Pharmacokinetics and relative bioavailability of heptabarbital and heptabarbital sodium after oral administration to man. A method has been developed for the quantitative determination of heptabarbital [5-(1-cyclohepten-1-yl)-5-ethylbarbituric acid] in human plasma after administration of single therapeutic doses of the drug. It involves a single extraction step followed by gas chromatography with alkali flame ionization detection, and the results were linear in the concentration range 0.125 - 5.0 mug/ml plasma. The pharmacokinetics and relative bioavailability of heptabarbital and heptabarbital sodium were studied in a crossover design in 7 healthy volunteers after oral administration of 20 tablets containing 200 mg heptabarbital and hard gelatine capsules containing an equivalent amount of its sodium salt. Heptabarbital concentrations in plasma were determined at regular intervals. The absorption of heptabarbital from the tablets absorbed more rapidly and peak concentrations occurred between 1/3 and 2 h. In all cases the elimination of heptabarbital could be described by a single first-order process with an average half-life of 7.6 h (range 6.1 - 11.2 h). The half-life of the drug in each individual was about the same in the two trials. The relative bioavailability in each volunteer was estimated by comparing the areas under the plasma concentration curves. The sodium salt had an average bioavailability of 83% relative to the free acid. In some volunteers urinary excretion of unchanged heptabarbital was measured; cumulative excretion amounted to 0.16 - 0.30% of the administered dose. Four volunteers received one tablet each night for eight or ten days, but no accumulation was found. In three volunteers the half-life of the drug prior to and after these experiments did not change, whereas in the other volunteer the half-life decreased from 7.1 to 4.6 h. The possibility of enzyme induction should be considered when heptabarbital is taken regularly. It was concluded that heptabarbital was a suitable drug for the treatment of insomnia, since its half-life was rather short. Heptabarbital sodium may be used for induction of sleep, whereas Medomin tablets, i.e. heptabarbital free acid, may be prescribed when the maintenance of sleep is the primary reason for treatment with a hypnotic drug.


The physiological disposition of etilefrine in man. Pharmacokinetic and metabolic studies with 3H-etilefrine were performed to assess the importance of a first-pass effect on the pharmacodynamic action of this sympathomimetic amine. Identical amounts of 3H-activity, ca. 80% of the dose, were excreted in the urine after intravenous or oral administration, which indicates complete enteral absorption of the drug. Comparison of the areas under the plasma curves of unchanged etilefrine after both routes of administration resulted in a bioavailability factor of 0.55, which can be explained by an extensive first-pass effect. The time curve of plasma levels of etilefrine was compatible with an open 2-compartment model characterized by a rather large volume of distribution (Vd, beta) of 160 1, and a predominant half life of 2 hours. The pharmacodynamic action corresponded to the amount of drug in the central compartment. The major pathway of metabolism of etilefrine was conjugation to form the phenolic sulphate, and a very minor proportion of the drug was excreted as the corresponding hydroxymandelic acid. This metabolic pattern seems to confirm our hypothesis that phenylalkylamines with hydroxyl group in the m-position of the benzene ring are predominantly conjugated in contrast to p-hydroxylated compounds which are mainly deaminated.


Long term treatment of moderate hypertension with penbutolol (Hoe 893d). II. Effect on the response of plasma catecholamines and plasma renin activity to insulin-induced hypoglycemia. The effect of insulin-induced hypoglycemia on the blood levels of catecholamines and renin activity has been studied in five patients with moderate hypertension before and after treatment for 3 - 8 months with penbutolol (PEN) 20 - 30 mg twice daily. Penbutolol caused no change in fasting blood glucose level. Insulin o.1 IU per kg body weight i.v. reduced blood glucose concentration by approximately 50 per cent after 30 - 45 min, both before and during treatment with penbutolol. Hypoglycemia prior to medication was accompanied by a marked increase in the production of adrenaline and a minor increase of noradrenaline in all five patients. During treatment the response of adrenaline to hypoglycemia was reduced in four patients and the data was inconclusive in one. Basal renin activity was rather low in three patients, within the normal range in one and relatively high in one. Before penbutolol the hypoglycemia-induced increase in catecholamine production caused no change in plasma renin activity in the three patients with low basal levels, whereas a marked increase was observed in the other two. During medication plasma renin activity remained unchanged on induction of hypoglycemia regardless of the catecholamine response. Despite the marked increase in plasma adrenaline following insulin-induced hypoglycemia, no statistically significant increase in pulse rate was recorded.


Gastrointestinal absorption and metabolism of two 35S-labelled ampicillin esters. Two ampicillin esters, 35S-pivampicillin and 35S-carampicillin and polyethylene glycol (nonabsorbable marker) were given orally to healthy subjects with gastrointestinal tubes. The cumulative absorption of radioactivity in both compounds (60-90%) was higher than (25-67%) previously found after administration of 35S-ampicillin. The pek plasma levels of radioactivity were reached earlier and were about twice as high as in the latter study. The amount of radioactivity excreted in urine was about the same as that absorbed from the proximal part of the gastrointestinal tract. Both 35S-pivampicillin and 35S-carampicillin were partly hydrolyzed in the stomach and upper small intestine and labelled ampicillin was released. They were also decomposed after absorption since all the radioactivity recovered from blood and urine appeared to be attached to ampicillin and ampicillin metabolites. Studies in vitro indicated that ampicillin esters absorbed intact may be hydrolyzed not only in the blood but also in the intestinal wall and the liver.


Brain homovanillic acid: regional changes over time with antipsychotic drugs. Acute administration of equivalent doses of either chlorpromazine, thioridazine, or clozapine, respectively, produced progressively smaller increases in brain homovanillic acid (HVA) in the rabbit; however, changes in HVA in three brain regions were of equal magnitude for a single dose of a given drug. Chronic administration of fluphenazine enanthate resulted in a decrease in HVA relative to acute treatment in caudate more than limbic regions. No differences between caudate and limbic regions were observed during daily chlorpromazine administration for 3 ro 8 days. Tolerance appeared to develop in approximately 1 week. Chronic treatment with clozapine produced no tolerance at one week but suggestive evidence of tolerance in caudate and limbic regions at two weeks. No tolerance was observed in the hypothalamus during chronic treatment with any drug used. Cisternal CSF HVA paralleled caudate HVA during acute and chronic treatments.


Hypoxia in fibroblast cultures. I. Changes in glucose consumption by 5 vol.-% oxygen concentration and reduced pH. In secondary cultures of rat embryonic fibroblasts the glucose consumption and lactate concentration were comparatively investigated in 5 and 21 Vol.-% O2 environment at pH 6.6 and pH 7.4 (HEPES-buffer). The results were correlated with cell density. The following observations were made: 1. At pH 6.6 the rate of cell proliferation was reduced to 81%; by additional hypoxia it was reduced to 71%. 2. Increase in cell density effectuated decrease of glucose consumption and lactate production at pH 7.4 and pH 6.6 in 5% as well as in 21% O2 environment. 3. At pH 7.4 enhancement of glucose consumption and lactate production due to hypoxia can be observed only at low cell density. 4. At pH 6.6 respiration of fibroblasts was little influenced by 5% O2 environment. 5. Transition from pH 7.4/21% O2 to pH 6.6/5% O2 effectuated decrease in glucose consumption and lactate concentration in tissues in hypoxic environment. This suggests a pH-governed feedback mechanism. Abbreviations used in the text: c-AMP = cyclic adenosine monophosphate; MPS = acid mucopolysaccharides (glycosaminoglycans).


Hypoxia in fibroblast cultures. 2. Influence of pH on the distribution pattern of acid mucopolysaccharides. Secondary cultures of embryonic rat fibroblasts [NEUPERT et al., Exp. Path. 7, 19-28 (1972)] were cultured in 21% and 5% O2 concentration at pH 6.6 and 7.4 for 6 or 8 days. The acid mucopolysaccharides were isolated and fractionated by alcohol precipitation, papain digestion, CPC-precipitation and fractionation after the microtechniques of SVEJCAR and ROBERTSON [see KITTLICK and NEUPERT, Exp. Path. 7, 7-18 (1972)]. Cells and medium were investigated together. The results were related to cell density and compared with the glucose and lactate values [see KITTLICK and NEUPERT, Exp. Path. 10, 109-114 (1975)]. Concerning the question of interrelations of MPS-synthesis and glycolysis a survey on literature is given. Our own test results were as follows: 1. Hypoxia (5% O2) did not influence MPS-total synthesis. 2. Depending on cell density the individual MPS fractions were different in their reaction to hypoxia. 3. Hyaluronic acid (and heparan sulphate) in the MPS-pattern showed other behaviour than chondroitin sulphate and dermatan sulphate, respectively. 4. At low cell density hypoxia effectuated increase in hyaluronic acid and decrease in chondroitin sulphate and dermatan sulphate, respectively. 5. At high cell density hypoxia effectuated decrease in hyaluronic acid and increase in chondroitin sulphate and dermatan sulphate, respectively. Possible relationship to processes in the tissue is discussed.


The extracellular protease from Pseudomonas fluorescens. An extracellular protease has been purified from cultures of Pseudomonas fluorescens. It is a metalloenzyme with a molecular weight of 37,000 +/- 3,700, able to digest casein, hemoglobin and gelatine.


Anionic activation of human salivary amylase. In all earlier studies on alpha-amylase, the influence of different ions were studied in phosphate buffer. The present report shows the effect of different ions individually with Tris and amino acid. Though it has been claimed recently that sodium ion is an activator of alpha-amylase, this study reconfirms that sodium ion does not activate human salivary amylase.


Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonoesterase activity in wheat leaves. Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme had KNADP value of 1.4 X 10(-4) M and a pH optimum of 5.9. In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinins (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.


Gastric mucus effusion elicited by oral copper compounds: potential anti-ulcer activity. In rats, both Cu(I) and Cu(II) show an irritancy profile not shared with Cu degrees or Zn(II) or Ni(II). The gastric response to Cu(II), i.e. copius fluid and mucus secretion, can protect the stomach from the acute ulcerative effects of aspirin or physical stress administered subsequently.


The effect of ion-exchange column chromatography on separation of X and Y chromosome-bearing human spermatozoa. Separation of X and Y chromosome-bearing spermatozoa has been attempted using ion-exchange column chromatography, with cation- and anion-exchange resins of low, intermediate, and high ionic strength. Examination of F-bodies on the Y chromosome of treated human sperm and progeny resulting from insemination of treated rabbit spermatozoa indicates that in none of the cases investigated did the treatment cause a separation of X and Y chromosome-bearing spermatozoa. The treatment does appear to filter out dead rabbit (and bull) spermatozoa, but the possible beneficial effects of this phenomenon are as yet uninvestigated.


Equality in survival of X and Y chromosome-bearing human spermatozoa. Human X and Y chromosome-bearing spermatozoa survived equally well during washing and resuspension in buffers of pH 5.2 and 8.0 and during incubation in these buffers for 11 hours at 37 degrees C. This suggests that alteration of the ratio of living X- and Y-bearing spermatozoa by direct treatment is not an effective method of sex ratio alteration.


Accumulation of nicotine in the uterine fluid of the six-day pregnant rabbit. In 6-day pregnant New Zealand White rabbits dosed intravenously with 3H-nicotine, the 3H-activity in the uterine fluid was approximately 5 to 11 times greater than that in the plasma at the corresponding times; 3H-nicotine itself accounted for most of this radioactivity. Dichlorodiphenyltrichloroethane (DDT) also accumulated in the uterine luminal fluid of 6-day pregnant rabbits, but to a lesser extent. However, nicotine or DDT accumulation did not occur in similarly treated, nonpregnant rabbits. The radioactivity in the uterine fluid of rabbits treated with 14C-isoniazid, salicylic acid, barbital, antipyrine, and caffeine was not different from that in the plasma (uterine fluid to plasma radioactivity ratios ranged between 0.67 and 1.85) in both 6-day pregnant and nonpregnant rabbits. No differences in regard to nicotine metabolism, volume of distribution, plasma disappearance, plasma protein binding, or urinary excretion were found between 6-day pregnant and nonpregnant rabbits. Accumulation of nicotine took place in the uterine luminal fluid of nonpregnant does pretreated with either progesterone or human chorionic gonadotropin, but did not occur in does pretreated with estrogen. It is possible that the accumulation of nicotine in uterine fluid of pregnant does and in human chorionic gonadotropin- or progesterone-pretreated nonpregnant does is due to the binding of nicotine to specific uterine fluid proteins.


Oxygen transport impairment in diabetes. Oxyhemoglobin dissociation curves (ODC) from zero to full saturation were developed from tests performed on whole blood from various groups of diabetic and nondiabetic healthy subjects. P50 at in-vivo pH was slightly but significantly lower than normal in ambulatory nonacidotic, uncomplicated juvenile diabetics (26.0 vs. 27.3 mm. Hg, P less than 0.001), despite increased red cell 2,3-diphosphoglycerate (2,3-DPG) concentrations in diabetic erythrocytes (15.0 vs. 13.7 mumole/gm. Hb, P less than 0.001). This combination of changes is in keeping with the presence of increased proportions of hemoglobin AIc in insulin-treated diabetics. The position of the ODC was positively correlated with the 2,3-DPG concentration (P less than 0.01), which varied in response to fluctuations in plasma concentration of inorganic phosphate (Pi) (P less than 0.001). Optimal metabolic control may lead to a normalization of the ODC in association with increased concentrations of red cell 2,3-DPG and P. When the diabetes was uncontrolled, the ODC was usually unchanged during the acidotic phase because the lowered pH balanced the effect of diminished 2,3-DPG concentration on the ODC. After correction of acidosis, the disproportion between erythrocyte 2,3-DPG and pH became quite prominent, accompanied by a corresponding fall in P50 (21.0 vs. 26.1 mm. Hg, P less than 0.001). Following ketoacidosis, with a persistently lowered Pi, it may take up to one week for 2,3-DPG to return to an approximately normal level, and the P50 will be impaired for the same period. A diphosphonate (EHDP) known to enhance tubular phosphate reabsorption in man was given to nonacidotic insulin-treated diabetic and healthy volunteers for 28 days. It caused a significant increase in mean Pi and P50 in both healthy and diabetic subjects (r = 0.58, P less than 0.01). When a dietary supplement of dibasic calcium phosphate was given to diabetic subjects for 28 days, a significant increase in P50 also occurred (25.2 vs. 27.2 mm. Hg, P less than 0.001). It is recommended that the diabetes diet be supplemented by dibasic calcium phosphate to prevent the inhibitory effect of a low concentration of Pi on red cell oxygen delivery.


Increased microvascular permeability to plasma proteins in short- and long-term juvenile diabetics. The present findings of increased microvascular protein passage are compatible with the hypothesis that the organic, histologically demonstrated diabetic microangiopathy is a long-term effect of periods of increased extravasation of plasma proteins, with subsequent protein deposition in the microvascular wall, i.e., the concept of plasmatic vasculosis. Arterial hypertension, frequently present in diabetes, enhances the development of arteriolar hyalinosis. Effective treatment of diabetes and hypertension arrests development of the microvascular lesions.


Muscle metabolism during rest and exercise: influence on the oxygen transport system of blood in normal and diabetic subjects. The oxygen dissociation curve shifted less to the right in venous blood draining from muscle in eight insulin-deficient diabetics working at a constant submaximal workload than in seven normal controls (28.7 mm. Hg vs. 30.8 mm Hg; P less than 0.05). This diminution of the in-vivo Bohr effect at the muscle tissue level during exercise in diabetics was due to a significantly smaller decrease of venous blood pH (down to 7.33 vs. 7.27 in normals; P less than 0.05), probably a consequence of an latered muscle metabolism in insulin deficiency. Although no glucose was taken up, even during exercise, and less lactate was produced by insulin-deficient muscle (P less than 0.05), the differences in venous blood pH appeared to be brought about mainly by a different CO2 production of the exercising muscle in the two groups. The response of Krebs cycle activity to exercise in insulin-deficient muscle might have been inadequate, as suggested by the increased 3-hydroxybutyrate/acetoacetate ratio in the venous blood observed in the normal controls but not in the diabetics. Furthermore, proportionally less of the arterial ketone body concentration was utilized by the working muscle in the insulin-deficient diabetics. Changes in erythrocyte 2,3-diphosphoglycerate did not contribute to the differences in the in-vivo Bohr effect.


Effect of duodenal acidification on gastric mucus and acid secretion in conscious cats. In cats with gastric fistulae and Heidenhein pouches, the effect of acid entering the duodenum on secretion of acid, pepsin, and mucus from the Heidenhain pouch during maximal acid stimulation with pentagastrin or histamine, was studied. Duodenal acidification produced stimulation of pepsin and mucus secretion comparable to that induced by exogenous hormones (secretin and the combination of secretin with cholecystokinin). In addition, duodenal acidification caused an increase in acid secretion, thus suggesting that, in addition to secretin and cholecystokinin, a factor that stimulates acid secretion was also released by acid.


Higher transpeptidation activity and broad acceptor specificity of gamma-glutamyltransferases of tumors. The gamma-glutamyltransferase (EC 2.3.2.2) (=gamma-glutamyltranspeptidase, gamma-GTP) activity in hepatoma induced by 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) was 120-fold higher than that of normal liver and high activity was also found in bovine hepatocellular carcinoma. gamma-GTPs from these malignant tissues responded more and showed broader specificity to gamma-glutamyl group acceptors than those from normal tissue such as bovine, rat, and mouse liver and bovine kidney. Three species of gamma-GTP were isolated from bovine kidney by DEAE-cellulose chromatography, whereas only two species were isolated from bovine hepatocellular carcinoma. The carcinoma lacked the least acidic enzyme species. Appropriate gamma-glutamyl group acceptors stimulated more-acidic enzyme species more than less-acidic species in both tissues. The fractions separated from the hepatoma were stimulated more than those of kidney by gamma-glutamyl group acceptor. The enzymes from normal tissues responded similarly to a gamma-glutamyl group acceptor irrespective of the difference in their activity. Thus, gamma-GTPs of malignant tissues appear to be more versatile for amino acid transport, both qualitatively and quantitatively. In these properties the enzyme of mouse fetal liver which showed the highest activity in the last period of pregnancy resembled the enzymes of malignant rather than normal tissues. The activity of hepatic gamma-GTP is not parallel with the rate of cell proliferation during normal development.


Ion selective effects of salicylate on antral mucosa. The effects of luminal salicylate, 5mM, were measured on rates of ion transport by isolated antral mucosa. At neutral luminal pH, salicylate increases Na and decreases Cl permeability. Salicylate does not alter net Na transport but decreases net Cl secretion. At luminal pH 4, the effects of salicylate can be arbitrarily divided into two phases. The intitial phase is associated with an increase in Na and marked decrease in Cl permeability. Subsequently, a greater increase in Na permeability and a marked increase in Cl permeability occurs. Active Na transport persists in the presence of salicylate at pH 4. Indirect evidence also suggests that Cl secretion persists under these conditions, buy at a reduced rate. The rate of luminal acid acid loss also increases in the presence of salicylate. A 4-fold increase in salicylate concentration or decrease in luminal pH from 4 to 3 did not appear to intensify the effects observed for 5 mM salicylate at pH 4. The increase in cation and decrease in anion permeability observed at pH 7 and initially at pH 4 are compatible with an influence of a negative charge of the salicylate anion. The subsequent changes observed in the presence of acid also are compatible with the concept that as the mucosa becomes overwhelmed with acid, a nonspecific increase in permeability occurs. However, the effect of salicylate on active Cl transport is largely independent of acid diffusing into the mucosa.


Mechanism of prevention of aspirin-induced gastric lesions by bile duct legation in the rat. Gastric reflux of bile has been reported to be essential for the production of acute gastric mucosal lesions by intragastric aspirin in the rat. The purpose of the present study was to determine whether bile duct legation of pylorus ligation in the rat inhibits asprin-induced gastric lesions, and, if so, what the protective mechanisms are. Operations were performed under ether anesthesia. Asprin, 200 mg per kg, was instilled into the stomach 1/2 hr postsurgery (bile duct ligation or pylorus ligation). Four hours later the rats were killed, the stomachs were examined, and mucosal lesions were scored. Bile duct ligation, but not pylorus ligation, significantly protected against aspirin-induced gastric-lesions. Bile duct ligation, in pylorus-ligated rats, inhibited gastric acid output by 78%. Instilling HCl + aspirin in bile duct-ligated rats restored lesion formation. Shunting bile to the colon (to prevent bile reflux) did not prevent aspirin lesions. Salicylate determination, to ascertain whether bile duct ligation altered asprin absorption, revealed no significant differences between bile duct ligation and aspirin, shunt + aspirin, and sham shunt + aspirin in plasma and gastric tissue salicylate concentrations. (1) Bile duct legation protects against aspirin-induced gastric mucosal lesions by inhibiting gastric HCl secretion. As a corollary, a certain amount of acid in the stomach is necessary for aspirin-induced gastric lesions to form. (2) Bile reflux is not necessary for aspirn-induced gastric lesions in the rat.


Gastric mucosal lesions produced by intravenous infusion of aspirin in cats. Aspirin was given by continuous intravenous infusion to 35 intact cats for 7 days in doses ranging from 25 to 200 mg kg-1 day-1. Gastric mucosal lesions occurred in 50 to 70% of the animals in the various dosage groups, including deep ulcers in 20%. All of the ulcers were in antral mucosa near its border with oxyntic mucosa. The incidence of lesions, including ulcers, showed no apparent relation to the dose of aspirin. With all but the highest dose, plasma salicylate levels were within or below what is regarded as the therapeutic range for man. Asprin, 100 mg kg-1 day-1, was given for 7 days to 4 cats with pouches containing all of the antral mucosa plus some oxyntic mucosa. One or more deep ulcers occurred in the antral mucosa of the pouches in each of these 4 cats. The electrical potential difference across the mucosa did not decrease, and net fluxes of hydrogen ions out of the pouch and of sodium ions into the pouch did not increase during the 7 days of aspirin administration despite the occurrence of ulcers in the pouches. It is concluded that intravenous aspirin, in doses giving plasma levels within or below the therapeutic range for man, causes gastric mucosal lesions including deep ulcers within 7 days in cats. These lesions occur without the changes in electrical potential difference and hydrogen and sodium fluxes that are regarded as characteristic of the "broken barrier."


Influence of acid secretory state on the gastric mucosal tolerance to back diffusion of H+. The effect of back diffusion of H+ PON THE POTENTIAL DIFFERence (PD), electrical resistance (R), and short circuit current (Isc) of spontaneously secreting and burimamide-inhibited amphibian gastric mucosae were studied in vitro. When back diffusion of H+ was induced by the passage of an electrical current of 510 mua per cm2 from the secretory (S) side of pH 2.25 to the nutrient side (N) for 30 min, the spontaneously secreting mucosae showed small decreases in PD (23.6 leads to 16.2 mv) and Isc (80 leads to 61 mua per cm2) but no significant change in R. In the burimamide-inhibited mucosae there were marked and significantly greater decreases of PD (26.8 leads to 3.4 mv), R (473 leads to 170 ohms cm2) and Isc (50 leads to 13 mua per cm2). When back diffusion of H+ was induced by establishing a concentration gradient of H+ from S leads to N or by exposing the secretory surface to sodium taurocholate with secretory pH 2.25, the burimamide-inhibited tissue demonstrated significantly greater depression of the electrical parameters than the secreting mucosae. Intracellular pH, measured by the 5,5-dimethoxyazolidine-2,4-dione method, was significantly higher in the histamine-stimulated tissues (7.28) than in the metiamide-inhibited tissues (7.11). These studies strongly suggest that the secretory status of the mucosa and hence its acid-base status are important determinants in the tolerance of the tissue to exogenous back diffusion of H+. The measure of absolute loss of H+ From the mucosal solution alone may not be an adequate assessment of the gastric mucosal barrier.


gamma-glutamyl transpeptidase of rat intestine: localization and possible role in amino acid transport. gamma-Glutamyl transpeptidase (gamma-GT), an enzyme possibly involved in amino acid transport, was investigated in rat small intestine using the synthetic substrate L-gamma-glutamyl-p-nitroanilide. Enzyme localization and characteristics were correlated with features of amino acid uptake. gamma-GT activity copurified with sucrase and alkaline phosphatase. Activity was maximal at pH 8.2 and was stimulated by monovalent cations. The relative specificity of the gamma-GT reaction with diglycine and eight essential amino acids as substrates correlated well with the rate of intestinal absorption of this dipeptide and these amino acids as observed by others. gamma-GT activity was 12-fold greater in the jejunum than in the ileum, again in agreement with relative rates of amino acid absorption along the length of rat intestine. The specific activity of gamma-GT in villus tip cells was 10 times greater than in crypt cells, and amino acid uptake was 2 to 6 times greater with villus tip than with crypt cells. Bromosulfophthalein, a noncompetitive inhibitor of gamma-GT, inhibited amino acid uptake. These studies support the concept that membrane gamma-GT may be involved in amino acid and dipeptide uptake, and indicate that further investigation of such involvement may be conveniently pursued using mammalian small bowel.


Association of inflammatory bowel disease and large vascular lesions. A case of inflammatory bowel disease with associated multiple large vessel vascular lesions similar to that seen in Takayasu's arteritis is described in a 15-year-old female. It is suggested that this type of vascular lesion may represent another rare systemic manifestation of inflammatory bowel disease.


Presence or absence of inhibitors of crystal growth in bile. 1. Effect of bile on the formation of calcium phosphate, a constituent of gallstones. When calcium and phosphate ions were mixed so that their final concentration was 4 mmol/1 and the pH was kept at 7-0, an amorphous precipitate immediately formed and this changed into crystalline material with an apatite-like structure after a period of time. The formation of either or both types of precipitate could be slowed down or prevented by adding to the crystallising medium trace amounts of pyrophosphate or citrate which are known inhibitors of the formation of calcium phosphate, or large quantities of sodium chloride which increased the ionic strength of the solution and hence the solubility of calcium phosphate, Both common duct and gallbladder bile from patients with gallstones composed of cholesterol and/or calcium carbonate had a very pronounced inhibitory action on the formation of these precipitates. Only very small amounts of bile were necessary to produce these effects, which therefore were not due to an increase in ionic strength. Ultrafiltration of bile showed that material with a molecular weight greater than 10 000 was mainly responsible for this activity. Because the inhibitor was present in both common duct and gallbladder bile, the liver is the likely source of origin. The possible identity of this material is examined. The powerful inhibitory effect of bile on the crystallisation of calcium phosphate is probably a contributory factor to the rare occurrence of the calcium phosphates, apatite and whitlockite, in gallstones.


[Interaction of dl-mandelamidine (Olimidine) with some antihypertensive drugs expressed in the blood pressure of conscious rats]. Hypotensive effect of dl-Mandelamidine (Olmidine, MA) in combination with some established antihypertensive drugs was studied in conscious normotensive rats. The mean blood pressure and heart rate were measured by means of a pressure transducer via a polyethylene tube inserted into the abdominal aorta of rat according to the method described by Weeks. The results obtained were as follows; 1) The hypotensive effects of guanethidine and hydrochlorothiazide were enhanced in combination with MA. 2) The hypotensive effect of reserpine was reduced by MA. 3) The hypotensive effects of clonidine, C6, propranolol and hydralazine were uneffected by MA. On the other hand, changes in heart rate induced by reserpine and C6 were increased by MA, however, those induced by guanethidine, clonidine propranolol and hydralazine were decreased by MA. The slight decrease in heart rate induced by hydrochlorothiazide was uneffected by MA. In view of our data, it is considered important that investigation of the interaction of antihypertensive drugs be done using conscious animals, as these drugs will be clinically prescribed.


[Metabolic fate of carteolol hydrochloride (OPC-1085), a new beta-adrenergic agent. (3) Autoradiographic total body distribution studies in mice]. Distribution of a new beta-adrenergic blocking agent, 3H-carteolol in mice was studied by whole body autoradiography. The distribution of radioactivity was observed in all organs except the eyes and brain, with particularly high specific activities in the kidneys, liver, gall bladder and content in the intestines within a short time after either oral or intravenous administration. The radioactivity was then promptly eliminated from all tissues and organs, and excreted almost entirely in the urine and bile. Propranolol is known to be distributed at a high concentration in the brain, whereas the concentration of (3H-) carteolol detectable in the brain was slight. In the adrenal gland, the radioactivity was localized in the medulla. Radioactivity was detected also in the stomach contents after the intravenous administration. The distribution of radioactivity in the fetus through the placenta was less than that in the major organs of the mother mouse, and the elimination of the activity was more rapid in the fetus than in mother. These findings indicate that carteolol and its metabolites do to some extent pass through the blood-brain barrier and placenta.


Biochemical basis of an animal model of depressive illness--a preliminary report--. Biochemical analyses of brain samples of an Animal Model of Depression indicate the state of motionlessness observed in response to a conditioned stimulus was due to an excess in functional activity of serotonin. An excess functional activity of serotonin may be directly responsible for human depressive illness. This conflicting conclusion to the currently popular theories of serotonin deficiency was discussed with reference to the animal and clinical data in the literature which are consistent with the conclusion.


[Current resistence situation in a surgical and urological department]. Spectrum and sensitivity of bacteria were studied at the Surgical (534 positive wound smears) and the Urological Clinics (7879 urine specimens). Krankenhaus Nordwest, Frankfurt/M., during the period of 1969-1971 and in 1973. The most common organisms identified in wound smears were E. coli, followed by Staph. areus, Aerobacter and Proteus species. E. coli were also predominant in urine, but followed by Enterococci, Proteus and Pseudomonas. E. coli, Proteus species and especially Pseudomonas increased in number whereas Enterococci decreased. There was no pronounced increase in resistance to 9 current antibiotics as well as to chemotherapeutics during the observation period which was particularly striking in the case of Ampicillin used on a large scale. The results of our study support the presently employed therapeutic method using bactericidal antibiotics of the penicillin group in strict indications.


[Newer aspects in the therapy of angina pectoris]. Progress in coronary surgery has led to new aspects in the treatment of angina pectoris. According to the coronar-angiographic findings, which make an exact identification of the coronary heart disease possible, and according to the ventricular function a therapeutic strategy can be determined today with adequate safety for each individual case. The most important criterium for a decision for surgical or conservative treatment is not only the improvement of the subjective symptoms, in special forms of coronary heart disease the bypass-operation not only results in improved quality of life but also in a considerably increased survival time.


[Inflammatory intestinal diseases: ulcerative colitis and Crohn's disease. Early diagnosis and treatment]. Early rather characteristic symptoms are found in ulcerative colitis as well as in Crohn's disease. In these patients, but also in an advanced stage, differential diagnosis between these two disorders is possible by means of endoscopic techniques and guided biopsy. In a high percentage of patients with ulcerative colitis good results are obtained with conservative therapy, using salazopyridine alone or in combination with cortisone. Combination therapy with salazopyridine and cortisone seems to offer the best results in Crohn's disease too, however, in a certain percentage of cases surgical intervention is unavoidable.


[Polygraphic recording of sleep under the influence of Plantival plus]. 10 patients with moderate non-psychotic sleep disturbances were investigated polygraphically for a total of 14 nights each. These 14 nights were subdivided into 4 test series: Placebo was given during the first, Plantival plus during the second and third and placebo again during the fourth series. The results showed that wakefulness decreased after the application of Plantival plus and that there was an increase in deep sleep. These effects could be observed mainly during the first hour of sleep and disappeared gradually during the later sleeping hours. REM-phases were not influenced by the medication. Due to these observations, Plantival plus should mainly be applicated when disturbances in falling asleep and problems in sleeping continuously occur. In case interrupted sleep in the early morning hours has been found, this preparation could be applicated only in combination with other kinds of treatment.


Poly(adenosine diphosphate ribose) is covalently linked to nuclear proteins by two types of bonds. (ADP-ribose)n residues formed by short-term incubation of adult rat liver and Ehrlich carcinoma nuclei with labeled NAD were analyzed by Cs2SO4/guanidinium chloride/urea density gradient centrifugation. Comparison with samples in which the protein had been completely digested revealed that most, or probably all, acid-insoluble (ADP-ribose)n chains are covalently bound to nuclear proteins, as is true for the short, acid-soluble (ADP-ribose)n chains. Complete release of (ADP-ribose)n chains is effected by dilute alkali. In contrast, NH2OH liberated only part of the long and the short (ADP-ribose)n residues from the protein conjugates, indicating two types of bonds, both alkali-labile, but only one susceptible to neutral hydroxylamine. Both types of bonds were equally distributed among acid-soluble and acid-insoluble (ADP-ribose)n chains. -Stability of the (ADP-ribose)n protein conjugates during isolation is only guaranteed at pH values below 7.


On the mechanism and some properties of vinylacetyl-CoA delta-isomerase of Clostridium kluyveri. Vinylacetyl-CoA delta-isomerase from Clostridium kluyveri grown on ethanol/acetate was purified 32-fold. The enzyme is rather labile. All experiments were conducted with the substrate analog thioester of vinylacetic acid and N-acetylchysteamine (vinylacetyl-SEtNAc (1 f)). 3-Butinoyl-SEtNAc is a strong inhibitor of the isomerase. The hydrogen transfer from the alpha-position of vinylacetyl-SEtNAc to the gamma-position of 2-butenoyl-SEtNAc (1f leads to 2 f) occurs partially intramolecularly (40-50%) as shown by experiments in 3HOH/H2O, 2H2O and 3HOH/2H2O as well as by experiments with [2,3-3H]vinylacetyl-SEtNAc. Only 0.07 atoms of tritium are incorporated into the gamma-position of 2f when the isomerisation takes place in 3HOH/H2O. The extent of intramolecularity is in agreement with results of experiments conducted in 2H2O with whole cells [4]. The reaction 1f leads to 2f shows no or only negligiebl reversibility and at least no considerable isotope effect.


A kallikrein-specific inhibitor in rat kidney tubules. A kallikrein inhibitor was found in tubules of the rat kidney and purified by chromatography on Sephadex G-100. The molecular weight of the inhibitor, estimated by gel filtration and dodecylsulfate electrophoresis, is about 4700. It inhibits the following kallikreins: porcine submanidbular and pancreatic kallikrein, rat kidney and urine kallikrein, and human urine and plasma kallikrein. An inhibition of bovine trypsin was not observed.


[Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)]. The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish  trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.


Identity of kynurenine: pyruvate aminotransferase with histidine: pyruvate aminotransferase. Kynurenine pyruvate aminotransferase was purified from rat kidney. The purified enzyme had an isoelectric point of pH 5.2 and a pH optimum of 9.3. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors. L-Amino acids were effective in the following order of activity: histidine greather than phenylalanine greater than kynurenine greater than tyrosine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values were about 0.63 mM, 1.4 mM and 0.09 mM for histidine, kynurenine and phenylalanine, respectively. Km values for pyruvate were 5.5 mM with histidine as amino donor, 1.3 mM with kynurenine and 8.5 mM with phenylalanine. Kynurenine pyruvate aminotransferase activity of the enzyme was inhibited by the addition of histidine or phenylalanine. The molecular weights determined by gel filtration and sucrose density gradient centrifugation were approximately 76000 and 79000, respectively. On the basis of purification ratio, substrate specificity, inhibition by common substrates, subcellular distribution, isoelectric focusing and polyacrylamide-gel electrophoresis, it is suggested that kynurenine pyruvate aminotransferase is identical with histidine pyruvate aminotransferase and also with phenylalanine pyruvate aminotransferase. The physiological significance of the enzyme is discussed.


Studies on the proteinase-A inhibitor I3A from yeast. The purification and some properties of the two inhibitors I2A and I3A of proteinase A from yeast have previously been described [Saheki et al, (1974) Eur. J. Biochem. 47, 325]. An improved method for the preparation of I3A which is less time-consuming and leads to higher yields is presented. Based on amino acid analysis, I3A contains 68 amino acids per molecule. The molecular weight was 7676. The inhibitor contained no proline, no arginine, no cysteine and no tryptophan, but did contain a large number of the polar amino acids glutamate + glutamine, aspartate + asparagine and lysine. Neither by dansylation nor by Edman degradation could an N-terminal amino acid be detected. Changes in the circular dichroism upon transition from pH 6.9 to 3.0 suggest different tertiary structures at these pH values. Experiments on the kinetics of inhibition of proteinase A revealed an apparent Ki value of 5.5 X 10(-8) M for I3A and 1.6 X 10(-8) M for pepstatin. A "non-stoichiometric inhibition" of a "pseudo-irreversible" type is concluded from the kinetic data. A hydrophobic type of binding of I3A to yeast proteinase A is suggested from experiments demonstrating a large decrease in the percentage of inhibition caused by addition of 2 M urea, 2 M guanidine hydrochloride, 0.125% Triton or 0.125% cholic acid.


Antigenicity of type-specific pneumococcal polysaccharides in rats. Hemagglutinating antibody responses of Lewis-Wistar and Sprague-Dawley rats to graded doses of type-specific pneumococcal polysaccharide were measured. Rats given a small dose (0.2 to 50 mug)of type 1 or 8 polysaccharide intraperitoneally developed type-specific hemagglutinating antibody. Rats given larger doses of polysaccharide (greater than or equal to200 mug) did not develop detectable hemagglutinating antibody, and they were unresponsive to a subsequence injection of a small (and normally antigenic) dose of polysaccharide. There was prolonged antigenemia in rats injected with a large dose of polysaccharide. There was prolonged antigenemia in rats injected with a large dose of polysaccharide, and the kinetics of antigen clearance in these animals resembled that reported for mice with polysaccharide immunological paralysis. These results indicate that a phenomenon resembling immunological paralysis with type-specific pneumococcal polysaccharides can be produced in rats.


In vitro differentiation and pH sensitivity of field and cell culture-attentuated strains of transmissible gastroenteritis virus. Characteristics of four transmissible gastroenteritis (TGE) virus field strains (Miller, Purdue, Bl, and V203) and four cell culture-attenuated strains (Purdue, SH, CKp, and Bl) were studied to find methods of differentiation between the two groups of viruses. TGE field virus strains did not replicate as well as attenuated strains at 37 C and could not be passaged serially for more than four to six passages at 33 C. There were clear differences in plaque size when the strains were compared. Field strains had average plaque sizes ranging from 3.59 to 3.15 mm, whereas attenuated strains induced plaques that were larger than 4.2 mm. Variations were observed in stability of strains at pH 3.0. Field strains and cell culture-attenuated strains CKp-270 and SH-114 were reduced in titer by about 1 log10. A reduction of about 3 log10, however, was obtained with cell culture strains B1-300 and Purdue-113.


Hemagglutination by equine infectious anemia virus. Equine infectious anemia (EIA) virus which was propagated on an equine dermal cell line agglutinated guinea pig erythrocytes. Viral fluids containing about 10(7.5) mean tissue culture infective doses/ml showed hemagglutinating (HA) titers ranging from 16 to 32 units/0.05 ml. Results of cesium chloride equilibrium density gradient centrifugation revealed that the hemagglutinin was inseparable from the virus particles. The hemagglutination reaction persisted over a wide range of temperature and pH, and the absence of divalent cations did not decrease its activity. The HA activity was stable at 4 degrees C but not at 56 degreesC. The activity was destroyed by virus-disrupting lipid solvents and moderately sensitive to a proteolytic enzyme. Neuraminidase enhanced HA activity slightly. Phospholipase C had no effect on HA titer, although it completely inactivated infectivity. It was relatively stable to ultraviolet irradiation. Thus, the hemagglutinin appears to be closely associated with virus particles, and its activity is dependent on the presence of its lipids and proteins. Hemagglutination was inhibited by sera from horses infected with EIA virus. Hemagglutinin receptors on the erythrocytes were inactivated by a proteolytic enzyme and formaldehyde but were not influenced by neuraminidase, sodium deoxycholate, or KIO4.


Humoral immunity to Streptococcus pneumoniae induced by a pneumococcal ribosomal protein fraction. Isolation of a protective subfraction of ribosomes from Streptococcus pneumoniae has been achieved, and the immune response it induces has been investigated. Mice immunized with pneumococcal ribosomes or purified protein extracted from the ribosomal preparation (2-CE protein) exhibit similar survival rates upon challenge by virulent S. pneumoniae. In contrast, recipients of purified ribosomal ribonucleic acid were never protected against pneumococcal challenge. Serum from mice immunized with pneumococcal ribosomes or 2-CE protein passively immunized syngeneic recipients against pneumococcal challenge, whereas spleen cells from the same donors were unable to transfer immunity. Passive immunization with antiribosome serum could be abrogated by absorption with whole ribosomes, 2-CE protein, or various serotypes of S. pneumoniae (capsular types 2, 3, 6, and 14). Antiribosome serum significantly enhanced clearance of S. pneumoniae from mouse blood in vivo and in vitro. This required phagocytic cells, since antiribosome serum alone, with or without complement, supported growth of S. pneumoniae to an extent comparable to normal serum. The data suggest that the primary immunogen of pneumococcal ribosomes resides in the protein fraction. Further, the immunity induced by the protein fraction is mediated by antibody that appears to function as an opsonin for S. pneumoniae.


Factors influencing heat-labile Escherichia coli enterotoxin activity. In this study, conditions for production, detection, and storage of heat-labile Escherichia coli enterotoxin (LT) in culture filtrates from E. coli H-10407 were defined by using the adrenal tumor cell assay system. An enriched medium containing 0.6% yeast extract, 2% Casamino Acids, and 0.25% glucose buffered at pH 8.5 produced the highest LT activity of the various test media. In E. coli strain H-10407, LT activity was markedly decreased if the initial pH of the culture media was reduced to pH 7.5 or less. In contrast to E. coli P-263, if strain H-10407 was grown in the presence of mitomycin C there was no increase in LT production. Crude-culture filtrates containing LT can be stored at 4 degrees C for several days without an appreciable loss of activity; however, for long-term storage lyophilization or freezing at -70 degrees C is recommended.


Factors influencing the immune enhancement of intrapulmonary bactericidal mechanisms. The effect of specific immunization on the antibacterial defense mechanisms of the murine lung was assessed against Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus aureus (Smith), Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Immunization by aerosol inhalation significantly enhanced the intrapulmonary killing of Pseudomonas aeruginosa and Proteus mirabilis but not the remaining organisms. With P. mirabilis, systemic immunization induced higher titers of specific serum agglutinins as compared with local respiratory tract immunization; however, local immunization was more effective in enhancing pulmonary bactericidal activity than was parenteral vaccination. Passive immunity against P. mirabilis or aerogenic challenge with preopsonized P. mirabilis significantly enhanced intrapulmonary killing of the homologous organism. With S. aureus, pulmonary bactericidal activity was not accelerated by aerosol challenge with the preopsonized organism, nor was it accelerated in passively immunized mice. These data demonstrate that the immune enhancement of pulmonary bactericidal activity is governed by the bacterium used for challenge and the route of immunization. The results further demonstrate that with P. mirabilis, antibody-mediated mechanisms are involved in the immune enhancement of pulmonary bactericidal activity.


Purification and properties of streptococcal hyaluronate lyase. Hyaluronate lyase (hyaluronidase) has been purified and characterized from a group A type 4 Streptococcus. Production of the enzyme was favored by growth in trypsinized veal infusion in the presence of hyaluronate oligosaccharide and tetrasaccharide. Detectable enzymatic activity was diminished in the presence of N-acetylglucosamine and glucuronic acid. Purification of hyaluronate lyase consisted of 40 to 60% ammonium sulfate precipitation, diethylaminoethyl A-50 Sephadex ion-exchange chromatography, gel filtration with G-200 Sephadex, and adsorption to Sepharose 6B. Purified enzyme was antigenically homogeneous and free of proteinase, deoxyribonuclease, streptolysin 0, and streptokinase. Active hyaluronate lyase was recovered from neutral polyacrylamide gels, and it appeared to be a glycoprotein. A single band was detected by sodium dodecyl sulfate-acrylamide electrophoresis, which had a molecular weight of approximately 50,000. A molecular weight of 70,000 was observed by gel filtration. The purified enzyme had a Km of 3.8 x 10(-4) and a pH optimum of 6.0. Reducing agents increased the activity of crude enzyme at least threefold and were necessary to prevent inactivation of the purified enzyme.


Hypnotic and minor tranquilizer use among inpatients and after discharge. A major concern in prescribing hypnotics and minor tranquilizers in the hospital is that long-term habituation might develop. Former psychiatric inpatients were surveyed to assess hypnotic and minor tranquilizer use following discharge. Patients who used flurazepam and chloral hydrate as inpatients were no more likely to use these hypnotics as outpatients than patients who received no hypnotics as inpatients. There was a significant association between inpatient and outpatient use of minor tranquilizers. Outpatient use of minor tranquilizers was also significantly associated with outpatient hypnotic use.


Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase. Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.


[A medical-legal question of hearing impairment following vaccination (author's transl)]. Three months following smallpox vaccination, a child was found to have profound sensory deafness. A medical-legal opinion in 1962 found no relationship between vaccination and deafness. However, when subsequent judgement appeal was made, the legality of the initial decision had to be justified. The consequence of the case, supported by available publication, indicated that a close temporal probability between vaccination and postvaccinial cerebral complication was required to justify any further legal claim. This was not considered to be a factor in the present case since the discovery of a goitre in the patient in association with an iodine dysfunction uncovered a Pendred's syndrome as the cause for the patient's hearing loss.


A pressure- and flow-insensitive reference electrode liquid junction. The design and construction of a pressure- and flow-insensitive reference liquid junction for use in ion concentration electrode measuring systems is described. The junction is inexpensive, is very easily and rapidly constructed, is rugged, and is adaptable to various applications. When used in a pH-measuring system, drift, pressure artifacts, and flow artifacts are negligible. The response time of the system appears to be less than 10 ms. Using the pH electrode device as described, the dissociation reaction rate constant of H2CO3 at 24 degrees C was determined to be 22 s-1.


Alkaline shift in lumbar and intracranial CSF in man after 5 days at high altitude. In six healthy male volunteers at sea level (PB 747-759 Torr), we measured pH and PCO2 in cerebrospinal fluid (CSF), and in arterial and jugular bulb blood; from these data we estimated PCO2 (12) and pH for the intracranial portion of CSF. The measurements were repeated after 5 days in a hypobaric chamber (PB 447 Torr). Both lumbar and intracranial CSF were significantly more alkaline at simulated altitude than at sea level. Decrease in [HCO3-] IN lumbar CSF at altitude was similar to that in blood plasma. Both at sea level and at high altitude, PCO2 measured in the lumbar CSF was higher than that estimated for the intracranial CSF. At altitude, hyperoxia, in comparison with breathing room air, resulted in an increase in intracranial PCO2, and a decrease in the estimated pH in intracranial CSF. With hyperoxia at altitude, alveolar ventilation was significantly higher than during sea-level hyperoxia or normoxia, confirming that a degree of acclimatization had occurred. Changes in cerebral arteriovenous differences in CO2, measured in three subjects, suggest that cerebral blood flow may have been elevated after 5 days at altitude.


Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae. The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.


Thiols of myosin. IV. "Abnormal" reactivity of S1 thiol and the conformational changes around S2 thiol. The flexibility of the tertiary structure around the active site of myosin ATPase [EC 3.6.1.3] was studied using the reactivity of two specific thiol groups, S1 and S2, as a structural probe. The following four maleimide derivatives were used as thiol-directed reagents: N-ethylmaleimide (NEM), N-(4-methoxy-2-benzimidazolyl methyl) maleimide (MBM), N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) and N-(4-dimethyl-amino-3,5-dinitrophenyl)maleimide (DDPM). 1. All the maleimide derivatives used activated the Ca2+-ATPase activity and inhibited the EDTA-ATPase activity, like NEM, indicating that they modified S1. The rate of modification of S1 by NEM and BIPM increased with increasing pH, while that by DDPM decreased. BIPM simultaneously modified S1 and S2. 2. S1 showed much higher reactivity toward the maleimides, except for BIPM, than did N-acetylcysteine (N-Ac-Cys) a low molecular-weight model compound. The extremely small pKa value of S1, 6.28, accounted for this high reactivity. In addition, the ATP-induced increase in its reactivity inducated that S1 was in a buried state. Kinetic analysis showed that the teritiary structure around S1 at alkaline pH differed from that at acidic pH. 3. The apparent rate constant of S2-modification with NEM was approximately one seven-hundredth and one four-hundredth of those of S1 and N-Ac-Cys, respectively. Fluorimetric studies using BIPM revealed that S2 in the buried state was exposed upon adding ATP; this was compensated by the burying of some other thiol group(s) (Sp). Non-linearity of the Arrhenius plots of the reaction rate of S2 suggested that the S2 region of myosin had different conformations at high and low temperatures, the transition temperature being 10--15degrees. This non-linearity completely disappeared in the presence of Mg2+-ATP. On the other hand, Arrhenius plots for the thiols reactive to BIPM did not show non-linearity in the presence or absence of ATP.


Stabilization of human serum alkaline phosphatase to histidine-induced heat inactivation by tryptic digestion. 1. Serum alkaline phosphatase [EC 3.1.3.1] was strongly inactivated by histidine during incubation at pH 8.0 and 45degrees; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further. 2. The effect of trypsin on the histidine-induced heat inactivation differed considerably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.


Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope. The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.


Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana. In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.


Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase. Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.


Incorporation of serine and ethanolamine into the phospholipids in rabbit retina. The incorporation of serine and ethanolamine into phospholipids in rabbit retinal subcellular fractions and in excised retinas was studied in vitro, and some enzymic properties of the incorporation of phospholipid bases by base exchange were examined in the microsomal fraction. The retina was found to have a higher rate of base exchange for the incorporation of phospholipid bases than other tissues. The retinal microsomal fraction possessed the highest specific activity of base exchange, while the rod outer segment had very little activity. These results suggest that the phospholipids in the rod outer segment may be transferred from the inner segment of the photorecepter cell. The apparent Km values for serine and ethanolamine in the microsomal fraction decreased with decreasing Ca2+ concentration. Although no further increase of incorporation of serine and ethanolamine occurred after 40 min in the microsomal fraction, continuous incorporation of both bases into phospholipids was seen for 3 hr in excised retina. Illumination did not significantly affect the incorporation of serine and ethanolamine in excised retina or in the rod outer segment fraction. Base exchange reaction thus may not play a direct role in the visual process.


The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors. Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.


Purification and properties of an enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62 strain. I. Splitting enzyme 1. An enzyme (S-1) which catalyzes the splitting of carbon-mercury linkages of organomercury compounds was purified about 24-fold from the cell-free extract of mercury-resistant Pseudomonas K-62 strain by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-150, DEAE-Sephadex, and DEAE-cellulose. A purified preparation of the enzyme showed a single band on polyacrylamide gel electrophoresis, and was colorless. The molecular weight of the enzyme was estimated to be 19,000, and Km was 5.3 X 10(-5) M for p-chloromercuribenzoic acid (PCMB). The temperature and pH optimum for the reaction were 50degrees and 7.0, respectively. The enzyme was capable of catalyzing the decomposition of methylmercuric chloride (MMC), ethylmercuric chloride (EMC), phenylmercuric acetate (PMA), and PCMB in the presence of a sulfhydryl compound to form a mercuric ion plus methane, ethane, benzene, or benzoic acid, respectively. The mercuric ion thus formed was reduced to metallic mercury by metallic mercury-releasing enzyme (MMR-enzyme).


Interaction of tropomyosin with troponin components. 1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.


Purification and characterization of beta-N-acetylhexosaminidases and beta-galactosidase from Streptococcus 6646 K. Three beta-N-acetylhexosaminidases [EC 3.2.1.52] and one beta-galactosidase [EC 3.2.1.23] were purified from the culture filtrate of streptococcus 6646 group K by a combination of column chromatographies on p-aminophenyl beta-D-thiogalactopyranoside-substituted Sepharose and N-(paminophenyl)oxamic acid-substituted Sepharose. These beta-N-acetylhexosaminidases showed optimal activities between pH 5.0 and 5.5 and could hydrolyze synthetic and glycopeptidic substrates. Glycolipids such as GM2, asialo-GM2, and globoside I were no susceptible to these beta-hexosaminidases. beta-Galactosidase, which was purified more than 11,000-fold, had a substrate specificity rather similar to that of beta-galactosidase from E. coli. This enzyme was inhibited by EDTA and activated by Mn2+, Ca2+, and Mg2+. Problems pertinent to the application of affinity chromatography to the purification of glycosidases are also discussed.


On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in "sonic" submitochondrial particles. A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.


3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli. The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.2.1.15) was purified to homogeneity from extracts of Escherichia coli K12. A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at 232 nm, was developed. The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-200, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This suggests either a rapid monomer-dimer equilibrium, or a very asymmetric shape for the native enzyme. The enzyme shows a narrow pH optimum around pH 7.0. The enzyme is stable for several months when stored at -20 degrees in phosphate buffer containing phosphoenol-pyruvate. Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction. Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex. The feedback inhibitor tyrosine at concentrations above 10 muM exhibits noncompetitive inhibition with respect to erythrose-4-P, and competitive inhibition with respect to the other substrate, P-enolpyruvate. In addition, tyrosine at concentrations of at least 10 muM causes an alteration of one or more than one kinetic parameter of the enzyme.


Human adenosine deaminase. Distribution and properties. Adenosine deaminase exists in multiple molecular forms in human tissue. One form of the enzyme appears to be "particulate". Three forms of the enzyme are soluble and interconvertible with apparent molecular weights of approximately 36,000, 114,000, and 298,000 (designated small, intermediate, and large, respectively). The small form of adenosine deaminase is convertible to the large form only in the presence of a protein, which has an apparent molecular weight of 200,000 and has no adenosine deaminase activity. This conversion of the small form of the enzyme to the large form occurs at 4 degrees, exhibits a pH optimum of 5.0 to 8.0, and is associated with a loss of conversion activity. The small form of the enzyme predominates in tissue preparations exhibiting the higher enzyme-specific activities and no detectable conversion activity. The large form of adenosine deaminase predominates in tissue extracts exhibiting the lower enzyme specific activities and abundant conversion activity. The small form of adenosine deaminase shows several electrophoretic variants by isoelectric focusing. The electrophoretic heterogeneity observed with the large form of the enzyme is similar to that observed with the small form, with the exception that several additional electrophoretic variants are uniformly identified. No organ specificity is demonstrable for the different electrophoretic forms. The kinetic characteristics of the three soluble molecular species of adenosine deaminase are identical except for pH optimum, which is 5.5 for the intermediate species and 7.0 to 7.4 for the large and small forms.


Electron paramagnetic resonance of the tungsten derivative of rat liver sulfite oxidase. Sulfite oxidase purified from livers of tungsten-treated rats has been used for EPR studies of tungsten substituted at the molybdenum site of the enzyme in a fraction of the molecules. The EPR signal of W(V) in sulfite oxidase is quite similar to that of Mo(V) in its line shape and in its sensitivity to the presence of anions such as phosphate and fluoride. Hyperfine interaction with a dissociable proton is also observed in both signals. The pH-dependent alteration in line shape exhibited by the Mo(V) EPR signal of the rat liver enzyme. Incomplete reduction of the tungsten center at pH 9 is indicated by attenuated signal intensity at this pH. The W(V) signal has g values lower than those of the Mo(V) signal, has a much broader resonance envelope, and is much less readily saturated by increasing microwave power. Kinetic studies on the reduction of the heme and tungsten centers of sulfite oxidase have shown that reduction of de-molybdo forms of sulfite oxidase by sulfite is catalyzed by the residual traces of native molybdenum-containing molecules. Reduction is accomplished by electron transfer involving intermolecular heme-heme interaction. The W(V) signal is generated only after all the heme centers are reduced. The rate and extent of heme reduction at pH 9 are the same as at pH 7. Studies on the reoxidation of W(V) and reduced heme by O2 and by cytochrome c suggest that the cytochrome b5 of sulfite oxidase is the site of electron transfer to cytochrome c, whereas oxidase activity is the property of the molybdenum center. It appears that the tungsten center in sulfite oxidase is incapable of oxidizing sulfite.


Tetramer-dimer dissociation in homoglobin and the Bohr effect. The pH dependence of the apparent tetramer to dimer dissociation constant has been determined at 20 degrees for both oxy- and deoxyhemoglobins A and Kansas. These measurements were made by three different procedures: gel chromatography, sedimentation velocity, and kinetic methods in either of three buffer systems: 0.05 M cacodylate, Tris, or glycine with 1 mM EDTA and 0.1 M NaCl between pH 6.5 and 11. The tetramer-dimer dissociation constant of human oxyhemoglobin A decreases from about 3.2 X 10(-6) M at pH 6.0 to about 3.2 X 10(-8) M at pH 8.5. The slope of this line indicates that the dissociation of tetramer to dimer is accompanied by the uptake of about 0.6 protons per mol of tetramer in this region. The corresponding dissociation constant for deoxyhemoglobin in the same pH region increases apparently almost linearly from 1.0 x 10(-12) M at pH 6.5 to about 1.0 x 10(-5) M at pH 11. To dimer is associated with the release of about 1.6 protons per mol of tetramer. Comparison of these data with the known proton release accompanying the oxygenation of tetramers confirms that the pH dependence of oxygen binding by dimers must be very small. The present data predict that the overall proton release or uptake per oxygen bound by dimer should be less than 0.1. The tetramer-dimer dissociation equilibria of oxy- and deoxyhemoglobins above pH 8.5 have identical pH dependences. In this range the dissociation constant of deoxy-Hb is about one-tenth that of oxyhemoglobin. Human oxyhemoglobin Kansas is known to have an enhanced tetramer-dimer dissociation compared with that of hemoglobin A. Below pH 8.5 the tetramer-dimer dissociation constant of Hb Kansas is about 400 times greater than that of HbA in the absence of phosphate buffers. In contrast, the tetramer-dimer dissociation constants of deoxyhemoglobins A and Kansas appear to be identical. These findings are consistent with previous structural observations on these hemoglobins. The data on the tetramer-dimer dissociation of human hemoglobin were used to calculate the total free energy of binding of oxygen to the tetramer and the median oxygen pressure on the basis of fundamental linkage relations and a pH-independent estimate of the total free energy of binding oxygen to dimer. Simulated oxygen binding curves were generated with the equations of Ackers and Halvorson (Ackers, G. K., and Halvorson, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4312-4316) by making two assumptions: (a) that the dimers are noncooperative and pH-independent in O2 binding and (b) that the distribution of cooperative energy in the oxygenation of tetramers is independent of pH. We have compared these simulations with experimental data obtained at low protein concentrations (30 to 124 muM heme) to show that the variation in oxygen affinity with pH can be described in terms of the subunit equilibria. We conclude that an accurate analysis of the contributions of individual oxygen binding steps to the Bohr effect cannot be made without considering the contributions of the dimers to oxygen binding...


Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase. Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.


ATP-dependent calcium transport in isolated membrane vesicles from Azotobacter vinelandii. Membrane vesicles from Azotobacter vinelandii O prepared by osmotic lysis of spheroplasts in tris (hydroxymethyl) aminomethane/acetate buffer (pH 7.8) contain a latent adenosine triphosphatase (ATPase). The ATPase can be activated when the vesicles are incubated in the presence of an electron donor (D-lactate) and a mixture of adenosine diphosphate and inorganic phosphate or by controlled treatment with trypsin. After the ATPase is activated, the membrane vesicles in the presence of adenosine triphosphate accumulate calcium but not glucose or rubidium (in the presence of valinomycin). ATP-dependent calcium uptake follows Michaelis-Menten kinetics with a Km of 48 muM and a Vmax of 20 nmol/min/mg of membrane protein and is highly specific for calcium over cations magnesium, barium, lanthanum, sodium, potassium, and lithium. The calcium accumulated in the presence of ATP is freely exchangeable with external calcium and is rapidly released in the presenceof uncouplers or ATPase inhibitors. Calcium uptake in the presenceof ATP is blocked by dicyclohexylcarbodiimide, ADP, p-chloromercuriphenylsulfonate, by the proton-conducting ionophores m-chlorophenylcarbonylcyanide hydrazone, nigericin, monensin, and gramicidin D, but not by potassium cyanide, anoxia, or valinomycin (in the presence of potassium). Measurements of the external pH of vesicle suspensions reveal that protons are actively taken up by the membranes during hydrolysis of ATP. These results suggest that vesicles prepared under these conditions have a topology which is inverted with respect to the intact cell and that calcium is accumulated by means of proton antiport.


Phosphofructokinase. I. Mechanism of the pH-dependent inactivation and reactivation of the rabbit muscle enzyme. The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase have been studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. From the enzyme concentration dependence, we conclude that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of one-half the molecular weight. Other data indicate a subsequent isomerization of the dissociated form. The pH and temperature dependence of the inactivation process shows that it is controlled by ionizable groups, and that the apparent pK for these groups is temperature-dependent in such a way as to make the enzyme show the characteristic of cold lability below pH 7. Reactivation of the inactive enzyme occurs by a kinetically different pathway involving deprotonation of an inactive, dissociated form to a form which may either isomerize to another inactive form, or dimerize to the active enzyme. A general mechanism is postulated in which the inactivation and reactivation processes are different aspects of the same mechanism. This mechanism assumes four species (two containing four subunits and two containing two subunits) each of which can exist in a protonated and unprotonated form. Inactivation or reactivation induced by changes in pH or temperature reflect the kinetic establishment of a new steady state between these forms. How the apparent pK values which control the distribution of the enzyme between protonated and unprotonated forms describe the pH-dependent characteristics of the enzyme is discussed in terms of the proposed mechanism.


Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme. The effect of ligands, including substrates and allosteric effectors, on the pH-dependent inactivation and reactivation of rabbit muscle phosphofructokinase has been examined in terms of the mechanism proposed previously (Bock, P.E. and Fireden, C. (1976) J. Biol. Chem. 251, 5630-5636). It is concluded thatt many ligands exert their effect by binding preferentially to either protonated or unprotonated forms of the enzyme and thus shifting an apparent pK for the inactivation or reactivation process. ATP and fructose 6-phosphate influence the apparent pK to different extents and in different directions, with ATP binding preferentially to the protonated forms and fructose 6-phosphate to the unprotonated forms. Enzyme inactivated by ATP can be reactivated by the addition of fructose 6-phosphate. The experiments indicate that inactivation and reactivation in the presence of these ligands can occur by kinetically different pathways as has been found for these processes in the absence of ligands. The results are discussed in relation to what might be expected for ligand binding properties of the enzyme as a function of pH, temperature, and enzyme concentration. The effect of ATP and MgATP is complex, perhaps representing more than one site of binding. Citrate appears to bind preferentially to protonated forms of the enzyme while fructose 1,6-bisphosphate and AMP bind preferentially to the unprotonated forms. ADP, K+, and NH4+ appear to have little or no preference in binding to different enzyme forms.


Phosphofructokinase. III. Correlation of the regulatory kinetic and molecular properties of the rabbit muscle enzyme. It is shown that the degree of regulatory kinetic behavior of rabbit muscle phosphofructokinase increases at a given pH and lower temperatures, as well as at a given temperature and lower pH values. It is also shown that the regulatory kinetic behavior which appears at lower pH values is inherent in the tetrameric (active) form of the enzyme. We conclude that a portion of the mechanism proposed previously (Bock, P.E., and Frieden, C. (1976) J. Biol. Chem. 251, 5630-5636) to describe the pH and temperature-dependent inactivation or reactivation may also be used to explain the pH and temperature-dependent regulatory kinetic behavior. According to this proposal, two rapidly equilibrating forms of the enzyme, which differ in the degree of protonation of specific residues, differ in their ability to bind substrates. While the protonated form of the enzyme subsequently becomes inactive by isomerization and dissociation, this process is too slow to affect the kinetic results, making direct comparisons between the association-dissociation behavior and regulatory kinetic behavior invalid. The time dependence of the processes of inactivation or reactivation in the presence or absence of ligands and of the appearance of regulatory kinetic behavior is discussed in relation to their possible role in metabolic regulation.


Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique. The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport.


Facile alkylation of methionine by benzyl bromide and demonstration of fumarase inactivation accompanied by alkylation of a methionine residue. Benzyl bromide is a selective alkylator of sulfur nucleophiles including methionine and cysteine. Only the mercaptide ion is a more efficient nucleophile than is the sulfur ether of methionine. Alkylation rates relative to methionine are 200: less than or equal to 0.03: less than or equal to 0.03: less than or equal to 0.02 for GS-, histidine, tryptophan, and GSH, respectively. Alkylation of methionine by benzyl bromide is more than 50 times faster than alkylation by iodoacetate. Fumarase is readily inactivated by exposure to benzyl bromide at pH 6.6 to 6.8 accompanied by alkylation of close to 1 methionine residue/subunit. Fumarase fully inactivated by exposure to benzyl bromide shows no detected alkylation of amino acid residues other than methionine. The rate of inactivation of fumarase by benzyl bromide is decreased about 4-fold by the presence of excess substrates. Denaturation of fumarase in 6 M urea at pH 6.5 exposes additional methionine as well as cysteine residues to alkylation.


Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme. The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.


Properties of a human liver ribonuclease. Inhibition by polynucleotides and specificity for phosphodiester bond cleavage to yield purine nucleosides at the 5' termini. A ribonuclease, purified 2500-fold from human liver, was found to be inactive against synthetic homopolynucleotides, whereas synthetic co-polymers containing adenylic acid were rapidly degraded. The specificity of the RNase is unique in that only purine residues, in a 5:4 ratio of guanylic to adenylic acid, are found at the 5' termini of the degradation products of yeast RNA. No specificity was observed at the 3' termini of the fragments. When analyzed by DEAE-cellulose chromatography, approximately 80% of the oligonucletoides were 4 to 11 residues in length. The hydrolysis of RNA by the liver enzyme, when examined in low ionic strength buffer, could be increased severalfold over control levels by the addition of polyamines. The enzyme was found to exist as two distinct species on sucrose gradients, with molecular weights of 128,000 and 14,000. However, the addition of spermidine to the gradients resulted in the recovery of all the enzyme activity as the smaller species. The polyamines were also shown to reverse the inhibition of the enzyme by the ordered polynucleotides, polyguanylic acid and polyadenylic acid. Inhibition of enzyme activity by the polyadenylic acid segment of various mammalian mRNAs was also demonstrated.


Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism. Reciprocal regulation of yeast acetyl-CoA carboxylase by fructose bisphosphate and citrate. The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.