lip on the crevice a clear N--F transition as well as the subsequent acid expansion are observed. These results indicate that the sulfhydryl is in the crevice, formed by the domains of albumin, which opens during the N--F transition. Further results indicate that bound fatty acids do not influence the integrity of the sulfhydryl environment at neutral pH.


Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer. Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging.


Lipoxygenation activity of purified prostaglandin-forming cyclooxygenase. Purified cyclooxygenase, a single enzyme which catalyzes the formation of endoperoxide from arachidonic acid (20:4) in a bis(dioxygenase) reaction, is capable of oxygenating eicosadienoic acid (20:2) at C-11 in a single dioxygenase reaction. The partial oxygenation of 20:2 resembles the formation of prostaglandin from 20:4, with both oxygenation reactions exhibiting similar pH optima, substrate Km values, and cofactor effects including a need for peroxide and an absolute requirement for heme. In addition, those processes known to destroy 20:4 oxygenase activity, such as heat inactivation, inactivation with anti-inflammatory drugs, and turnover-mediated inactivation, have equally destructive effects on 20:2 oxygenase activity. Thus, both oxygenations are catalyzed by one enzyme. All of the above similarities for 20:2 and 20:4 oxygenation demonstrate that C-11 oxygenation is an integral rate-limiting step of cyclooxygenase action rather than a separate reaction resembling that of plant lipoxygenase.


The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition. 1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution).


The effect of calcium on the respiratory responses of corn mitochondria. Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.


Modes of reduction of nitrogen in heterocysts isolated from Anabaena species. N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.


The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces. The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.


Effect of nucleotides on potential and pH changes across the thylakoid membrane of spinach chloroplasts. With appropriate preparations of spinish chloroplasts we observe three distinct effects of the nucleotides: 1. An accelaration of the dark decay of the light induced 520 nm absorbance change after ATP addition. 2. An acidification of the internal space of the thylakoids after ATP addition in darkness. 3. A dark ATPase activity which is regulated by the deltapH across the membrane. We conclude that the effect of the nucleoside triphosphates on the 520 nm signal is linked to a change of the proton conductivity of the membrane, induced by the formation of a deltapH across the membrane in consequence of the dark ATPase activity. The mode of action of the nucleoside diphosphates in the presence of inorganic phosphate on the 520 nm signal is discussed. It is proposed that the effects observed are linked to the hydrolysis of the newly formed nucleoside triphosphates.


Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans. Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.


Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin. In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (lambda greater than 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 leads to P380 and P380 leads to rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 leads to P380 and P380 leads to rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 leads to P380.


The stimulation of Na+ uptake in frog skin by uranyl ions. 1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane.


Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles. Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.


Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes. Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.


Influence of (DL)-propranolol and Ca2+ on membrane potential and amino acid transport in Ehrlich ascites tumor cells. (1) (DL)-Propranolol and Ca2+ are shown to alter the transmembrane potential difference of Ehrlich ascites tumor cells as measured by means of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide, whose fluorescent intensity changes as a function of membrane potential. (2) The changes in membrane potential elicited by these agents are dependent of the external K+ concentration in a manner which suggest that the potential changes result from a specific increase in the permeability of the plasma membrane to K+. (3) Na+-dependent amino acid transport in the presence of propranolol can be modulated by varying the external K+ concentration (K+o). The initial rate of uptake is stimulated by propranolol at low K+o and inhibited at high K+o. The change in transport rate is nearly directly proportional to the natural logarithm of [K+]o in the presence of propranolol. (4) ATP depletion of the cells by preincubation with rotenone abolishes the changes in fluorescence and amino acid uptake seen with propranolol as a function of K+o. Restoration of cellular ATP with glucose in presence of Ca2+ restores both fluorescence and amino acid transport changes which occur in response to propranolol. (5) The fluorescence changes and amino acid transport changes in response to propranolol are pH dependent, with little effect seen at pH6. (6) It is concluded that the rate of Na+-dependent amino acid uptake is a function of membrane potential and is dependent on the electrochemical potential difference for Na+.


Transport of alpha-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens. The transport of alpha-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent Km values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for alpha-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting alpha-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using 137Cs in the presence of valinomycin. Direct attempts to demonstrate a protonsymport mechanism for alpha-aminoisobutyrate transport were negative.


How much is secondary structure responsible for resistance  of double-stranded RNA to pancreatic ribonuclease A? 1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA.


Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B. The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation.


Hydroxymethylglutaryl-coenzyme A reductase. Purification and properties of the enzyme from Fusarium oxysporum. The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature.


Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.


Characterization of guanylate cyclase of rod outer segments of the bovine retina. Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.


Isoelectric focusing of carboxypeptidase N. Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing.


Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12. Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.


Effects of thiol inhibitors on hepatic guanylate cylase activity. Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues.


Lipoxygenase-like enzyme in rat testis microsomes. Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers.


Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys). Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A.


A proton NMR investigation of proline-24 cis-trans isomerism in corticotropin 1-32 and related peptides. 250 MHz 1H-NMR studies performed on aqueous solutions of corticotropin1-32, corticotropin1-24, corticotropin15-32, corticotropin20-32 and corticotropin15-24 have allowed the location and the subsequent assignment of the signals of Tyrosine-23 aromatic protons and valine-22 methyl protons. These signals are sensitive to the geometry of proline-24, clearly transcribe its isomerism and yield the ratio of the cis-trans conformers. It is concluded that for large peptides in specific cases, the proton signals of side chains can be used to probe the backbone conformation.


Role of Bohr group salt bridges in cooperativity in hemoglobin. Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity.


Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes. Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate.


Proton NMR study of the thermodynamics and kinetics of the acid in equilibrium base transitions in reconstituted metmyoglobins. Optical and proton NMR pH titrations of sperm whale metmyoglobin (metMb) in its native form and reconstituted with chemically modified hemes reveal that the pKa for the acid in equilibrium base transition decreases as the heme 2,4-substituents are made more electron withdrawing. The proton NMR spectra yields resonances which are averaged over the acidic and basic forms of the protein, but still exhibit significant exchange line broadening. Analysis of this exchange contribution to the linewidth is consistent with the simple kinetic scheme metMb+H2O + OH- k2 in equilibrium k1 metMbOH + H2O with k2 = 1.3 +/- 0.5 . 10(10) M-1 . s-1 and k1 = 1.6 +/- 0.6 . 10(5) s-1 for the native protein. The effect of electron-withdrawing substituents on the heme increase k2 and decrease k1.


The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis. The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-biphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4]. In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities. The thermostability of phosphofructokinase (at 53 degrees C) increased, while that of aldolase (at 48 degrees C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52 degrees C) was also unchanged.


Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain. The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.


Binding of polyanions to carrier ampholytes in isoelectric focusing. The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.


[Acetate kinase chromatography on agarose derivatives]. Acetate kinase from E. coli K-12 was studied chromatographically on omega-aminoalkyl polysacharide sorbents. The dependence of the protein sorption-desorption on ionic strength and the effect of pH on the acetate kinase sorption were studied. The increase in the ionic strength caused a decrease in the amount of protein sorbed on the hexamethylenediamine- and chlorotriasinehexamethylenediamine sepharoses. On hexamethylenediamine-, octamethylenediamine- and dimethylhexamethylenediamine agaroses acetate kinase was adsorbed within the pH range of 6.5-9.0, whereas on the chlorotriasinehexamethylenediamine sepharose--at pH 6.5-8.0. The active protein was eluted at ionic strength of 0.14-0.17 M. Acetate kinase was not adsorbed on the carboxypropyonylaminohexyl sepharose within the pH range studied, i.e. 5.0-9.0 and was not adsorbed on hexamethylenediamine agarose at pH 4.0 and on chlorotriasinehexamethylenediamine sepharose--at pH 9.0. The mechanism of the enzyme-adsorbent interaction is discussed.


[Molecular weight and tertiary structure of transketolase from baker's yeast]. Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5.


[Properties of the high energy phosphate bonds of triphosphoinositide]. High heat of enzymatic hydrolysis of triphosphoinositide phosphate bonds and of high-energy phosphates is observed using microcalorimetry. Heats of hydrolysis of triphosphoinositide, ADP and ATP sharply increase with increasing pH values from 6.6 to 7.4. Heat of hydrolysis of diphosphoinositide correlates with that of low-energy phosphates, pK4 and pK5 values for triphosphoinositide are found to be 7.4 and 9.3 respectively by means of potentiometric titration deltaGo' values for diphosphoinositide and triphosphoinositide are -3.5 and -7.1 kcal/mole respectively, taking into consideration the correction for heat neutralization-ionization during hydrolysis. Rapid triphosphoinositide hydrolysis takes place in 1% aqueous pyridine solution at 100 degrees C. In contrast to diphosphoinositide and monophosphoinositide, infrared spectra of triphosphoinositide have an additional absorption band at 930 cm(-1). 31P NMR method has revealed the presence of one diester and two monoester groups in the molecule of triphosphoinositide. The differences described between triphosphoinositide and other compounds with phosphomonoester groups are suggested to be due to electrostatic nonbounded interaction of vicinal diequatorial phosphate groups.


[Indirect immunoprecipitation by rat liver polyribosomes using antibodies to tyrosine aminotransferase]. A fraction of rat liver polyribosomes is isolated, which in its immunochemical characteristics considerably enriched with polyribosomes capable to synthesize hydrocortisone-induced liver tyrosine aminotransferase isoenzyme. This specific polyribosome fraction was purified by immunochemical fractionation of total liver polyribosomes using indirect precipitation. The content of polyribosomes in immunoprecipitates comprise 0.4-0.8% of its initial amount (before immunochemical fractionation). The ratio of specific polyribosomes in immunoprecipitates varies from 20 to 45%, which corresponds to 25-100-fold purification. The data obtained suggest that the method of indirect precipitation can be an efficient step in the isolation procedure of individual mRNA.


[Light activation of NADH and NADPH].

[Light activation of NADH and NADPH]. Illumination of NADH and NADPH by UV-light in the absence of oxygen resulted in the reduction of ferredoxin or methyl-viologen to cation-radical and under prolonged illumination to dihydrodipyridyl. The reaction may by accompanied by triplet and singlet exitation of NADH. It was shown that hematoporphyrin in aqueous solution photosensitized the reaction of NADH oxidation by ferredoxin and methylviologen to the visible region of the spectrum. Under light excitation the redox potentials of NADH and NADPH were increased up to the level exceeding the potential of hydrogen electrode. Illumination of NADH and NADPH by UV-light in the presence of bacterial hydrogenase resulted in hydrogen evolution. The reaction of hydrogen evolution could be sensitised towards the visible region of the spectrum by chlorophyll or chloroplasts.


[Ribosomal proteins of pea seeds behaving like anions at pH 8.6]. A group of proteins migrating to the anode at pH 8.6 under polyacrylamide gel electrophoresis was revealed in the total protein of non-dissociated KCl-washed pea seed ribosomes. No proteins with an isoelectric point below pH 4.2 Were found. The presence of acidic proteins in 80 S ribosomes is due to the presence of a specific set of relatively acidic proteins in the total protein of large (5 major and 10 minor components) and small (2 major and 4 minor components) subunits. The mostly acidic proteins are located in the large subunit. The acidic proteins of 60S and 40S subunits are represented by the polypeptide chains with molecular weights from 48 000 to 13 000. The acidic proteins are present in the ribosomes studied in considerably less number than the basic proteins, and the former produce a very weak staining under electrophoretic analysis as compared with the latter. The data obtained suggest that 80S ribosomes of higher plants differ from animal ribosomes by a higher content of relatively acidic proteins.


[Effect of N-alkylimidazoles on oxidation of o-dianizidine catalyzed by horseradish peroxidase]. Effect of a number of N-alkylimidazoles (from N-methyl to N-octylimidazole) on peroxidase oxidation of o-dianizidine at pH 8.0 is studied. Alkylimidazoles studied are found to activate the reaction under given conditions, the activation type being non-competitive KA and (alpha) values are similar for all the activators, which suggests a similar mechanism of their action. Similar KA values suppose an insignificant role of hydrophobic interactions in the binding of N-alkylimidazoles with the enzyme.


[Interaction of intracellular RNAase from Aspergillus clavatus with derivatives of its substrates]. Using spectrophotometric and kinetic methods and also the methods of protection of Aspergillus clavatus RNAse (EC 3.1.4.23) by adenine nucleotides and their components against inactivation by means of acylation or heating, it was found that RNAse-nucleotide complex was formed by association of one enzyme molecule with one nucleotide molecule. It was also shown that all components of nucleotides (base, ribose and phosphate) take part in the formation of such complex and the removal of one of them (base or phosphate) lead to loosening of bindings of remaining fragments (ribose-5'-monophosphate, adenine) with the active site of RNAse, and to disappearance of bends within the pH range of 3.0-4.0 on the plot of pKi (5'-MP) versus pH, within the pH range of 5.5-7.0 on the plot of oKi (Ado) versus pH. The possibility of participation of associative pair RNAse imidasole groups - nucleotide phosphate groups and RNAse carboxylic group - nucleotide base in the mechanism of formation of enzyme-nucleotide (enzyme-substrate) complexes is postulated.


[Subcellular distribution and several properties of the cAMP enzyme system of phototrophic bacteria]. In the cells of the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris the two enzymes of the cAMP system enzymes - adenylate cyclase and cAMP phosphodiesterase (PDE) exist in a soluble and membrane-bound forms. After mild disruption of the cells (sonication up to 3 min) the activity of both enzymes is found in the chromatophores. In the cells of the two types of bacteria grown under anaerobic conditions soluble adenylate cyclase is predominant. In the cells of R. rubrum the soluble form of PDE posesses higher activity, whereas in the cells of Rh. palustris a higher activity is observed in the membrane-bound form. In addition to their different localization in the cells, the PDE forms of Rh. rubrum differ in their ratios to the concentrations of hydrogen ions and bivalent metals; the latter difference, however, may be accounted for by the effect of a protein modulator of PDE. The pH optimum of membrane-bound PDE is 9.15. Soluble PDE has two activity maxima at pH 7.5 and 8.7. It is probable that similar to the animal tissue enzyme, PDE from Rh. rubrum exists in the soluble phase in at least tw forms. Close pH optima for soluble adenylate cyclase and for one of the soluble PDE forms (about 8.5) may indicate the unidirectional control of these enzymes by hydrogen ion concentration.


[Irreversible inactivation of Chlorella glutamine synthetase by urea]. The effect of urea on Chlorella glutamine synthetase (E. C. 6.3.1.2) activity and tertiary structure is investigated. Urea is found to inhibit the activity of glutamine synthetase, the inhibitory effect being independent on the time. The enzyme molecule relax and changes its affinity to ammonium under the effect of urea at concentrations of 1.0-4.0 M. Higher concentrations of urea (5,0 M and more) produce a dissociation of the enzyme molecule into monomers without any intermediate forms. Monomers do not possess any synthetase and transferase activities. Substrates and cofactors do not protect the enzyme from the effect of urea and do not stimulate the emzyme reactivation and reaggregation after its dissotiation. The data obtained are discussed from the viewpoint of the regulation of Chlorella glutamine synthetase activity in vivo.


Influence of pH and temperature on behaviour of surfactant from human neonatal lungs. The surface pressures of human surfactant films were measured under static conditions and under dynamic (expansion-compression) conditions. The results from both methods showed that the maximum surface pressures generated by surfactant films in vitro were strongly dependent on temperature throughout the range 29-41 degrees C, higher pressures being achieved at the lower temperatures. In contrast, increasing the pH of the subphase stepwise over the range 7.1-7.7 produced a modest fall in the maximum pressures exerted by the films. The possibility that surfactant films may behave in a similar manner in vivo is discussed.


In vitro effects of inosine-pyruvate-phosphate on P50 values and DPG contents of fresh and stored blood from healthy neonates, symptom-free premature infants and premature infants with respiratory distress syndrome. A study was made of the effect of a 3-hour incubation with inosine-pyruvate-phosphate (IPP) on the P50 values and 2,3-DPG contents of fresh blood and blood which had stood for 96 h, from healthy adults, term neonates, 1-2-week-old symptom-free premature infants and RDS premature infants. It was found that the P50 value and the 2,3-DPG content could be increased considerably by IPP treatment in both the fresh and the stood blood in the various groups of neonates. The question remains open as to whether the effect of IPP, in significantly improving the O2 transport in neonates, is a consequence of the 2,3-DPG alone or of some other metabolite. In RDS the stimulating effect of the IPP mixture is appreciably lower.


[Effect of heparin polyanion on chromatin preparations obtained in solutions of low ionic strength]. DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b.


[Property of a heat- and acid-stable inhibitor of serine proteinases from the blood serum to inhibit lymphcyte transformation stimulated by mitogens]. Thermo- and acid-stable serine proteases inhibitor from the rabbit blood serum (TASPI) was shown to inhibit the human peripheral blood lymphocytes transformation stimulated by phytohemagglutinin (PHA) or concanavalin A. The extent of inhibition depended on the concentration of the preparation and its specific activity. The maximal inhibition of lymphocytes proliferation constituted 50 to 70%. TASPI displayed no cytotoxic activity. Considerably more effective inhibition was demonstrated by TASPI addition to the culture medium 24 hours after the addition of PHA. The antiprotease activity of crude human serum and that inactivated under different conditions is described. The results obtained suggest the participation of TASPI in the control of biological activity of the lymphoid tissue cells.


Effects of oxypertine on the isolated vas deferens of the rat. 1 The isolated vas deferens of the rat was used to examine the peripheral action of oxypertine, a psychotropic-anxiolytic drug. 2 Oxypertine (4.4 X 10(-10) M to 2.6 X 10(-5) M) antagonized competitively the effects of noradrenaline (pA2 = 7.2), 5-hydroxytryptamine (pA2 = 8.6) and dopamine (pA2 = 9.8). 3 Oxypertine (8.8 X 10(-9) M to 2.6 X 10(-5) M) antagonized the effects of low concentrations of acetylcholine and enhanced the contractions elicited by high concentrations of acetylcholine. 4 The contractions evoked by transmural stimulation of the vas deferens were reduced by oxypertine. 5 Oxypertine failed to antagonize the responses to potassium chloride. 6 These findings are compared with the effects of other antidepressant drugs.


Investigation of the role of histamine in antigen-induced bronchoconstriction in the ascaris-hypersensitive dog. 1 Aerosol administration of ascaris antigen to the airways of ascaris-hypersensitive dogs provoked increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). These changes in pulmonary mechanics were not inhibited by the histamine H1-receptor antagonists, diphenhydramine or mepyramine. 2 Increases in Rp and decreases in Cdyn induced by a histamine aerosol were markedly or totally inhibited by comparable doses of these H1-antihistamines. 3 Doses of antigen which produced pathophysiological pulmonary responses failed to produce a detectable histamine release from the cardiopulmonary system in vivo. Aerosol antigen provocation, equivalent to 5 to 9 times greater than that which produced substantial pathophysiological pulmonary responses, did cause histamine release in vivo. 4 The canine cardiopulmonary system showed only a modest ability to remove and/or degrade circulating histamine. 5 It is concluded that histamine may not play a major role in mediating the acute antigen-induced bronchoconstriction in the ascaris-hypersensitive dog.


Characterization of receptors mediating 5-hydroxytryptamine- and catecholamine-induced platelet aggregation, assessed by the actions of alpha- and beta-blockers, butyrophenones, 5-HT antagonists and chlorpromazine. 1 Blood platelets from normal human volunteers were isolated and aggregated in vitro with 5-hydroxytryptamine (5-HT), noradrenaline (NA), dopamine and N-dimethyldopamine (DMDA). 2 The effects of 5-HT antagonists, alpha- and beta-adrenoceptor blocking agents, butyrophenones and chlorpromazine upon aggregation induced by the four amines were investigated. 3 Only the the 5-HT antagonists, chlorpromazine and spiroperidol were potent inhibitors of 5-HT-induced aggregation, and only phentolamine was a potent inhibitor of the catecholamine-induced aggregation. 4 Evidence was obtained for two populations of receptors, one for 5-HT and one for the three catecholamines.


Biochemical evidence for the dual action of labetalol on alpha- and beta-adrenoceptors. 1 Labetalol (AH 5158A) inhibited the adrenaline-stimulated adenylate cyclase activity of rat liver and heart. This drug had no effect on basal or guanosine triphosphate (GTP)-activated adenylate cyclase activities. 2 Labetalol displaced the binding of the specific ligands [3H]-dihydroergocryptine and (-)-[3H]-dihydroalprenolol from their respective alpha and beta-adrenoceptors in rat heart and liver. The affinity of labetalol was 10 fold higher for the beta- than for the alpha-adrenoceptor. It appeared to be 10 to 100 times less potent than phentolamine in blocking alpha-adrenoceptors and 5 to 10 times less potent than propranolol in blocking beta-receptors. 3 It is concluded that labetalol exerts its dual alpha- and beta-antagonism by acting directly on the plasma membranes, where it binds competitively to alpha- and beta-adrenoceptors.


The treatment of frigidity: a comparative study of group and individual desensitization. Forty patients took part in this study. Twenty patients treated by group desensitization were compared with twenty treated by individual desensitization. Group desensitization appeared the more effective treatment if outcome was measured on the patients' self-rating scale, but the less effective if outcome was measured by the sexual interest questionnaire. Of the two, the group method took longer to administer, but more patients treated in this way completed the course of treatment.


Antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. Report of a study group on bacterial resistance. Twenty laboratories in England and Scotland took part in 1977 in a survey of antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. In Str pneumoniae 59 (6.8%) of the 866 strains studied were resistant to tetracycline and three to chloramphenicol, and one strain showed a decreased susceptibility to penicillin. The prevalence of resistance to tetracycline was lower than that found in a similar study performed in 1975. Nine hundred and fifty-two strains of H influenzae were examined: 15 (1.6%) were resistant to ampicillin (all were beta-lactamase producers) and 26 (2.7%) to tetracycline. Only two strains were resistant to chloramphenicol and two to trimethoprim. Sixty-three H influenzae strains were capsulated. Thirty-four of these were of Pittman type b, and antibiotic resistance, particularly to ampicillin, was more common in these than in other serotypes or non-typable strains. Some variation was seen in the resistance rate of both H influenzae and Str pneumoniae to tetracycline in strains from different centres, but too few were isolated to assess whether this represented a true geographical difference.


Recurrent genital candidosis in women and the effect of intermittent prophylactic treatment. A study of clotrimazole for the treatment of recurrent genital candidosis unexpectedly showed that symptoms and infection can be dissociated. The aim of the study was to see if intermittent antifungal treatment would reduce symptoms in women constantly distressed by recurrent genital candidosis. Forty women seriously affected by the condition were initially given oral and local antifungal treatment. When the patients were symptom-free and the vagina was free of yeasts, they were entered into a double-blind clinical trial and were treated prophylactically for four months with either intermittent clotrimazole pessaries and cream or a placebo. The prophylactic treatment kept symptoms below a critical level but did not affect the return of the yeasts to the vagina. This dissociation between symptoms and vaginal yeasts was unexpected. Rectal yeast carriage was unaffected by prophylactic vaginal treatment. Male contacts and patients both showed a high incidence of non-specific genital infection. This association has seldom been reported. A few patients cultured yeasts from their homes but this environment was not considered a major source of reinfection. The vaginal pH did not appear to be altered by the presence of yeasts. The results of the study suggested that symptoms in women with recurrent genital candidosis were not caused by yeasts alone, and possibly the reason for recurrences might lie not in constant reinfection by yeasts, but in failure to recognise and remove a primary underlying factor, perhaps infection with other sexually transmitted agents. The question of a synergistic action between yeasts and other organisms is discussed.


[Nycthemeral plasma and testicular androgen contents in guinea pigs at puberty]. A study based upon transversal sampling involving 13 series of 7 animals each does not show a significant circadian rhythm of testosterone and delta4 androstenedione concentrations in the plasma and testis of the guinea-pig at the age of puberty.


Energy-driven uptake of humic acids by Aspergillus niger. The uptake of humic acids by mycelia of Aspergillus niger was demonstrated to be energy-dependent with a sensitivity to sodium azide and to 2,4-dinitrophenol. Greater uptake of humic acids by submerged mycelium occurred at pH 3.0 and at 32 degrees C. The rate of uptake was influenced by the concentration of humic acids with an apparent Km of 0.2 grams/ml and with a Vmax of 0.13 mg humic acids per gram mycelial dry weight.10 min-1. In the absence of added energy source, Vmax of 0.05 mg humic acids per gram mycelial dry wt.10 min-1 was obtained; however, the affinity for humic acids by this uptake system was the same as for the energy-driven process. Apparent binding of humic acids to cell structures was indicated because only 41.8% of the humic acids taken up by the energy-dependent system could be recovered.


Induction of autolysis in Bacillus subtilis by ochratoxin A. Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.


Antifungal properties of 2-alkynoic acids and their methyl esters. Thirteen 2-alkynoic acids and their methyl esters (C3--C12, C14, C16, and C18) were tested against Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 4.0 and 5.6. Toxicity to Candida albicans, Trichophyton mentagrophytes, and Mucro mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. The fungitoxicity of the acids was influenced by chain length, pH of the medium, and absence or presence of adsorbents. The toxicity of the esters was influenced primarily by chain length and to a lesser extent by the pH of the medium and the presence of beef serum. The order of activity of the 2-alkynoic acids is C10=C11=C12 greater than C14=C16 greater than C9 greater than C8 greater than C7. When compared with other fatty acid analogs, the order of fungitoxicity on a weight basis is 2-alkynoic acids greater than 2-alkenoic acids greater than alkanoic acids greater than 2-bromoalkanoic acids greater than 2-fluoroalkanoic acids. There is an inverse relationship between chain length and pKa of the acids, suggesting that partition behavior is a fundamental determinant of fungitoxicity along with the effect of adsorbents.


Prescribing of psychoactive drugs for chronically ill elderly patients. The prescribing of psychoactive drugs for 1431 chronically ill elderly patients being assessed for long-term institutional or community care was surveyed. Psychoactive drugs had been prescribed for about one quarter of the patients; benzodiazepines were the most frequently prescribed group. Judging from the extensive prescribing of flurazepam and chloral hydrate, commonly used hypnotics, the main reason psychoactive drugs were prescribed was to provide night-time sedation. Antidepressants and drugs promoted as useful in improving cognitive function were infrequently prescribed. Commendable prescribing practices included the infrequent use of "cerebral vasodilators" and barbiturates. Questionable prescribing practices included the infrequent use of tricyclic antidepressants in severely depressed patients and the use of tranquilizers in patients described by their attending physician as markedly or extremely withdrawn.


Clinical pharmacology and therapeutics of benzodiazepines. Benzodiazepines are among the most commonly prescribed drugs in the world. In contrast to their extensive use, the therapeutic indications and potential of benzodiazepines are limited. All benzodiazepine derivatives available in Canada are similar structurally and in their pharmacologic actions. Few have specific advantages over any others. For example, no benzodiazepine has been shown to be superior to chlordiazepoxide in the treatment of acute anxiety, chronic anxiety neurosis or insomnia. Barbiturates should not be prescribed for these problems since benzodiazepines are just as effective and are safer. Persons more than 70 years old should receive initial doses of benzodiazepines 50% less than those prescribed for younger persons, and individuals with cirrhosis should receive chlordiazepoxide or diazepam in one third the usual dose; oxazepam or lorazepam should be considered for these two groups of patients. Diazepam and chlordiazepoxide should not be given intramuscularly. Benzodiazepines should be prescribed only when clearly indicated and only for the necessary length of time.


Heat fractionation and thermotolerance: a review. A rational approach to the design of clinical protocols combining fractionated hyperthermia plus X-irradiation or hyperthermia plus chemotherapy requires an understanding of the biology of fractionated heat alone. Mammalian cells growing in vitro can dramatically increase their tolerance to thermal damage (i.e., reduce the cellular inactivation rate) after prior heat conditioning. Although the mechanism(s) for this cellular thermotolerance is still unknown, it is apparent that the thermal history, the heat fractionation interval, and the recovery conditions all modify significantly the degree of thermotolerance subsequently exhibited. At the tissue level, the role of cellular thermotolerance is further complicated by host physiological mechanisms. Few data are available on heat fractionation in vivo, and the relative importance of physiological versus cellular effects remains to be defined.


Purification and properties of acid ribonucleases in human serum and leukocytes. Acid RNase was purified from normal human serum about 2400-fold by chromatography on phosphocellulose and Sephadex G-75 and rechromatography on Sephadex G-75. Assayed with yeast RNA as substrate, the enzyme showed the maximal activity at about pH 6.5 with sodium phosphate buffer. The reaction was activated by Na+, K+, and spermine, but it was not affected greatly by Mg2+, Co2+, and EDTA. Ca2+, Fe2+, Zn2+, and Cu2+ inhibited the reaction. Among the synthetic substrates examined, the enzyme preferentially hydrolyzed pyrimidine nucleotides, with a higher affinity for polycytidylate than for polyuridylate. The enzyme was thermolabile, but it stabilized with bovine plasma albumin. The molecular weight was approximately 15,000, estimated gel filtration on Sephadex G-75, and its isoelectric pH was above 11.0. From normal human leukocytes, acid RNase was purified about 400-fold by the same procedure described previously except that rechromatography on Sephadex G-75 was omitted. The properties of leukocytic RNase were found to be similar to those of serum acid RNase, but the latter enzyme differed in substrate specificity substantially from leukocytic RNase, preferring polyuridylate to polycytidylate. This evidence shows that serum RNase is not of leukocytic origin under normal physiological conditions.


Serum acid ribonuclease in myelogenous leukemia. Acid and alkaline RNase activities in serum were measured with yeast RNA as the substrate in normal subjects and in leukemic patients pretreatment and posttreatment, and the acid/alkaline ratios of activities were 0.63 +/- 0.08 (S.D.) (N, 12), 2.28 +/- 0.82 (N, 8), and 0.60 +/- 0.13 (N, 9), respectively. The mean value for the ratio in the pretreated leukemia was significantly higher than that in the other 2 groups (p less than 0.01). By separating these acid and alkaline RNases from normal and leukemic sera by phosphocellulose chromatography, it was further confirmed that acid RNase alone increased markedly in leukemic serum. From serum and leukocytes of leukemic patients, acid RNases were purified about 2000-fold and 300-fold, respectively, by phosphocellulose and Sephadex G-75 chromatography. Both enzymes displayed properties nearly identical with those of normal serum and leukocytes, except that leukemic serum acid RNase had about a 2.4-fold greater affinity for polyuridylate than for polycytidylate as substrate, in contrast to normal serum acid RNase that degraded polycytidylate exclusively. On the other hand acid RNases from serum leukocytes of leukemia showed a similar substrate preference. These results suggest that the high RNase levels of leukemic sera are due to an excessive leakage of acid RNase into the blood stream from abnormal leukocytes.


Changes in small vessel blood content of the rat heart induced by hypercapnic, hyperoxic or asphyxic conditions. The small vessel blood content (SVBC) of the ventricular walls of the heart of anesthetized closed chest rats was determined using 59FeC13 to label the plasma siderophilin. SVBC was measured breathing air, 100% O2, 5% CO2 in 21% O2 and during asphyxia. The average SVBC could be ranked: air less than 100% O2 less than 5% CO2 less than asphyxia. Only the '100% O2' values were not significantly above the 'air' values. Under control conditions, SVBC of the rat heart averaged 6.77 +/-(SE) 0.23 ml blood/100 g tissue. Inhalation of 5% CO2 increased this to 8.68 +/- 0.34, while asphyxia produced a maximal response to 14.40 +/- 0.77. Comparison of SVBC were made between various regions of the ventricular walls. The only significant difference was that the right side of the septum had a lower SVBC than the left ventricular subepicardium under all conditions except CO2. These differences in SVBC are related to the number of open capillaries in the myocardium. Thus inhalation of CO2 decreases intercapillary distance while 100% O2 has no effect.


[Purification of rat brain guanylate cyclase]. The purification of guanylate cyclase has been achieved. After electrophoresis on polyacrylamide gel only one protein band was observed. The low factor of purification must therefore be ascribed to the loss of an activator of guanylate cyclase. Stimulation of the activity by nitroprusside is also lost during purification. The purified enzyme follows Michaelis--Menten kinetics, it is activated by Mn++ ions and inhibited by triphospho nucleotides.


Isolation of gamma-glutamyltransferase from human liver, and comparison with the enzyme from human kidney. We isolated gamma-glutamyltransferase [(gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2] from human liver and compared some of its properties with the same enzyme prepared from human kidney. The enzymes from these two sources are very similar with respect to initial velocity kinetic constants, pH optima of the transpeptidation and autotransfer reactions, heat stability, competitive inhibition by glutathione of the colorimetric assay in which gamma-glutamyl-4-nitroanilide is substrate, stability of catalytic activity to trypsinization, and relative rates of transfer of the gamma-glutamyl moiety from gamma-glutamyl-4-nitroanilide and L-[glycine-2-3/]glutathione to some amino acids and small peptides. The kidney enzyme is inhibited more by the gamma-glutamyl acceptor substrate, glycylglycine, as reflected in a sevenfold lower value for the inhibition constant KiA. Major differences were observed in the lectin-binding properties of liver gamma-glutamyltransferase compared to the kidney enzyme. Lectin-binding property differences are retained for the trypsinized form of the liver and kidney enzymes, although the degree of precipitation was less for certain lectins as compared to the untreated enzyme. Lectin-binding properties were reversed by carbohydrates specific for each lectin. We adapted the histochemical staining technique of Rutenberg et al. [J. Histochem. Cytochem. 17, 517 (1969)] to the detection of gamma-glutamyltransferase activity in acrylamide gels; diffusion artifacts are minimized and the color produced is stable for several days. Untreated and trypsinized forms of the liver enzyme both migrated faster in acrylamide gels (as single bands) than did the corresponding forms of the kidney enzyme.


Biochemical characterization of an aryl acetic ester hydrolase isolated from human monocytes. A carboxylic-ester hydrolase was isolated from the leukocytes of a patient with myelomonocytic leukemia. Its relative molecular mass as estimated by sucrose density-gradient sedimentation is about 70 000. The purified enzyme is specific for acetyl esters of aromatic alcohols. It is inhibited by fluoride, but insensitive to eserine or p-chloromercuriphenylsulfonate. Hydrolysis of 1-naphthyl acetate was optimal above pH 6.0; of o-nitrophenyl acetate, above 8.0. The common catalytic site for the two types of substrates on the enzyme was confirmed by competitive inhibition data.


A novel gamma-glutamyl transpeptidase in renal carcinoma in comparison with normal kidney enzyme. A novel gamma-glutamyl transpeptidase was found in a renal carcinoma tissue. This enzyme electrophoresed more quickly than that of normal kidney, but more slowly than normal liver enzyme. Their Rf values are 0.46, 0.33, and 0.52 for the renal carcinoma, normal kidney and normal liver enzyme, respectively. After treatment of the renal carcinoma and normal kidney enzyme with neuraminidase, the renal carcinoma gamma-glutamyl transpeptidase still electrophoresed slightly faster than that of normal kidney. The catalytic properties of the renal carcinoma gamma-glutamyl transpeptidase were almost the same as those of normal kidney except for molecular weight; the molecular weight for the renal carcinoma was estimated to be about 130 000, while that of normal kidney was about 90 000. These results may mean that the enzyme of the renal carcinoma is different from that of normal kidney in chemical constituents other than sialic acid. Of 10 patients with renal carcinoma examined electrophoretically, 5 posessed this novel enzyme in their renal carcinoma tissues.


A simple radioimmunoassay for plasma cortisol. A simple radioimmunoassay (RIA) for plasma cortisol is described which combines the advantages of (i) direct analysis of untreated plasma samples, (ii) use of solid-coupled anti-cortisol antibodies and (iii) use of a gamma-labelled radioligand. The reagents are relatively easily prepared and stable, and the analysis can be completed in 4 h. Inter-assay precision (C.V.) is 8-11%. Critical examination of specificity using high pressure liquid chromatography showed that 23-35% of the immunoassayable material in plasma was not cortisol. RIA results on samples collected under basal conditions were on average 40 nmol/l lower than fluorimetric results, while in insulin hypoglycaemia and synacthen (ACTH) stimulation tests, this difference increased to over 100 nmol/l. The RIA is technically more simple than fluorimetric, competitive-protein-binding, and many RIA methods, and can be used with advantage in the routine investigation of adrenocortical function. However, using the present antiserum, the RIA is not applicable to investigations on patients receiving metyrapone, nor in suspected cases of congenital adrenal hyperplasia.


Purification and serological studies of human alpha-L-fucosidase in the normal and fucosidosis states. An antiserum has been raised to purified alpha-L-fucosidase. Levels of cross-reaction with serum of two unrelated fucosidosis patients and normal individuals with low activity are consistent with the presence of very low amounts of normal enzyme. Similarly no cross-reacting material could be found in cultured fibroblasts from fucosidosis patients. It is deduced that in these cases there is no production of mutant enzyme in quantities comparable to normal levels. Some observations on the interrelations of fucosidases I and II are reported.


Sphingomyelinase activity levels in human peripheral blood leukocytes, using [3H]sphingomyelin as substrate: study of heterozygotes and homozygotes for Niemann-Pick disease variants. Patients and heterozygous carriers of Niemann-Pick disease types A and B as well as the primary (genetic) sea-blue histiocyte syndrome were investigated for their leukocyte sphingomyelinase activity. In parallel, glucocerebrosidase activity was determined in all cases studied. [3H]Sphingomyelin and [14C]glucocerebroside served as substrates for sphingomyelinase and glucocerebrosidase activity measurements, respectively. Conditions for these enzymes' assays are discussed. Sphingomyelinase activity was completely absent in three cases of Niemann-Pick disease type A and significantly diminished in one patient with Niemann-Pick disease type B and two with the sea-blue histiocyte syndrome. Sphingomyelinase activity in obligatory heterozygotes of all variants investigated represented about 40 to 70% of normal activity. Nevertheless, some overlapping with normal values occasionally occurred. Interestingly, glucocerbrosidase activity was elevated in patients with Niemann-Pick disease variants.


Serum alanine assay with an enzymic micromethod (L-alanine dehydrogenase). L-Alanine was measured by an enzymic micromethod in serum after treatment with perchloric acid, as well as after ultrafiltration through collodium membrane filters. Serum L-alanine was also determined by an automated column chromatographic technique. Using the enzymic method, spuriously increased L-alanine levels could be obtained due to the absorbent reaction between hydrazine and NAD-containing reagents. It could be shown that this absorbent reaction depends on the pH value of the test solution. In contrast to supernatant diluted by perchloric acid, ultrafiltrates gave L-alanine data equivalent to those by column chromatography, since conditions of reaction time and pH value could be kept identical for blank, standard and ultrafiltrate solutions.


Interaction of [14C]acetylsalicylic acid with normal human peripheral blood lymphocytes. Therapeutic concentrations of acetylsalicylic acid (ASA) have strikingly inhibited in vitro and in vivo mitogen- and antigen-induced blastogenesis by human lymphocytes. These observations may be pertinent to the anti-inflammatory actions of ASA. To investigate further the possible effects of ASA on cellular responses, we studied the in vitro interaction of [14C]ASA with lymphocytes. Results indicated that the [14C]ASA association with cells was (a) proportional to ASA concentrations, (b) non-saturable at high concentrations of ASA, (c) dependent on pH, (d) independent of temperature, (e) dependent on cell concentration, (f) not consistently displaced by unlabelled ASA or other drugs, (g) rapid and unchanged over 1 min to 72 hr incubations and (h) reversed by repeated cell washing. These data confirmed that ASA indeed interacted with lymphocytes. The association was rapid, reversible, pH-dependent and not demonstrably specific under these experimental conditions.


Clorazepate kinetics in treated epileptics. Clorazepate is decarboxylated to form desmethyldiazepam and is a convenient way of administering it. Its kinetics were investigated in epileptic patients after single oral and multiple oral doses. Peak serum concentrations of demethyldiazepam occurred in 0.5 to 1 hr. There appeared to be a brief lag before rapid absorption. Because of the rapid absorption with resulting high serum levels, daily doses should be divided. Serum concentration/time curves were best fitted by the two-compartment open model. The apparent t1/2 of the distribution phase was 1.28 +/- 0.44 hr and the t1/2 of the disposition phase was 40.8 +/- 9.96 hr. Serum concentrations rose after meals. Whole body apparent volume of distribution (VB/F) was 1.63 +/- 0.24 L/kg. Total plasma clearance was 34.4 +/- 7.2 ml/min, which is greater than clearance levels for desmethyldiazepam in normals and reflects the greater hepatic metabolism which occurs in treated epileptics. The discrepancy illustrates the hazards of extrapolating data collected in normals to patients with multiple drug exposures.


Barbiturate physical dependence in mice: effects of neuroleptics and diazepam on the withdrawal syndrome. Diazepam (8 mg/kg) quickly and effectively reduced the incidence of withdrawal symptoms in phenobarbital dependent C57BL/6J male mice. In contrast, the neuroleptic agents employed (chlorpormazine, haloperidol, or reserpine) or alpha-methyl-p-tyrosine tended to exacerbate the withdrawal syndrome. Chlorpromazine and haloperidol were approximately equally potent in their effects; this would suggest that their antidopaminergic effects were not the primary mechanism of action for the increased incidence of withdrawal symptoms.


The cardiovascular effects of intraventricular 5,6-dihydroxytryptamine in conscious hypertensive rats. 1. Conscious experimental hypertensive rats injected intraventricularly with 5,6-dihydroxytryptamine (50 microgram) responded with a rapid and long-lasting (4 days) fall in blood pressure and this was accompanied by a bradycardia of similar duration. 2. The acute actions of 5,6-dihydroxytryptamine on the cardiovascular system were not modified by pretreatment with bromolysergide (0.5 mg/kg, i.p.), methysergide (2.5 mg/kg, i.p.) or intraventricular phentolamine (200 microgram). 3. The results suggest that central serotonergic neurones play an important role in the regulation of blood pressure. However, the site of action remains to be investigated.


Comparative trial of two antihistamines, mequitazine and brompheniramine. A double-blind trial was carried out in 48 patients with allergic disorders to compare the efficacy and tolerance of mequitazine and brompheniramine. Patients received either 10 mg mequitazine or 24 mg brompheniramine daily, divided into 2 doses, for 14 days. The results, analyzed from patients' answers to a questionnaire recorded on a daily basis, showed that mequitazine was at least as effective as brompheniramine and caused significantly less drowsiness throughout the trial period. Few other side-effects were reported.


Comparative trial of an antihistamine, mequitazine, and placebo. Forty patients suffering from a dermatological condition suitable for treatment with an antihistamine were studied in a double-blind randomized trial to compare the effectiveness and tolerance of mequitazine and placebo. Over a period of 2 weeks, patients received either 5 mg mequitazine twice daily or placebo. Where justified, patients were also given concomitant topical treatment but only with a hydrating cream or ichthyol paste. Patients' overall response to treatment was significantly better in the mequitazine group, whether the results were analyzed for all patients or those receiving just the trial drugs without topical treatment. Few side-effects were reported and there was no significant difference between the levels of drowsiness experienced with mequitazine compared with placebo.


Double-blind clinical comparison between a gastrin-receptor antagonist, proglumide, and a histamine H2-blocker, cimetidine. A double-blind trial was carried out in 30 patients with peptic ulcers to assess the effects of treatment with a gastrin-receptor antagonist, proglumide, compared with a histamine H2-blocker, cimetidine. Patients received either 1200 mg proglumide or 1200 mg cimetidine per day for 28 days. The results showed that both drugs significantly reduced clinical symptoms and gastric secretion. In patients treated with cimetidine there was a significant increase in blood gastrin levels and marked hypertrophy and hyperplasia of the antral mucosa was observed in almost all patients. No such changes were found in the patients treated with proglumide.


Dose-ranging study of the new beta-adrenergic antagonist nadolol in the treatment of essential hypertension. A preliminary, single-blind, dose-ranging study was carried out in 30 patients with essential hypertension to assess the efficacy of nadolol, a new beta-adrenoceptor blocking agent without intrinsic sympathomimetic action and with an extremely long plasma half-life. After a 2-week period on placebo, patients were treated for 14 weeks with daily doses of 40 mg nadolol (20 mg twice daily). Dosage was increased every second week up to a maximum of 560 mg daily or until the patient was stabilized at an effective normotensive dose level. The results showed that at the end of the trial period there was a significant reduction in both systolic and diastolic blood pressure (approximately 34/21 mmHg) at an average daily dose of 110 mg nadolol. Apart from a tendency to bradycardia, explained by the drug's lack of sympathomimetic action, no other side-effects attributable to treatment were reported and no patient complained of sleep disturbance.


Comparison of the new beta-adrenoceptor antagonist, nadolol, and propranolol in the treatment of angina pectoris. A randomized, double-blind study was carried out in 24 patients with stable angina pectoris to compare the efficacy of nadolol, a new beta-adrenoceptor antagonist, and propranolol. After a period on placebo, 14 patients received nadolol once daily and 10 patients propranolol 4-times daily over a 10-week dose-ranging period followed by a maintenance period of 4 weeks. Optimal daily dosage for nadolol was 100 mg, and 112 mg for propranolol. Parameters used for evaluation of therapeutic effects included the number of anginal attacks, number of nitroglycerine tablets needed, time before onset of chest pain during exercise test, exercise time, and overall clinical impression of response. The results indicated that nadolol given once daily was equally as effective as propranolol 4-times daily in treating angina pectoris.


Cutaneous vasculitis.

Cutaneous vasculitis. Cutaneous vasculitis is a common but confusing clinical syndrome. We report three illustrative cases demonstrating the variable nature of the process. Renal disease is generally a poor prognostic sign; therefore, the renal status should be evaluated early. The process probably represents an immune complex disorder. Although various therapies have been used, an aggressive corticosteroid regimen is the preferred mode of treatment at the current time.


Metabolism of 5-isopropyl-1-methyl-2-nitro-1H-imidazole. Identification of some urinary metabolites in the dog. The metabolism of 5-isopropyl-1-methyl-2-nitro-1H-[2-14C] imidazole in dogs has been investigated after oral administration of 50 mg/kg. Three main metabolites, still containing the nitro group and accounting for about 50% of the total radiocarbon, together with a small amount of the unchanged drug, were isolated from the urine within 48 hr. The structures were determined by mass, infrared, and nuclear magnetic resonance spectroscopy. The biotransformations giving rise to the metabolites isolated involve the isopropyl chain of the molecule, either at the tertiary carbon atom or at one of the two methyl groups, or both. Thus, the metabolic behavior of this 2-nitroimidazole derivative appears to be similar to that previously demonstrated for the class of the isomeric 5-nitroimidazoles.