A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability. Incubation of synaptosomes under conditions which result in complete phosphorylation of membrane bound accepter proteins does not affect the permeability to Na+ or K+ as measured by a spectrophotometric method. This technique was not, however, sensitive enough to determine permeability to Ca2+ which was thus estimated, using 45Ca2+. It was found that although phosphorylation did not affect the equilibrium binding of 45Ca it did lower the rate of both Ca2+ uptake and efflux. The most likely interpretation of these results is that phosphorylation of proteins in the synaptic membrane lowers the permeability of the membrane to Ca2+. This could have a role in the regulation of synaptic transmission.


Irreversibel inactivation of the membrane-bound enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus by triton X-100 and protection by substrates. Enzyme IIlac, the membrane-bound component of the lactose phosphotransferase system of Staphylococcus aureus, catalyzes the phosphorylation-transport reaction below: (see article).  (The sugar can be lactose or one of its analogs.) The effects of the non-ionic detergents Triton X-100, Brij 35, and Tween 40 on the activity of Enzyme IIlac were studied. Especially striking effects were observed using Triton X-100, a detergent previously used to solubilize and isolate this enzyme. A systematic study of Triton effects over a range of concentrations and temperatures demonstrated three aspects of Triton-membrane interaction. At 0.1% Triton and 25 degrees C Enzyme IIlac is activated, but remains particulate. At 0.5% Triton and 0.5% Triton and 37 degrees C, it is rapidly and irreversibly inactivated. Sugar substrates and inhibitory sugar analogs protect Enzyme IIlac against inactivation; the effect is specific for beta-galactosides. The other substrate of Enzyme IIlac, phospho-Factor IIIlac, does not affect Triton inactivation, and the product analog galactose 6-phosphate slightly enhances the inactivation rate.


Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium. Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.


Carnitine uptake into human heart cells in culture. The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carinitine is 3-10-fold higher in heart cells than in fibroblasts (pmol - mug DNA-1). The uptake of carnitine increases with temperature coefficient KT of 1.6 in the interval 10-20 degrees C and with a negligible uptake at 4 and 10 degrees C. The uptake of carnitine follows Michaelis-Menten kinetics with a KM of 4.8 +/- 2.2 muM and V = 8.7 +/- 3.2 pmol - mug DNA-1 - H-1. Carnitine uptake is suppressed 90% by NaF (24MM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a KM of 5.7-17.3 muM and V = 8.7-9.3 pmol - mug DNA-1 - h-1. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.


Kinetics of 42K and 86Rb loss from the crayfish retina in the dark and the effect of light on the rate of isotope loss. 86Rb and 42K loss from non-illuminated crayfish retinas is described by a sum of two exponential functions with rate constants lambda1 approximately 0.12 min-1 and lambda 2 approximately 0.006 min-1. 86Rb and 42K movements distinguish mainly by the lambda2 rate constants, lambda2Rb/lambda2K = 0.7. Oubain causes a delayed increase in the rate of isotope loss from non-illuminated retinas (lambda approximately 0.009 min-1 after 40 min). Light stimuli evoke a temporary increase in the rate of both 86Rb and 42K loss by the same factor (2.7 under these conditions). In the presence of ouabain the light-induced isotope loss is abolished after 40 min if the retina is periodically illuminated but not if the retina is kept in the dark.


The effect of S-adenosylhomocysteine on DNA methylation in isolated rat liver nuclei. DNA methylation was studied in vitro using whole nuclei from regenerating rat liver. Methyl incorporation from S-adenosyl-[Me-3H]methionine in nuclei from regenerating liver was four times higher than that of normal liver. The effect of S-adenosylhomocysteine on DNA methylation was examined, and it was found at equal molar concentrations of S-adenosylhomocysteine to to S-adenosylmethionine that DNA methylation was competitively inhibited 50%.


Dependence of the composition of the protein moiety of nuclear ribonucleoprotein particles on the extent of particle purification as studied by electrophoresis including a two-dimensional procedure. Extraction with 0.1 M NaCl in 0.01 M Tris-HCl buffer, pH 8.0 releases from liver nuclei 30-40-S ribonucleoprotein particles containing newly synthesized RNA. Separation of the protein moiety of these particles by acid-urea gel electrophoresis depends on the concentration of beta-mercaptoethanol in the buffer used for the solubilization of the particles. At low concentration or with short time of solubilization, only a polypeptide chain with apparent molecular weight 38 000 penetrates into the gel and can be detected by electrophoresis. By introduction of two-dimensional polyacrylamine gel electrophoresis, we succeeded to separate the protein moiety of these particles into a core group of 4 major and 6 minor polypeptides with molecular weights ranging from 38 000 to 50 000 and a second group of 19 polypeptides ranging in molecular weight from 50 000 to 120 000. The composition of the protein moiety of these particles is dependent on the extent of purification. Polypeptides with molecular weight below 50 000 represent 55% of the total protein of particles purified only by centrifugation through a 15-30% sucrose gradient. If the particles were first  purified by gel filtration through Bio-Gel A-50m followed by centrifugation in sucrose gradient, the low molecular weight proteins represent 80% of all the proteins of the particles. The purification removed selectively the minor high molecular weight polypeptides without resulting in any extensive release of the four major polypeptides with molecular weight below 50 000 which form a stable core particle. By repeated purification it is possible to strip the particles of the high molecular weight polypeptides even further. An increase in the NaCl concentration of the extraction buffer to 0.35 M will extract additional 30-40-S particles associated with a newly synthesized RNA from the cell nucleus. These particles contain the same polypeptides as particles extracted at lower salt concentration. Extraction with 0.1 M and 0.35 M NaCl at pH 8.0 removed from the nucleus approximately 55% of all RNA labeled in 30 min after intraperitoneal injection of [3H] orotic acid to the rats.


Isolation of biologically active polysomal messenger ribonucleoprotein by centrifugation in cesium sulfate. The ribosomal fraction from HeLa cells treated at elevated temperature was centrifuged in Cs2SO4. 1. The bulk of the RNA labeled in the presence of a low dose of actinomycin D banded at p = 1.36 g/cm3. 2. This fraction contained heterogeneous RNA and proteins with molecular weights close to those of proteins found in polysomal messenger ribonucleoproteins. 3. This ribonucleoprotein bound ribosomes in conditions were deproteinized RNA did not. 4. The binding was inhibited by aurintricarboxylic acid, ethylene diamine tetraacetic acid and sodium deoxycholate, but not by Triton X-100.


Actinomycin D inhibits hemoglobin synthesis in rabbit reticulocyte lysates and binds to a 15 S cytoplasmic particle involved in the initiation step of translation. Actinomycin D inhibits hemoglobin synthesis in rabbit reticulocyte lysates by affecting initiation of globin polypeptide chains. To obtain information on the distribution of actinomycin-sensitive structures among the components of the translation machinery, a reticulocyte lysate is incubated with [3H]actinomycin. The tritium label is found associated with ribosomes actively engaged in protein synthesis and with a 15 S cytoplasmic particle. During cell-free incubation or when initiation is inhibited by aurintricarboxylic acid, the labeling of the 15 S particles increases, suggesting that they are relased from ribosomes into the cytoplasm and play a role in the initiation process. The 15 S cytoplasmic particles contain a DNAase-sensitive component, probably complexed with ribonucleic acids and proteins. A possible role of these DNA-containing particles in the initiation step of translation is discussed.


Inhibition of hexokinase by multisubstrate analogs. The compounds P1-(adenosine-5')-P3-(glucose-6) triphosphate (Ap3 glucose) and P1-(adenosine-5')-P4-(glucose-6)tetraphosphate (Ap4 glucose) were synthesized as possible transition-state analogs for hexokinase (ATP: D-hexose 6- phosphotransferase, EC 2.7.1.1). Both compounds were inhibitors of this enzyme, competitive against ATP and apparently uncompetitive against glucose. The inhibition constants for Ap3 glucose and Ap4 glucose were 0.43 mM and 0.37 mM, respectively. These results indicate that the inhibitors do not appreciably bind to hexokinase until the glucose binding site is filled. The sugar portion of the inhibitors therefore does not contribute to binding, and the compounds are acting as ATP analogs, Ap3 glucose and Ap4 glucose are also slow substrates for glucose-6-phosphate dehydrogenase.


Glucose-phosphorylating enzymes of Candida yeasts and their regulation in vivo. Three glucose-phosphorylating enzymes having different specificities for glucose and fructose were separated from the cell-free extract of Candida tropicalis by means of ammonium sulfate fractionation and chromatography on DEAE-cellulose and Sephadex G-100. Two of them, which phosphorylated fructose 1.5 times faster than glucose, were designated as hexokinase I and II (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1.), and the other with very low or no fructose-phosphorylating activity, as glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2). Km values for glucose with both hexokinase I and glucokinase were 0.3 mM, and that for fructose with hexokinase I was 2.2 mM. Time-course changes in the levels of these enzymes in C. tropicalis growing on glucose and on n-alkane revealed that hexokinase was induced specifically by the sugars, while glucokinase was a constitutive enzyme. Addition of cycloheximide to the culture medium prevented the increase in the hexose-phosphorylating activity and in the Fru/Glu ratio (the ratio of enzymatic phosphorylation of fructose to that of glucose) in the cells. Although Candida lipolytica also contained hexokinase and glucokinase, both enzymes seemed to be constitutive.


Purification of acetylcholinesterase from house fly brain by affinity chromatography. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the heads of house flies (Musca domestica L.) was purified by affinity chromatography. The enzyme was adsorbed from the crude extracts on an affinity column containing trimethyl(p-aminophenyl) ammonium chloride hydrochloride (Ki approximately 1.7 - 10(-4) M), covalently linked to Sepharose 4B, then eluted with a solution of a selective reversible inhibitor, 1,5-bis (4-allyl dimethyl ammoniumphenyl)-pentan-3-one dibromide (BW 284C51; Ki approximately 1 - 10(-7) M). The enzyme was purified 1223 times in one step and had a specific activity of 752 units/mg protein. Disc gel electrophoresis in polyacrylamide gel revealed five protein bands, four corresponding to the enzyme activity bands and one devoid of enzyme activity. On the basis of periodic acid-Schiff stain intensity, the slower moving isozyme I and the contaminating band appear to be rich in carbohydrate. The purity of the enzyme estimated by disc gel electrophoresis was 94%. Density gradient centrifugation in sucrose showed two major species each of which ran as a single band on disc gel electrophoresis. The average molecular weights were 306000 (+/- 11 150) for heavy (s20,w = 11.5 S) form and 143000 (+/- 4700) for light (s20,w = 6.9 S) form.


Inactivation of ethanolamine ammonia-lyase by 5,5'-dithiobis(2-nitrobenzoic acid). Further evidence for the involvement of sulfhydryl groups in adenosylcobalamin-dependent rearrangements. Treatment of the adenosylcobalamin-requiring enzyme ethanolamine ammonia-lyase (EC 4.3.1.7) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) discloses three classes of -SH groups: a rapidly reacting class (2 -SH groups/enzyme molecule), a slowly reacting class (6 -SH groups/molecule) and a class which does not react unless the enzyme is denatured (7 -SH groups/molecule (Kaplan, B.H. and Stadtman, E.R. (1968) J. Biol. Chem. 243, 1794)). The enzyme is inactivated by Nbs2 at a rate similar to the rate at which Nbs2 reacts with the slowly reacting class of -SH groups. Inactivation of the enzyme is retarded by adenosylcobalamin but not by ethanolamine. Once inactivated, the enzyme cannot be reactivated with mercaptoethanol. These observations provide further evidence for the importance of -SH groups in catalysis by adenosyl-cobalamin-requiring enzymes.


Magnitude of the equilibrium isotope effect on carbon-tritium bond synthesis. The exchange of tritium from 3HOH into the methyl group of pyruvate catalyzed by 6-phospho-2-keto-3-deoxygluconate aldolase (6-phospho-2-keto-3-deoxy-D-gluconate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.14) of Pseudomonas putida shows an equilibrium isotope effect of 0.78. From this value and the deuterium effect on the fumarase equilibrium (Thomson, J.F. (1960) Arch. Biochem. Biophys. 90, 1), one can calculate by use of the relative fractionation factors of Hartshorn and Shiner (Hartshorn, S.R. and Shiner, Jr., V.J. (1972) J. Am. Chem. Soc. 94, 9002), fractionation factors for transfer of deuterium or tritium from water to a number of organic molecules of interest.


Radioimmunoassay for canine creatine kinase isoenzymes. Antibodies specific for the B and M subunits of creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) isoenzymes were obtained by immunization of rabbits with canine BB and MM creatine kinase. A radioimmunoassay capable of measuring picomolar amounts of creatine kinase isoenzymes has been developed which measures concentrations of intact isoenzymes rather than activity. It should aid in elucidating the metabolism and breakdown of creatine kinase isoenzymes.


Rat liver microsomal palmitoyl-CoA synthetase: subunit structure. Long chain fatty acyl-CoA synthetase (acid:CoA ligase (AMP-forming), EC 6.2.1.3) from rat liver microsomes was shown to dissociate completely into one polypeptide chain in 75% 2-chloroethanol in water. The presence of one amino and one carboxy terminal was established. The molecular weight of the subunit as deduced from sedimentation equilibrium as well as quantitative carboxy terminal analysis agrees with the value of 28 000 +/- 1000 as reported previously (Bar-Tana, J. and Rose, G. (1973) Biochem. J. 131, 443--449). Hence, the catalytic unit of 168 000 daltons appears to be composed of identical subunits.


Raman spectra of some saturated, unsaturated and deuterated C18 fatty acids in the HCH-deformation and CH-stretching regions. We have analyzed the Raman spectra of stearic acid and two deuterated derivatives thereof, 2,2-dideutero stearic acid and 18,18,18-trideutero stearic acid, as well as of four unsaturated, C18 acids, (cis,-9; cis, cis-9,12, cis, cis, cis-9,12,15; and trans, trans-9,12) in their solid and liquid states. The different spectra of stearic acid and the two deuterated derivatives exhibit distinctive HCH-deformation and CH-stretching features for CH2 residues adjacent to CO2H or CH3 residues. Unsaturated hydrocarbon chains reveal additional features attributed to CH2 residues adjacent to one or two HC=CH groups. The spectra of the various fatty acids in their solic or liquid states show that both the HCH-deformation and CH-stretching regions are very sensitive to chain architecture, the CH-stretching region most dramatically so. Evaluation of the spectral features in the two regions indicates that the detailed structure of the CH-stretching region depends strongly upon interaction, enhanced by Fermiresonance, between CH-stretching fundamentals and HCH-deformation overtones. The data further suggest that the differences between HCH-deformation, in the solid and liquid states, and the resulting altered interactions between CH-stretching fundamentals and HCH-deformation overtones, produce the large modifications of the CH-stretching region that accompany solid-liquid transitions. The results thus provide a basis for the changes in the CH-stretching region, that accompany order-disorder transitions in model and biological membranes.


Mitochondrial biogenesis in cultured mammalian cells. III. Synthesis of mitochondrial phospholipids by subcellular fractions isolated from normal and chloramphenicol-treated BHK-21 cells. The capacity of subcellular fractions isolated from chloramphenicol-treated BHK-21 cells to synthesize various mitochondrial phospholipids in vitro and examined. Both mitochondria and microsomes showed the capacity to acylate sn-glycerol 3-phosphate and dihydroxyacetone phosphate to lysophosphatidic acid and acyldihydroxyacetone phosphate and subsequently to phosphatidic acid. Both processes are inhibited in mitochondria from chloramphenicol-treated cells. The synthesis of CDPdiacylglycerol in mitochondria or microsomes, and the synthesis of phosphatidylinositol and of phosphatidylcholine in microsomes were stimulated in treated cells. A slight stimulation was also observed in the synthesis of phosphatidylglycerol and diphosphatidylglycerol when the labelled precursor was sn-glycerol 3-phosphate in treated cells, although the process was inhibited with labelled glycerol as the precursor. Conversion of phosphatidylglycerol phosphate to phosphatidylglycerol by mitochondria was rate limiting unless the post-microsomal supernatant fraction was added. These results are discussed in regard to the observed inhibition of phospholipid synthesis in BHK-21 cells in culture by chloramphenicol.


Phospholipids of subcellular organelles isolated from cultured BHK cells. Mitochondrial and nuclei were purified from cultured hamster fibroblasts (BHK21 cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver.


Effects of 25-hydroxycholesterol and 7-ketocholesterol, inhibitors of sterol synthesis, administered orally to mice. When 25-hydroxycholesterol or 7-ketocholesterol was fed to mice with the diet, growth was suppressed and mature mice lost weight. The effect of the 7-ketone upon body weight was effectively counteracted by cholesterol whereas cholestanol and beta-sitosterol were ineffective. Growth repression due to 25-hydroxycholesterol was only partially relieved by cholesterol. The effects of 25-hydroxycholesterol and 7-ketocholesterol upon body weight were related to an apparent effect upon appetite. However the sterols were not unpalatable since diets containing them were not rejected in favor of control diet. Intestinal sterol synthesis was inhibited soon after the administration of dietary 7-ketocholesterol or 25-hydroxycholesterol but inhibition decreased with prolonged feeding. When fed by gavage, the sterols suppressed intestinal sterol synthesis as soon as 2 h after administration. In contrast, cholesterol administered by gavage did not affect intestinal sterol synthesis during a 24 h test period. When fed with the diet 25-hydroxycholesterol and 7-ketocholesterol did not depress hepatic cholesterol synthesis beyond the low levels found in pair-fed controls. Inhibition of intestinal sterol synthesis was accompanied by a decrease in the concentration of cholesterol in the intestinal mucosa and, usually, by a drop in the molar ratio of cholesterol to phospholipids.


Site of synthesis of phosphatidic acid and diacyglycerol in spinach chloroplasts. The enzymatic synthesis of lysophosphatidic acid, phosphatidic acid, monoacylglycerol and diacylglycerol from sn-[14C]glycerol 3-phosphate occurs in purified chloroplasts. The results indicate that: (1) the chloroplast extract contains a soluble acylase (acyl-CoA: sn-glycerol 3-phosphate acyltransferase); (2) the envelope fraction contains an acyl-CoA synthetase, a bound acylase (acyl-CoA: acyl-sn glycerol 3-phosphate acyltransferase) and a phosphatidic acid phosphatase; without chloroplast extract in the incubation medium, the envelope is unable to incorporate sn-glycerol 3-phosphate into phosphatidic acid and diacylglycerol; addition of chloroplast extract to the incubation medium induced a fast increase of the incorporation of sn-glycerol 3-phosphate into phosphatidic acid and diacylglycerol; thylakoids being unable to incorporate sn-glycerol 3-phosphate (in presence or absence of soluble chloroplast extract in the incubation medium) our results indicate that the envelope of spinach chloroplast is the site of phosphatidic acid and diacylglycerol synthesis; (3) diacylglycerol actively synthesized by the envelope is also the substrate for the first galactosylation enzyme.


Rate constants for the uptake of cholesterol from various intestinal and serum lipoprotein fractions by the liver of the rat in vivo. The increase in the mass of cholesterol esters in the liver was used to estimate hepatic net uptake rates of cholesterol from various serum and intestinal lipoprotein fractions. Initial uptake rates equalled essentially zero for high density serum lipoproteins and for large chylomicrons while administration of both low density serum lipoproteins and smaller chylomicrons produced a significant increase in hepatic cholesterol ester levels. The rate of uptake of both serum lipoprotein fractions did not change over a 5 h interval after injection: in contrast, the rates of uptake of the intestinal fractions increased 10-25-fold during this interval. Circulation of large chylomicrons in functionally eviscerated rats markedly increased the rate of hepatic cholesterol uptake when these metabolized lipoproteins were reinjected into recipient animals. Uptake of cholesterol from intestinal lipoproteins was essentially a linear function of the amount of chylomicrons administered to the animals and was independent of the level of circulating serum cholesterol and the rate of hepatic cholesterogenesis. These observations are consistent with the view that the liver is capable of taking up cholesterol from chylomicron remnants and, at significantly lower rate, low density serum lipoproteins.


Cholesterol beta-D-glucoside-6'-O-palmitate, a metabolite of Pythium sylvaticum. Cholesterol beta-D-glucoside-6'-O-palmitate has been identified as a polar metabolite in the mycelium of mated cultures of Pythium sylvaticum grown in the presence of cholesterol. The structure was confirmed by synthesis of the metabolite. Similar steroid beta-D-glucoside-6'-O-palmitates were obtained from beta-sitosterol and campesterol when these sterols were added to cultures of P-sylvaticum. Corresponding esters of myristic and stearic acids were also detected.


Partial characterization of the bile salt-dependent triacylglycerol lipase from the leopard shark pancreas. Leopard shark triacylglycerol lipase has been characterized as a crude pancreatic preparation. The enzyme demonstrated an absolute requirement for trihydroxy bile salts for activity with natural bile salts of the shark giving a 4-fold greater stimulation of activity than pure sodium taurocholate. Bile salts also protected the enzyme from apparent inactivation by p-chloromercuribenzoate and trypsin treatment. The shark lipase demonstrated a temperature optimum of 36 degrees C and was rapidly inactivated at 50 degrees C even in the presence of bile salts. Divalent metal ions were required for activity with Ca2+ providing the greatest stimulation. At 22 degrees C, pH 8.5 and in the presence of natural bile salts, the apparent V was about 0.6 mumol fatty acid released/min per mg protein. The shark enzyme hydrolyzed over 90% of the fatty acids from trioleovylglycerol and methyl esters of pancreatic lipase-resistant fatty acids were hydrolyzed at the same rate as typical fatty acid methyl esters. Hydrolysis of triacylglycerol proceeded about ten-times faster than wax ester hydrolysis. The kinetic properties of the leopard shark enzyme were compared to other bile salt-dependent lipolytic enzymes. Pancreatic lipase activity was not detected.


The effect of palmitoyl-coenzyme A on rat heart and liver mitochondria. Oxygen consumption and palmitoylcarnitine formation. Rat heart and liver mitochondria, respectively, oxidized palmitoyl-CoA and palmitoylcarnitine optimally at 20-30 and 10-20 nmol substrate/mg. The oxidation of palmitoyl-CoA was accompanied by a lag in State 3 respiration that was proportional to the palmitoyl-CoA concentration. The delay in State 3 rates was more prolonged in liver than in heart at comparable palmitoyl-CoA levels. A similar range of palmitoyl-CoA concentrations produced significant inhibition of respiration in mitochondria oxidizing glutamate-malate. The inhibition was not due to a detergent effect of palmitoyl-CoA since addition of carnitine restored State 3 rates. Electron microscopic examination of mitochondria at low palmitoyl-CoA levels revealed normal ultrastructure. At comparable concentrations of palmitoyl-CoA, formation of palmitoylcarnitine by mitochondria from rat heart and liver followed first-order kinetics. The apparent first-order rate constants decreased with increasing palmitoyl-CoA. These results suggest that substrate inhibition may influence the rate of palmitoyl carnitine formation even at physiological concentrations of palmitoyl-CoA. The apparent first-order rate constant at palmitoyl-CoA levels (12 nmol palmitoyl CoA/mg) optimally oxidized by liver mitochondria, was one-third the value of the apparent rate constant measured in heart mitochondria at the identical substrate level. The prolongation in time to reach equilibrium may acocunt for the relatively greater respiratory sensitivity of liver mitochondria to increasing levels of palmitoyl-CoA.


Specific requirement of lysophosphatidylcholine for palmitoyl-CoA synthetase of chicken intestine. The presence of palmitoyl-CoA synthetase (EC 6.2.1.3) in the brush border-free particulate fraction of chicken intestinal mucosa is demonstrated. The enzyme was dependent on the simultaneous presence of lysophosphatidylcholine and Triton X-100 as well as ATP, CoA and Mg2+ for maximal activity. Lysophosphatidylcholine could not be replaced by other lipids. Enzyme preparations solubilized by Triton X-100 or lysophosphatidylcholine were still dependent on the presence of detergents for maximal activity.


Heterogeneity of membrane-bound delta5-3-oxosteroid isomerase. Studies on bovine adrenocortical microsomes. There are conflicting reports concerning the number of distinct delta5-3-oxosteroid isomerases present in various steroid producing tissues; this problem was reexamined by two techniques: (1) kinetics measurements with a mixture of androst-5-ene-3,17-dione and pregn-5-en-3, 20-dione. (2) thermal inactivation of the androst-5-ene-3,17-dione isomerase and pregn-5-ene-3,20-dione isomerase activities. The results are the following: (a) Kinetic data support the concept of a single delta5-3-oxosteroid isomerase with a low substrate specificity in the microsomes. (b) Thermal inactivation experiments also support the one enzyme theory, but strongly suggest an heterogeneity of the delta5-3-oxosteroid isomerase in the bovine adrenocortical microsomes. In view of these results, a tentative explanation of the conflicting reports is proposed.


Rapid zymogen activation and isolation of serine proteases from an individual mouse pancreas by affinity chromatography: genetical heterogeneity of chymotrypsins of Mus musculus. A rapid method to prepare homogeneous fractions of the various chymotrypsins and trypsins from a single mouse pancreas (130-150 mg wet weight) is described. The method was applied to investigate intra-species variation on a molecular level using chymotrypsins as biochemical indicators. The conditions for optimal extraction of the zymogens in the homogenized pancreas have been studied. DNA had to be removed from the homogenate to obtain maximum chymotrypsin yields (approximately 1% of the wet weight of the pancreas). The activation was initiated by immobilized bovine trypsin that was removed by filtration. Then chymotrypsinogens in the homogenate were activated by mouse trypsin. After completed activation homogeneous chymotrypsins (one anionic and one cationic form) could be isolated in an one step analytical affinity chromatographic separation, using soybean trypsin inhibitor bound in Sepharose as a protease specific adsorbent. The end products were characterized by isoelectric focussing, amino acid composition, enzymatic parameters, molar extinction coefficient, and the number of polypeptide chains. Hereby, the existence of two chymotrypsinogen loci in the mouse genome could be demonstrated. Differences in structure and function between the corresponding enzymes from the two strains were found. This allelomorphism was verified in the crossing of the off-spring.


Binding properties of purified adult and fetal bovine serum albumin. Fetal and adult bovine serum albumin were purified by standard techniques and then treated with activated charcoal at pH 2.75 to remove small molecular weight legands. The two proteins were compared and found to be identical by the following criteria: isoelectric focusing, electrophoresis on cellulose acetate and polyacrylamide gel, by the binding of bromcresol green, salicylic acid, diphenylhydantoin, and six different fatty acids (palmitic, stearic, oleic, elaidic, linoleic, and arachidonic). Reports of marked differences in the capacities of fatal and ault plasma to bind bilirubin and various drugs have usually been attributed to the presence of a "fetal" albumin. Our results, however, are consistent with the view that there is no difference between adult and fetal albumin and that the observed altered binding properties of fetal plasma are due to the tight binding of a ligand to fetal albumin in blood, which modifies the protein's affinity for lipophilic compounds.


Comparative immunochemistry of bacterial, algal and plant ferredoxins. 1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 [4Fe-4S] ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green algia Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris. The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins. 2. Antibodies to the bacterial ferredoxins reacted to a significant extent only with their homologous proteins. On the other hand, antibodies to the plant and algal ferredoxins showed cross-reaction with other ferredoxins. There was a correlation between the degrees of immunoprecipitation and the similarity in amino acid sequences. These results suggest that the method can be used as a marker in taxonomic studies. 3. The interaction of the antibodies with the five native ferredoxins was compared with the reactions with their apoproteins. In each case the degree of interaction was different. This behaviour was interpreted as due to an influence of tertiary structure on the antibody-antigen interaction.


The hemoglobin-oxygen equilibrium associated with subunit dissociation. The effect of oxygen-linked tetramer-dimer dissociation on oxygen equilibrium of hemoglobin was investigated by measuring the equilibrium curves over a wide range of protein concentration. A Hill scheme which takes the subunit dissociation into account describes well the overall concentration dependences of the oxygen pressure and slope of the Hill plot at half saturation. Values of dissociation constant for oxyhemoglobin estimated from the equilibrium data agree with the vaues measured by other methods for phosphate-free and diphosphoglycerate-added hemoglobin. The present results indicate that oxygen equilibrium properties are only slightly influenced bysubunit dissociation in the concentration range above 60 muM (as heme) at which most equilibrium experiments have been carried out.


Cleavage of the S-S bond in 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence of a cationic detergent: an approach to the cleavage of the S-S bond in bovine plasma albumin. The hydrolysis of 5,5'-dithiobis-(2-nitrobenzoic acid) was studied at pH 9.20 and 25 degrees C in presence of tetradecyltrimethylammonium bromide or micellar tetradecyltrimethylammonium bromide. The reaction was pseudo-unimolecular reaction with regard to the concentration of dithionitrobenzoic acid. The rate constant k1 depended on the tetradecyltrimethylammonium bromide concentration and on the ionic strength of the buffer solution. At a constant ionic strength, the value of k1 increased with the increase in the tetradecyltrimethylammonium bromide concentration, attained maximum at a certain concentration above the critical micelle concentration, and then decreased. The value of k1 was larger when the ionic strength was lower. The rate of hydrolysis at the ionic strength 0.1 was the same as that without tetradecyltrimethylammonium bromide. On the contrary, sodium dodecyl sulfate or micellar sodium dodecyl sulfate had no effect on the rate of hydrolysis. These results lead to the conclusion that the S-S bond is cleaved easier, when it is surrounded by the cationic detergent. The SH/S-S exchange reaction of bovine plasma albumin in the presence of cationic detergent was slower when the ionic strength was higher. The fact could be explained by assuming that the exposed S-S bond is surrounded by the cationic detergent. Further, it was speculated that some S-S bonds in albumin are surrounded by the positively charged basic amino acid residues.


Degradation of TN-C component of troponin by trypsin. The peptide pattern obtained during degradation of TN-C component of troponin by trypsin markedly depends on the concentration of calcium ions. In the presence of calcium higher than 0.1 mM a peptide with a mass of about 16 000 daltons is formed, followed by its splitting into two peptides (8500 and 7500 daltons), which accumulate during further digestion. When calcium is sequestred by EGTA the degradation of TN-C is much faster. The first products of digestion are the 11 000 and 7500 dalton peptides. The former one is degraded to 9700 dalton peptide and, subsequently, both peptides are split to small fragments. In the presence of calcium only three -NH2 groups per molecule of TN-C appear during digestion, whereas in its absence almost all peptide bonds available for trypsin are cleaved. The fragments of TN-C obtained in the presence of calcium are still able to interact with TN-I component as judged by disc-electrophoresis performed in the presence of calcium. During digestion in the presence of calcium only a slight decrease of tyrosyl intrinsic fluorescence occurs and the further decrease after removal of calcium bound to TN-C is fully reversible. Similarly, the content of alpha-helix decreases only slowly during digestion in the presence of calcium. All the results suggest that calcium stabilizes the structure of TN-C molecule, so that trypsin in the presence of calcium releases one small peptide from N-terminal not involved in the first "EF-hand" and the other from the region between pairs 1-2 and 3-4 of "EF-hands" in the nomenclature of Kretsinger (see Kretsinger and Barry [16]). The obtained fragments, containing almost exclusively pairs of "EF-hands", restore most of the properties of original TN-C molecule.


Critical appraisal of electronic structure of metmyoglobin. 14N and 57Fe hyperfine interactions. The electronic structure of metmyoglobin is subjected to a critical examination by comparison of results of recent ENDOR measurements of nitrogen hyperfine interaction constants on the porphyrin ring and on the proximal histidine linking the heme to the protein with theoretical values for these properties from calculated electronic wavefunctions for this molecule. The observed interesting trends of the 14N hyperfine data as well as available 57Fe data are both successfully explained showing that theory has provided a satisfactory description of the electronic distribution in this important molecule.


Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Standard experimental procedures for continuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were modified to give more effective separation and improved resolution of myofibrillar proteins. The system utilizes a running gel consisting of 10% acrylamide with 0.1% bisacrylamide crosslinker (100:1) incorporating 400 mM Tris/glycine (pH 8.80), 0.1 mM ethylenediaminetetraacetate, 5% glycerol and 0.1% sodium dodecyl sulfate. Electrophoresis was performed at 1 mA per gel with corresponding running times of 4-6 h. The myosin heavy chain enters and migrates as a narrow symmetrical band while the smaller regulatory proteins of the myofibril are resolved. The utility of the procedure in relation to the study of protein structure is detailed.


The cross-linking of 125I-labelled transferrin to rabbit reticulocytes. 125I-labelled transferrin has been covalently linked to proteins of rabbit reticulocyte membranes using three different cross-linking reagents: (a) bis(methyl) suberimidate (b) 4-methyl mercaptobutirimidate and (c) Cu-o-phenanthroline. Analysis of the products of the cross-linking reactions by SDS polyacrylamide gel electrophoresis revealed three radioactively labelled components. The major component corresponds to non-cross-linked transferrin while a second smaller component represents a complex (125 000 daltons) of membrane protein and transferrin. A third component (210 000 daltons) was observed when 4-methyl mercaptobutirimidate was used as a cross-linking reagent. The possibility that the two higher molecular weight complexes contain the receptor protein for transferrin is discussed.


Tyrosyl fluorescence in hemocyanin from the scorpion Leirus quinquestriatus. The fluorescence properties of hemocyanin from the scorpion Leirus quinquestriatus were studied. Emission and excitation spectra were determined for protein in both its oxygenated and deoxygenated forms. Oxygenation was found to bring about a large blue shift in the position of the fluorescence maximum, in addition to a marked quenching of the fluorescence intensity as noted before for another hemocyanin. An appreciable tyrosyl contribution to the fluorscence of oxyhemocyanin was inferred from the wavelength dependence of its emission and excitation spectra. No tyrosyl fluorescence could be observed in either apo- or deoxyhemocyanin. It was concluded that the inability to observe tyrosyl emmision in deoxyhemocyanin is due to the dominating emission to tryptophan. The implication of the findings to the often noted failure to detect tyrosyl emission in proteins containing both tyrosine and tyrptophan is discussed.


Mössbauer investigation of deoxymyoglobin in a high magnetic field. Orientation of the electric field gradient and magnetic tensors. We have examined the Mössbauer spectra of deosymyoglobin in a 6 T magnetic field in the temperature range 4.2-195 K. Spectra were fitted by the least-squares method using a phenomenological model in which the internal magnetic hyperfine field was assumed to be related to the applied field by a temperature dependent tensor õmega. The results indicate that õmega has axial symmetry and a principal axis system aligned with that of the electric field gradient (efg). The principal component of the latter is negative and lies on the symmetry axis of õmega. Our fits indicate that of efg asymmetry parameter is eta = 0.7, with no appreciable temperature dependence. Both axial and transverse components of õmega have the expected 1/T temperature dependence for T  greater than 20 K. Our experimentally determined value of eta, combined with published single crystal zero-field measurements, constrains the efg-heme relative orientation to two possibilities. In neither of these is an efg principal axis near to the heme normal.


Isolation of the high molecular form of bovine factor X and some of its physical properties. A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO4 absorption, exhaustive washing with 0.001 M BaCl2 and chromatographed on DEAE-cellulose column employing a linear salt gradient. This isolated factor X showed a single protein band on analytical polyacrylamide gel disc electrophoresis. Only one single protein peak was observed in the chromatogram of DEAE-Sephadex A-50 chromatography conducted at 3 degrees C. Sedimentation equilibrium analysis of this bovine factor X revealed no apparent heterogeneity or self association-dissociation phenomena. It yielded a weight-average molecular weight of 74 000 for the native factor X. In the absence of any reducing agent, factor X migrated in dodecyl sulfate gel electrophoresis as a single component with an estimated molecular weight of 74 300. Both dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol and agarose gel chromatography in 6 M guanidinium chloride revealed that this native factor X is composed of two polypeptide chains of molecular weights of 56 000 and 22 100. Factor X can be converted to the enzymatically active factor Xa by Russell's viper venom and in the presence of Ca2+. Factor Xa was purified by DEAE-cellulose chromatography. This Russell's viper venom activated factor Xa also showed a single protein band upon analytical polyacrylamide gel disc electrophoresis. Sedimentation equilibrium analysis of this factor Xa yields a weight-average molecular weight of 59 000 with no apparent heterogeneity or self-association phenomena. In the absence of any reducing agent, factor Xa migrated as a single component in dodecyl sulfate gel electrophoresis with an estimated molecular weight of 58 500. From the results of dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol as well as agarose gel chromatography in 6 M guanidinium chloride, factor Xa is also composed of two polypeptide chains of molecular weights of 36 700 and 22 800. Therefore, the heavy and light chains of both native factor X and factor Xa are linked together by disulfides. Great care was taken in washing the BaSO4 precipitate and it is this effective washing which enabled us to isolate the higher molecular from of bovine factor X.


Purification of human immunoglobulin M by affinity chromatography on protamine-Sepharose. Human IgM has been isolated from plasma by a simple procedure in high yield. The first step was adsorption to protamine-Sepharose and elution by increasing the ionic strength with NaCl. This was followed by two gel filtration steps resulting in a 98% pure IgM in about 30% yield. A somewhat modified procedure could also be used for purification of IgM from Cohn fraction II + III. The purified IgM was found to have a sedimentation constant in agreement with reported values. In immunoelectrophoresis and isoelectric focusing, purified IgM showed the same behaviour as IgM in plasma. Different fragments of IgM were tested for binding to the protamine-Sepharose adsorbent. IgM and Fab were not bound unlike (FC)5, indicating that the sites responsible for binding are located in the Fc part and that several Fc parts are necessary for sufficiently strong binding for adsorption.


A glycoprotein of high molecular weight in the urine of germ-free rats. A carbohydrate-rich glycoprotein fraction was obtained by purification of the non-dialysable material of germ-free rat urine. This procedure involved proteolytic digestion with pronase, presipitation of the acidic glycoprotein with cetyltrimethyl-ammonium bromide (CTAB), and chromatography on DEAE-Sephadex and finally on Sepharose 4B from which it was partly excluded. The purified glycoprotein contained fucose (10.1%), galactose (12.2%).N-acetylglucosamine (16.6%), N-acetylgalactosamine (19.8%), sialic acid (11.9%), and sulphate ester groups (0.7%). The amino acid content was 19%, threonine, serine and proline being the predominating amino acids. Gel electrophoresis and gel chromatography revealed a pronounced poly-dispersity of the material. Treatment of the glycoprotein with alkali in the presence of borohydride led to partial degradation of the polymer with the formation of fragments containing N-acetylgalactosaminitol. The chemical properties of the purified glycoprotein are characteristic of a mucin. The presence of this glycoprotein could not be demonstrated in conventional rat urine.


Disulfide-linked cyanogen bromide peptides of bovine fibrinogen. II. Isolation and sequence analysis of the chain constituents from the amino terminal region. Bovine fibrinogen was cleaved with CNBr and the peptide F-CB1 which originates from the amino end of the molecule was purified by chromatographic methods. After reduction and alkylation of F-CB1 three main polypeptide chains could be identified. They were derived from the A alpha chain (F-CB1 alpha), Bbeta chain (F-CB1 beta) and gamma chain (F-CB1 gamma) of fibrinogen and consisted of 54, 143 and 78 amino acid residues, respectively. Thrombin digestion released fibrinopeptide A from F-CB1 alpha and smaller amounts of the peptide Gly-Pro-Arg while fibrinopeptide B was released from F-CB1 beta. Fragment F-CB1 gamma was resistant to thrombin. The sequences of 35 amino acid residues in the portion of F-CB1 alpha adjacent to fibrinopeptide A and of 15 residues in F-CB1 beta beyond fibrinopeptide B were determined. They differ by six and two substitutions, respectively, when compared with the homologous part in human fibrinogen. However, the first 29 amino acid residues in bovine and human F-CB1 gamma were identical. The data show that the cysteine-containing regions and the portions to the right of the site of thrombin cleavage in fibrinogen show much less interspecies variability than that known for the fibrinopeptides.


Glycosylation of hemoglobin S by reducing sugars and its effect on gelation. The binding of various reducing mono- and disaccharides to hemoglobin S has been measured both before and after treatment of the sugar-protein adducts with NaBH4. Incubation of 0.3 M solutions of D-glucose, D-galactose, D-maltose, and lactose, with 2% hemoglobin for 2 h at 37 degrees C, pH 7.2, leads to the incorporation of 1.1, 1.8, 1.8, and 3.3 mol of sugar, respectively, into 1 mol of hemoglobin tetramer (either A or S). Exposure of these aldose-protein adducts to NaBH4 for an additional hour at 10 degrees C increases the binding to 2.0, 3.3, 2.5, and 4.1 mol per mol tetramer, as would be expected if Schiff base linkages were involved in this protein modification reaction. The data suggest a stereochemical requirement for enhanced binding. The dependence of the pre-reduction binding of glucose on the sugar concentration, and on the oxygenation state of hemoglobin has also been examined. Glycosylation of hemoglobin significantly increases the minimum gelling concentration of the deoxy conformation, as measured by sedimentation  equilibrium ultracentrifugation. Of the sugar derivatives of hemoglobin S examined by this method, those modified by D-galactose or lactose have minimum gelling concentrations (in the absence of 2,3-diphosphoglycerate) which are comparable to, or greater than, that of fully carbamylated hemoglobinS.


Interaction of progesterone receptor with immobilized adenosine triphosphate. Affinity chromatography has been used to study the binding of ATP to cyto-plasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCl (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80% recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-cellulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors.


Liquid-phase method for peptide synthesis utilizing photolytic cleavage from a new o-nitrobenzoyl polyethylene glycol support. Photolysis as a method for removal of a tert-butyloxycarbonyl-protected peptide possessing a free C-terminal carboxyl group from a polystyrene support in the solid-phase method of peptide synthesis was introduced by Rich, D. H. and Gurwara, S. K.((1975) J. Am. Chem. Soc. 97, 1575-1578). Using this basic concept we prepared a photosensitive 3-nitro-4-bromomethylbenzoyl polyethylene glycol support for use in the liquid-phase method of peptide synthesis. Photolytic cleavage of a protected tetrapeptide possessing a free C-terminal carboxyl group from the polyethylene glycol support resulted in a 98% yield compared with a 69% yield for the photolytic leavage from the polystyrene support. This application of photolysis as a cleavage method in liquid-phase peptide synthesis avoids the low yields and rather drastic conditons needed to remove a peptide attached directly to the poly-ethylene glycol support in the conventional liquid-phase method.


Differences in the sites of iodination of proteins following four methods of radioiodination. The rate of deiodination of radioiodinated proteins varies with the method of iodination. To elucidate differences in the iodinated protein labeled by various methods, we have hydrolyzed fibrinogen and several small peptides iodinated by the iodine monochloride, chloramine-T, electrolytic and enzymatic methods. Under conditions of either acidic or basic proteolysis, extensive deiodination occurred and the major product was I-. When a protease of Streptomyces griseus was used, radio-iodinated fibrinogen and other polypeptides were degraded to single iodinated amino acid residues and only a small yield of I-. The iodinated amino acids resulting from proteolysis were separated by ion-exchange chromatography. The iodine monochloride and enzymatic methods yielded largely iodotyrosine with small amounts of other iodinated amino acids. The chloramine-T product spectrum varied with the chloramine-T:protein ratio, whereas the electrolytic method yield was a complex function of the reaction conditions. The different methods of iodination lead to some differences in the site of iodination which correlate with stability of the protein-iodine bond.


The possible pathways of self-organization of immunoglobulin domains. It was proposed that the most probably germ of the beta-structure in globular proteins would be an antiparallel beta-hairpin initiated by the flexibility and/or bend formation of the polypeptide chain between the connected strands. The possible pathways of self-organization of immunoglobulin domains from antiparallel beta-hairpins are computed using well-known kinetic limitations. The most favourable structures corresponding to the greatest number of dehydrated bulky hydrophobic groups are selected. One of the two most favorable structures obtained in such a way coincides with the native tertiary structure of the domains.


Reinvestigation of the primary structures of red deer and roe deer pancreatic ribonuclease and proline sites in mammalian ribonucleases. The sequences of amino acid residues 15-23 of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) pancreatic ribonuclease and the identity of residue 99 in roe deer ribonuclease are corrected. Earlier results are explained by the cleavage of an Asp-Pro bond in both enzymes during the treatment with CNBr in 70% formic acid and by wrong interpretations of amino acid analyses. Proline residues, which occur at a number of positions in several mammalian ribonucleases, can be accommodated in a model of bovine ribonuclease S without disrupting the conformation of the main chain.


The isolation and identification of 2-methyl-2,4-thiazolidine dicarboxylate as a by-product in the conversion of cysteine to glucose in the perfused rat liver. Cysteine is one of the more toxic amino acids, however the toxic agent associated with cysteine toxicity has not been identified. Recently it was shown that 2-methyl-2,4-thiazolidine dicarboxylate (MTD) was formed fromcy steine by rat liver and would be toxic to the rat. This suggested that MTD formed by the rat liver can be produced both enzymatically and by chemical interaction between cysteine and another compound, possibly pyruvate. When MTD (1 mmol/rat) was injected into 6 rats, two of them died. Only 70-80% of the MTD was excreted within 24 h, suggesting an accumulation of MTD. It is possible the MTD could accumulate in the tissues until it reached a toxic concentration. Whether this could account for the death associated with cysteine toxicity is unknown.


A dialysis procedure for loading erythrocytes with enzymes and lipids. A dialysis procedure for hypotonic hemolysis has been developed in which erythrocytes can be loaded with water-soluble enzymes, detergent-solubilized enzymes (glucocerebrosidase) and detergent-dispersed glycolipid (glucocerebroside). The procedure allows approx. 40-50% of the added enzyme or glycolipid to be encapsulated. The final intracellular concentration of enzyme or glycolipid is about to the extracellular concentration. The loaded cells can be ingested by macrophage in vitro and the glucocerebroside partially degraded by lysosomal glucocerebrosidase. The use of this procedure for the investogation of Gaucher's disease is discussed.


Phytohaemagglutinin stimulation of human lymphocytes: effect of fatty acids on uridine uptake and phosphoglyceride fatty acid profile. 1. When added to cultures of human peripheral lymphocytes, saturated (palmitate, stearate, heptadecanoate) and unsaturated (oleate, linoleate, arachidonate) fatty acids bound to albumin at an acid-albumin ratio of 2:1, inhibited the phytohaemaegglutinin-stimulated uptake of [14C]-uridine. Uridine uptake in unstimulated cells was not affected by any of these fatty acids. 2. When saturated and unsaturated acids were present simultaneously in the incubation mixture the inhibit but relieved the inhibitory effects of both saturated and unsaturated fatty acids. 4. Stimulated and unstimulated cells incorporated exogenous fatty acids into membrane phosphoglycerides. Details of the fatty acid profiles are given. 5. Evidence is presented that the inhibition results, at least in part, from modification of phosphoglyceride fatty acid profile.


Protein-catabolic stage of isolated rat hepatocytes. Isolated rat hepatocytes in suspension are in a protein-catabolic state (negative nitrogen balance), as measured by the continuous release of nitrogen in the form of amino acids and urea. The nitrogen loss corresponds to a protein degradation rate of 3-4% per h, while the rate of protein synthesis is negligible. Cells prepared from fasted, fed to regenerating livers are all highly protein-catabolic. The nitrogen balance is unaffected by insulin or amino acids (physiological mixture), and various metabolites and sera have only moderate effects. However, incubation of the cells for 2-4 h in a tissue culture medium (Dulbecco's) reduces the nitrogen loss dramatically, suggesting the formation of an anticatabolic factor under these conditions.


Inhibition of virus-induced plaque formation by atoxic derivatives of purified cobra neurotoxins. Venom from Naja naja siamensis was resolved into 16 toxic and nontoxic fractions by chromatography on SP-Sephadex, C-25. The principal neurotoxin preparations were chromatographically and electrophoretically homogeneous. Of the purified constituents, only the principal neurotoxin and minor neurotoxins were precursors of inhibitors of plaque formation among baby hamster kidney fibroblasts infected with Semliki Forest Virus.


Application of polarography to the study of chemical radiosensitization of DNA components. The half-wave reduction potentials of a range of nitroheterocyclic compounds were measured polarographically and compared with their radiosensitizing abilities in a simple radiation biochemical assay for damage to 5'-GMP. The chemical enhancement ratio for sensitization of inorganic phosphate release from irradiated 5'-GMP, increased with sensitizer electron affinity, but in a non-linear manner. An onset of sensitization was observed for nitroheterocyclics with half-wave potentials less negative then -0.05V vs. standard calomel electrode and maximum sensitization, approaching the oxygen effect, was obtained at a sensitizer half-wave potential of -0.25 V vs. standard calomel electrode. Nitroheterocyclic sensitizers with half-wave potentials in this range may be potential clinical radiotherapeutic agents.


Ammonia inhibits protein secretion in isolated rat hepatocytes. The general secretion of proteins by isolated rat hepatocytes in suspension is inhibited by colchicine, anoxia, or ammonia (NH4Cl). The inhibition by ammonia is accompanied by the cytoplasmic retention and swelling of protein secretory vesicles, suggesting that ammonia accumulates in the vesicles and thereby prevents their translocation to the cell periphery. General protein synthesis appears to be relatively unaffected by ammonia.


Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver. We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 +/- 2 mg/h per 100 g liver, hemopexin at 5.0 +/- 0.7, haptoglobin at 3.2 +/- 0.6 and transferrin at 5.1 +/- 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 +/- 0.1, and that of lactate: pyruvate 27 +/- 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies. A minimal effect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3-4-fold increase in hemopexin but not in albumin and transferrin synthesis. A striking 2-3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.


Sources of ammonia for urea synthesis in isolated rat liver cells. L-Leucine inhibits urea synthesis in rat hepatocytes from a number of nitrogen sources, including ammonia. The inhibition by L-leucine is largely overcome by addition of 1 mM L-ornithine, suggesting that the main site of L-leucine action is at ornithine transcarbamylase, rather than at glutamate dyhydrogenase. L-Norvaline is a more potent inhibitor of urea synthesis than is L-leucine, but again the inhibition is largely counteracted by L-ornithine. Addition of aminooxyacetate and L-norvaline strongly suppresses the formation of glucose and lactate from L-asparagine, suggesting that an alternate pathway of aspartate metabolism, the purine nucleotide cycle, in not a major pathway. Hadacidin, an inhibitor of adenylosuccinate synthetase, an enzyme of the purine nucleotide cycle, has no effect on urea synthesis in rat liver cells.


Biosynthesis and metabolism of cystathionine in Astragalus pectinatus. Metabolism of L-[35S]cystathionine, L-[35S]cysteine and L-[35S]homocysteine has been investigated in Astragalus pectinatus. The results indicate that cystathionine undergoes both beta and gamma cleavage to give homocysteine and cysteine. Results also show that cystathionine is synthesized from both cysteine and homocysteine. Furthermore, in addition to the incorporation of 35S into cystathionine, incorporation of 35S from cysteine into methionine and from homocysteine into S-methylcysteine is not only in agreement with the above cystathionine cleavage activities, but also suggests, that transsulfuration in A. pectinatus proceeds in both directions, eg. cysteine leads to cystathionine leads to homocysteine and homocysteine leads to cystathionine leads to cysteine. It is suggested, that the latter reaction may be contributing to the net synthesis of cysteine.


Metabolic effects of 2-deoxy-D-glucose in isolated fat cells. The uptake and phosphorylation of 2-deoxy-D-glucose by isolated adipocytes of the rat was determined by a method of rapid flotation through oil coupled with separation of sugar from sugar phosphate by chromatography on Dowex-1-formate. Uptake of the sugar is rapid and linear over 5 min, with a gradual decline thereafter; by 1 h, no further uptake is observed. Initially only 2-deoxy-glucose phosphate is observed within the cells; by 1 h, however, free 2-deoxy-glucose accumulates to levels approximately those in the medium. Phosphorylation ceases when intracellular levels of 2-deoxyglucose phosphate are about 50 mM regardless of the medium concentration of 2-deoxyglucose; this does not represent feedback inhibition of hexokinase, since the enzyme in fat cell homogenates is not inhibited by 50 mM 2-deoxyglucose 6-phosphate. Accumulation of deoxyglucose 6-phosphate is associated with a marked decline in intracellular ATP levels. Fat cell respiration is also depressed by approximately 50 per cent after a 1 h preincubation with 10 or 20 mM 2-deoxyglucose. Intracellular ATP levels and O2 uptake are only partially corrected by the addition of pyruvate to the incubation medium. Since no glucose was present in the medium, and intracellular concentrations of glycogen are known to be small in adipose tissue, it is proposed that accumulation of 2-deoxyglucose 6-phosphate within fat cells has a direct inhibitory effect on cell respiration unrelated to inhibition of glycolysis. No increase in intracellular free fatty acids was observed to explain this, and under the conditions of the incubations it is unlikely that Pi availability was rate limiting. The exact locus of inhibition is unknown.


Glucocorticoid receptors in corticosensitive and corticoresistant thymocyte subpopulations. II. Studies with hydrocortisone-treated mice. In vitro studies of the residual thymocyte population isolated 48 h after in vivo hydrocortisone injection showed: 1. These cells are partly sensitive to steroid as demonstrated by uridine incorporation inhibition. 2. This residual cell fraction appears heterogeneous after centrifugation on a bovine serum albumine gradient. 3. These cells exhibit low steroid binding and DNA synthesis capacities.


Influence of amytal and menadione on high-energy phosphates and acid secretion in frog gastric mucosa. Measurement of ATP and creatine phosphate in isolated frog gastric mucosa showed that amytal depressed the level of both high-energy compounds and inhibited acid secretion. Addition of menadione restored the ATP level to control values and partially restored the creatine phosphate levels but did not support acid secretion. Menadione was found to support that portion of active chloride transport which is not associated with acid output. It is concluded that an amytal-sensitive reaction, presumably mitochondrial coupling site I, is required for acid secretion to occur and that ATP is not a sufficient source of energy for this secretory process.


Plasminogen activator from human embryonic kidney cell cultures. Evidence for a proactivator. The nature of the trypsin-activatable plasminogen activator produced by kidney cell cultures (Bernik, M.B (1973), J. Clin. Invest. 52, 823-834) was investigated using human embryonic kidney (HEK) cell cultures in serum-free medium. Plaminogen activator activity ratios (trypsin-activated/ untreated controls) in HEK cell-conditioned media were maximal (up to 3) during the first week of culture and remained nearly constant at approximatley 2 for the next 3-5 weeks, while the total plasminogen activator titer increased in a nearly linear manner. Therefore, coincident with progressive cell degeneration and death, the ratios decreased to near unity due to "spontaneous" activation of the enzyme, which was inhibited in cell-free conditioned media by the pancreatic trypsin inhibitor Kunitz and benzamidine. Since the activator is not inhibited by the trypsin inhibitor, it is concluded that a protease other than the plasminogen activator is responsible for the activation. Increases in the plasminogen activator titers (about 2-fold) were similarly obtained by culturing the cells in medium containing low concentrations (0.05-0.10 mug/ml) of trypsin for up to about 6 weeks. The presence of the trypsin inhibitor in HEK cells cultures decreased the rate of activation, resulting in higher activity ratios (up to 6), and the total plasminogen activator activity was reduced only minimally (less than 20%), if at all, by the highest concentration of the trypsin inhibitor (100 mug/ml) tested. Affinity chromatography of conditioned media with activity ratios of 1.6--2 separated the plasminogen activator into an active fraction and a fraction which was activated a minimum of 200-fold by trypsin and contained no measurable activity prior to activation. Gel filtration of crude conditioned media or partially purified activator separated the plasminogen activator into two peaks; both were trypsin-activatable, and their relative proportions varied with the isolated conditions. The results indicate the occurrence of a proenzyme form of the plasminogen activator in the culture media.


A tryptic digestion fragment of nerve growth factor with nerve growth promoting activity. A peptide, isolated from the acid-insoluble portion of a tryptic digest of cyanogen bromide cleaved nerve growth factor, favors life maintenance of sensory target cells and promotes rapid neurite outgrowth from 7-day-old chick embryo sensory ganglia. The fragment has been identified as a 30 amino acid peptide consisting of two linear oligopeptides linked by a disulphide bridge and corresponding to residues 10-25 and 75-88 of the amino acid sequence of nerve growth factor. On a molar basis the fragment is about 100 times more effective than intact nerve growth factor. Other peptides isolated from the digest are biologically inactive.


Preparation and biochemical characterisation of isolated parenchymal cells from adult sheep liver. 1. A simple procedure for the isolation of morphologically intact, metabolically viable sheep liver parenchymal cells is described in detail. 2. The method is based on the initial treatment of fresh liver slices with collagenase and hyaluronidase. 3. The cell preparation was studied with respect to membrane permeability, potassium content, ATP/ADP ratio, adenylate content, and gluconeogenic capacity with respect to various substrates. 4. Data are present with respect to the distribution of phosphoenolpyruvate carboxykinase in isolated cells and whole sheep liver. 5. The results are compared, where possible, with data currently available from isolated perfused sheep liver and multi-catheterised animals.


Distribution of mitochondrial enzymes between the perikaryal and synaptic fractions of immature and adult rat brain. The subcellular distribution of mitochondrial enzymes was studied in cerebral hemispheres of 15-day-old and adult rats. At both ages the synaptosomal fraction contained very little glutamate dehydrogenase (EC 1.4.1.2) but significant amounts of succinate dehydrogenase (EC 1.3.99.1), glutaminase (EC 3.5.1.2), hexokinase (EC 2.7.1.1), malate NADP dehydrogenase (EC 1.1.1.40) and beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30). In immature brain, in the fraction enriched with free (perikaryal) mitochondria, the concentrations of these enzymes were 9.5, 1.8, 2.0, 0.92, 1.5, and 2.1 times higher, respectively, than in the synaptosomes. The increase with age in succinate dehydrogenase and glutaminase was restricted to free mitochondria while hexokinase and malate NADP dehydrogenase accumulated and beta-hydroxybutyrate dehydrogenase diminished in both fractions. In adult brain, too, where the above ratios became 7.5, 5.2, 3.5, 0.84, 1.4, and 2.0, respectively, the concentrations of enzymes relative to each other distinguished clearly between free and synaptic mitochondria. The results substantiate previously noted signs of mitochondrial heteroeneity in adult brain, and extend them to immature brain. The chemical composition, the quantitative pattern of enzymes, of free and synaptic mitochondria is clearly different, and undergoes separate changes during postnatal differentiation.


Studies on the receptors to 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol in sow nasal mucosa. The presence of receptors to the "boar taint" pheromones 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol has been demonstrated in sow olfactory mucosa. Binding studies indicated that a sufficiently low concentration of olfactory tissue homogenate exhibited saturation of binding of 5alpha-androst-16-en-3-one, and this was of high affinity compared with control tissues of non-olfactory and heated olfactory tissues. Analysis of receptor binding of 5alpha-androst-16-en-3-one gave a value for the affinity constant (Ka) of approx. 8.3-10(8) M-1 and the value for the molar concentration of binding sites (n[M]) was approx. 3.3 pmol/mg protein. Almost identical values of Ka and n [M] were obtained when receptor binding of 5alpha-[5alpha-3H]androst-16-en-3alpha-ol was investigated (Ka 8.4-10(8) M-1; n [M] 3.7 pmol/mg protein). This suggests that the same receptor binds both 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol with equally high affinity. In a preliminary investigation to establish the specificity of the receptor, the binding of 17beta-hydroxy-5alpha-androstan-3-one was assayed; this steroid is odourless but has a similar structure except in ring D to 5alpha-androst-16-en-3-one. Binding was of the low affinity, non-specific type only, indicating that the sow olfactory receptors are not sensitive to this androgen.


Purification of rat intestinal receptor for intrinsic factor-vitamin B-12 complex by affinity chromatography. Rat intrinsic factor was bound to vitamin B-12-Sepharose to produce intrinsic factor-vitamin B-12-Sepharose. Intestinal receptor for intrinsic factor-vitamin B-12 complex was purified from rat ileal extract by affinity chromatography using the intrinsic factor-vitamin B-12-Sepharose as an affinity adsorbent with recovery of 48.5% and specific activity increased 335 fold of original sample.


Localization and characterization of luminescent cells in ophiopsila californica and Amphipholis squamate (echinodermata: ophiuroidea). 1. The distribution of luminescence in Ophiopsila californica and Amphipholis squamata is described on the basis of image intensification, fluorescence microscopy, and histological techniques. 2. Luminescence appears to be intracellular. 3. The photogenic cells, termed photocytes, can be identified in histological sections by observation of 460 nm excited fluorescence and appear to have two components: varicosities and processes. The processes are morphologically similar to the neurons of the peripheral nervous system and the radial nerve cord. 4. The theory of gland cells as sites of luminescence in Ophiopsila spp. and Amphipholis squamata is not supported. 5. The emission spectra of luminescence in O. californica and A. squamata are broad with a half band width of 71 nm and an approximate emission maximum at 510 nm. 6. Fluorescence appears only after the onset of luminescence.


Genic similarity of American and European species of the lobster Homarus. European lobsters (Homarus gammarus) from the Norway coast and from the Irish Sea are examined for electrophoretically detectable genetic variation in seventeen functionally different proteins. Forty-one loci encoding these proteins are homologous with loci studied in a previous survey of eight populations of H. americanus. Progeny hatched from ovigerous Norway females show variation in three enzymes, but Mendelian inheritance is confirmed only for triosephosphate isomerase and for one of the phosphoglucose isomerases. Complex PGI phenotypes are described. The average amounts of genetic variability in European and American lobster populations appear to be equivalent. More than one allele is detected at 20% of the loci, the average number of alleles detected per locus is 1.2 and the average proportion of loci heterozygous per individual is 4.0%. While much less genically variable than other invertebrates, Homarus is not atypical when compared with eleven decapod species that average 5.8% heterozygosity. This is consistent with hypotheses relating genetic variability to adaptive strategy. At thirty loci H. gammarus is monomorphic for the common H. americanus allele. Two acid phosphatase systems are fixed or nearly fixed for alternative alleles in the two species while the remaining polymorphic loci show various degrees of interspecific divergence. Unique H. gammarus alleles are detected at five loci but only contribute significantly to species differences at the Acph-5, Me, and Pgi-4 loci. Acph-1, Est-2, Pgi-3, and Pgm-1 are polymorphic for the same alleles in both species, but again, with various differences in allelic frequencies. In sum, average genetic identity and average genetic distance are: I = 0.896 +/- 0.007 and D = 0.110 +/- 0.007, respectively. Compared to the values for conspecific population comparisons, I = 0.994 +/- 0.001 and D - 0.006 +/- 0.001, it is clear that a small but significant amount of genetic divergence separates the European and American lobster. Based on the premise that protein differences between existing species reflect the amount of time since they shared a common ancestor, it can be speculated that the European and American lobsters were isolated during the Pleistocene. The apparent weakness of reproductive isolating barriers suggests that these populations have evolved allopatrically. Finally, quantification of species' genetic differences, together with recent successes in interspecific laboratory matings, implicates species hybridization as a potentially important breeding practice in lobster aquaculture.


Morphological and cytochemical studies on the secretory granules of the pyloric caeca of the starfish, Asterias amurensis. A fraction rich in secretory granules was prepared from the pyloric caeca of Asterias amurensis by sucrose density gradient centrifugation. The freshly prepared fraction exhibited no casein-hydrolyzing activity, but showed nine times as much specific activity as that of tissue homogenates after incubation at 37 degrees C for thirty minutes. Electron microscopy showed that the secretory granules were membrane-bound granules measuring 0.5-2.0 mu in diameter and contained dense and/or light amorphous substances.


Development of the dimorphic claw closer muscles of the lobster Homarus americanus: L Regional distribution of muscle fiber types in adults. 1. The closer muscles of the dimorphic claws (chelipeds) were studied for the presence and location of fast and slow muscle fibers. 2. Cutter claws were composed of about 60-70% short sarcomere (less than 4 mum) fast fibers; the remainder was longer sarcomere (greater than 6 mum) slow and intermediate (4-6 mum) fibers. 3. Crusher claws were composed of a uniform population of long sarcomere (6-13 mum) slow and intermediate (4-6 mum) fibers. 4. There was a regional distribution of fibers in the cutter claw. Ventral fibers were predominantly slow. Dorsal fibers and central medial fibers were fast. Proximal and distal fibers in the medial section were usually mixed. 5. The regional distribution of cutter fibers correlates with previous physiological studies on the distribution of the fast and slow motor axons to these muscle fibers.


The larval development of Cliban vittatus (Bosc) (Crustacea: Decapoda; Diogenidae) reared in the laboratory. 1. A table of previous descriptions of larvae of Diogenidae is given. 2. Larvae of the hermit crab Clibanarius vittatus were reared on a diet of Artemia nauplii. Five, rarely four, zoeal stages and megalopa were obtained. Duration, mortality and sizes of larvae from two hatches are listed. 3. Detailed descriptions and figures for each larval stage are presented.


Calcium and electroconvulsive therapy of severe depressive illness. In severely depressed patients with primary affective illness, consistent decreases in total calcium concentration have been demostrated in both CSF and serum following successful ECT treatment. The hypocalcemia does not appear to occur after the initial ECTs, but develops after three to five treatments, coincident with an acceleration in clinical antidepressant effects, and is not an artifact of the anesthetic premedications or mechanical ventilation of the patients. Evidence linking alterations in calcium metabolism to mood is reviewed and a number of mechanisms by which the calcium change might result from ECT and mediated its effects on mood are discussed.


Average evoked responses in a rapidly cycling manic-depressive patient. A patient with unusually regular and rapid switches from mania to depression was studied for 113 consecutive days through five switches. Average evoked responses (AERs) to four intesities of light were recorded from vertex and occipital leads; telemetered activity records and behavioral ratings were also collected. Late AER components (P200) tended to change amplitude synchronously with the switches, vertex P200 amplitude decreased and occipital P200 amplitude increased in mania. Urinary MHPG changes paralleled the changes in P200 amplitude. Early AER components (P100) and especially the amplitude/intesity slope measures for P100 decreased about 8-10 days before a switch from depression to mania. Cross-spectral and linear regression analysis helped confirm these observations. The results, taken together with AER data in recent L-dopa studies, were consistent with catecholamine potentiation prior to the switch process from depression into mania.


Asymmetric charge distributions in planar bilayer systems. Using the simple argument based on irreversible thermodynamics and the Gouy-Chapman theory of the double layer, we show that the equilibrium distribution of charged lipid molecules between the two surfaces of a bilayer is asymmetric if the two solutions bathing the surfaces have the same ionic strength but contain ions of different valencies. For example, if one bathing solution contains 0.10 M NaCl and the other contains 0.70 M NaCl and 0.10 M CaCl2, the ratio of charged lipid molecules of the two surfaces in a membrane that contains 50% total negative lipids is 1.46, leading to a transbilayer potential of 18 mV. A complete set of such numerical results is presented in four figures.


Theoretical calculation of the dielectric constant of a bilayer membrane. The dielectric constant (epsilon) and refractive index (n) of a bilayer lipid membrane is determined from the known values of the polarizabilities of the carbon-carbon and carbon-hydrogen bonds. It is assumed that the hydrocarbon chains are hexagonally arranged in an all-trans conformation perpendicular to the plane of the membrane. The only variable in the calculation is the average separation between the chains and the theory relates epsilon to this separation. The calculation and results differ significantly from those presented in a 1968 publication by Ohki. It is shown that a thin membrane is not homogeneously polarized by the applied field. This effect is analysed and the dependence of epsilon on the membrane thickness is determined. The theoretical results are in good quantitative agreement with experimental measurements on bulk paraffins and on oriented multilayers of saturated fatty acids. The most important conclusion is that the dielectric constant for an applied field perpendicular to the membrane (which is the appropriate value for capacitance measurements) differs by only a few percent from the value for the macroscopic (bulk) liquid hydrocarbon. Thus the dielectric constant of a bilayer membrane can be approximated by the value for the appropriate bulk hydrocarbon.


The cylindrical cell with a time-variant membrane resistance. Measuring passive parameters. The passive electrical properties of a cable can be measured by injecting a step of current at a point and fitting the resulting potentials at several positions along the cable with analytic solutions of the cable equation. An error analysis is presented for this method (which is based on constant membrane resistance) when the membrane resistance is not constant, but increases linearly with time. The increase of rm produces a "creep" in the membrane potential at long times, as observed in cardiac, skeletal, and smooth muscle. The partial differential equation describing the time-varying cable was solved numberically for a step of current and these "data" were fit by standard constant-resistance methods. Comparing the resulting parameter values with the known true values, we suggest that a correction of the standard methods is not satisfactory for resistance changes of the kind observed; instead, the cable equation must be solved again for the particular form of rm(t). The practical implementation of a method by Adrian and Peachey for measuring the membrane capacitance and an approximate method for estimating the rate-of-change of membrane resistance are discussed in appendices.


Nanosecond decay studies of a fluorescence probe bound to apomyoglobin. Excited state interactions of N-(p-tolyl)-2-aminonaphthalene-6-sulfonate (2, 6 p-TNS) bound to apomyoglobin were studied by nanosecond time-resolved emission spectroscopy. A dynamic interaction of the excited dye molecule with its binding site, associated with a significant change in the emission energy with time, was observed. The decay kinetics were found to be complex and consistent with the kinetic model for solvent relaxation as proposed by Bakhshiev et al. (Opt. Spectrosc. 21:307. 1966). The behavior of 2, 6 p-TNS bound to apomyoglobin was found to be qualitatively similar to that of the dye dissolved in a viscous solvent such as glycerol or adsorbed to egg lecithin vesicles. The detailed information obtained by following the changes in emission spectra of fluorescent probes on the nanosecond time scale leads to a better understanding of their interactions with biological systems.


Viscoelastic properties of the human red blood cell membrane. II. Area and volume of individual red cells entering a micropipette. Previous work demonstrated that human red cells can be drawn into cylindrical glass micropipettes of internal diameter approximately 2.0 mum without lysing. For pipettes of less than approximately 2.9 mum inside diameter, the red cell must become less spherical, that is, reduce its volume-to-area ratio. In this work measurements were made from 16-mm film records that allowed the determination of the cellular area and volume of individual erythrocytes as they were drawn into a 2.0-mum pipette with negative pressures. The results showed that the total surface area of the membrane remains constant and that the cell endures the passage into the pipette by losing volume. The volume loss was interpreted to be due to cell water and solute loss when the membrane is under stress. The loss of cell volume, rather than the stretching of the membrane, adds confirmation that although it is very deformable, the membrane is very resistant to two-dimensional strain.


The quantum efficiency of the bacteriorhodopsin photocycle. The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.


The quantum efficiency for the photochemical conversion of the purple membrane protein. The quantum efficiency for the formation of M(412), an intermediate product in the photoconversion of the purple membrane protein of Halobacterium halobium, was determined to be 0.30 +/- 0.03 at -40 degrees C. This photochemical reaction was photoreversible to the original pigment and the ratio of the quantum efficiencies gamma PM(568 leads to M(412)/gamma M(412) leads to PM(568) was 0.39 +/- 0.02. No change was seen in either value when exciton interaction between chromophores was eliminated. The sum of gamma PM(568) leads to M(412) plus gamma M(412) leads to PM(568) was 1.07 +/- 0.10, approximately 1, suggesting that the pigment and its primary photoproduct share a common excited state.


Microwave dielectric properties of tissue. Some comments on the rotational mobility of tissue water. Dielectric permitivity and conductivity data are reviewed for tissue over the frequency range of 0.1-10 GHz. The conductivity of muscle increases quadratically with frequency above 1 GHz, suggesting a Debye relaxation for tissue water centered at 20 GHz at room temperature, the same as for bulk water. Approximate mixture equations suggest that this "free" water accounts for about 70% of the tissue weight, showing that most of the tissue water has rotational mobilities similar to those in the bulk fluid.


125I-UDR induced division delay.

125I-UDR induced division delay. Mitotic selection for cell cycle analysis was used to investigate the effects of (3)H and (125)I, incorporated into DNA, on the cell cycle progression of Chinese hamster ovary (CHO) cells. The results indicate that S-phase cells were delayed and G(2) cells were not.


Use of new silylating agents for identification of hydroxylated steroids by gas chromatography and gas chromatography mass spectrometry. Differences in methylene unit values were used for the determination of the hydroxyl group number of a steroid by means of gas chromatography. This index is defined as the difference in the methylene unit value between trimethylsilyl and other dimethylalkylsilyl (DMAS) ether derivatives of hydroxylated steroids, namely dimethylethylsilyl (DMES) and dimethyl-n-propylsilyl (DMPS) ethers. The reactivities of DMES and DMPS imidazoles as silylating agents were nearly equal to that of TMS-I. Mass spectra of these derivatives were characterized by the molecular ion cluster, [M]+., [M-15]+ and [M-29]+ (or [M-43]+). The molecular ion cluster of these derivatives is most useful for estimating the molecular weight. Therefore, these DMAS ethers provide valuable information for structural elucidation of hydroxylated steroids by gas chromatography electron impact mass spectrometry.


Single and multiple ion recording techniques for the analysis of diphenylhydantoin and its major metabolite in plasma. A method has been developed for single ion monitoring of diphenylhydantoin and its major metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin in plasma. A plasma extract is reacted with N,O-bis(trimethylsilyl) acetamide and single ion recording is carried out using a gas chromatograph mass spectrometer system. The mass value selected, m/e 254, is common to the TMS ethers of diphenylhydantoin and its principal metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin. The result indicate that one cause of an adverse reaction to diphenylhydantoin could be a reduced ability to hydroxylate the drugmquantitative methods for the analysis of the drug and its major metabolite have also been developed. Diphenylhydantoin and 5-(p-hydroxyphenyl)-5-phenylhydantoin can be analysed in plasma after addition of deuterium labelled internal standards and conversion to volatile derivatives for mass fragmentographic analysis. Diphenylhydantoin and its internal standard are analysed as the N,N-dimethyl derivative, and the hydroxylated metabolite and its internal standard are converted to a petrimethylsilyl compound by reaction with N,O-bis-(trimethylsilyl)acetamide.


Determination of phenylbutazone, tolbutamide and metabolites in plasma and urine using chemical ionization mass spectrometry. Quantitative analytical procedures for the analysis of phenylbutazone and tolbutamide levels in plasma have been developed which involve the addition of deuterium labeled internal standards to plasma followed by extraction and direct sample insertion into a mass spectrometer operating under chemical ionization conditions. Peak height ratios used to calculate plasma levels were determined by using either selected ion monitoring or repetitive scan data. The scan approach was used in a related procedure for the simultaneous determination of tolbutamide and two metabolites from urine. The accuracy, precision and sensitivity of the direct sample insertion approach to drug level measurement has been determined. Examples are given of data obtained in the course of pharmacokinetic studies in which this analytical approach appears to offer advantages in the analysis of multicomponent mixtures encountered in drug-drug interaction studies.


Mass spectrometry of cyclopenta[alpha]phenanthrenes. Metabolites formed in vitro and in vivo from the carcinogen 15,16-dihydro-11-methyl-cyclopenta[alpha]phenanthren-17-one. The mass spectra of a number of synthetic cyclopenta[alpha]phenanthrenes are described, and identification of an in vitro metabolite of a carcinogen of this series is reported. The correlation of the mass spectra of the major urinary metabolite, its dehydration product and its triacetate with the structure assigned to the metabolite is described.


A novel inexpensive device for the electrochemical generation of metallic emitters for field desorption. Details for the construction of a novel, inexpensive device for the electrochemical generation of metallic emitters for field desorption mass spectrometry are described. Use of the device for the generation of cobalt and nickel emitters is demonstrated.


An emission-controlled field desorption and electron impact spectrometry study of some N-substituted propane and butane sultams. It is shown that the production of field desorption mass spectra with an emission-controlled emitter heating device is a useful tool in obtaining information about molecular structures. Since the structurally significant signals are sometimes missing in the corresponding electron impact spectra but not in the field desorption spectra the use of field desorption mass spectrometry appears to be more advantages. This will be especially true for substances that are thermally labile, unvolatile or unstable upon electron impact (both at high and low electron energy), the latter being the case for some of the compounds presently investigated which are derivatives of a drug with antitumour and antiepileptic effects. Further, emission-controlled desorption improves the reproducibility of the spectra obtained and thus appears to be a prerequisite for quantification in biomedical and pharmacokinetic studies.


Some simple modifications to the LKB 9000S to achieve improved selected ion monitoring and repetitive scanning performance. Modifications are described to the electron multiplier pre-amplifier, selected ion monitor, magnet current supply and scan controller of the LKB 9000S gas chromatograph mass spectrometer. The modifications described have secured an improved performance for specific ion monitoring and fast repetitive scanning gas chromatography mass spectrometry.


Multiple myeloma: quantitative staging and assessment of response with a programmable pocket calculator. Studies of M-component synthesis and metabolism, as well as clinical staging of multiple myeloma, have provided the basis for calculation of the total body tumor cell number and quantitation of response to treatment. Many of the calculations involved have been complex and have previously required the availability of a computer. The advent of programmable pocket calculators has permitted simplification of these programs for general clinical and investigative applications in studies of myeloma and related monoclonal gammopathies. In this paper we have included the background information and a clinical example of the use of the programmable calculator, as well as the logic steps and the calculator programs themselves, for several of the most useful programs for clinical staging and quantitation of response to therapy in IgG myeloma.


Polycythemia vera: hormonal modulation of erythropoiesis in vitro. We performed in vitro culture studies examining the interaction of erythropoietin with red cell progenitors in polycythemia vera. Bone marrow was obtained from five patients with typical disease and from five healthy volunteers, and assayed for erythroid colony formation (CFU-E) by the methylcellulose technique. In cultures without added erythropoietin, a mean eightfold greater cloning efficiency was noted with the polycythemia vera marrows, as compared to normal. There was prominent stimulation of colony formation by erythropoietin, and the shape of the erythropoietin dose-response cruves appeared to be similar in both patients and controls. Anti-erythropoietin antibody reduced the number of CFU-E in cultures not containing added erythropoietin, but did not eliminate them. Dexamethasone (10(-9) M) caused a consistent increase in CFU-E in the patients' cultures. These studies provide evidence for functional erythropoietin and glucocorticosteroid receptor mechanisms on erythroid precursors in polycythemia vera. The observations are consistent with a concept of this disease as a disorder of hematopoietic stem cells in which peripheral erythrocytosis is caused by an expanded erythroid progenitor compartment which maintains responsiveness to hormonal modulation.