Purification of terminal riboadenylate transferase from calf thymus gland.

A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.


Cholesterol esterase in rat adipose tissue and its activation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

A high level of cholesterol esterase activity, comparable to that of hormone-sensitive triglyceridase, has been demonstrated in rad adipose tissue. Essentially all of the activity was in the isolated adipocytes, primarily in the 100,000 times g supernatant fraction of the adipocytes. Cholesterol esterase activity in the 100,000 times g supernatant fraction was increased 40 plus or minus 16% by incubation with ATP (0.5 mM), Mg-2+ (1.25 mM), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) (10 muM), conditions which also activated hormone-sensitive triglyceridase. Protein kinase inhibitor (rabbit skeletal muscle) blocked activation, and activation was restored by the addition of excess protein kinase (bovine skeletal muscle). In extracts prepared from adipocytes first incubated for 5 min with 10 muM epinephrine and 1 mM theophylline, there was no cyclic AMP-dependent cholesterol esterase activation, implying that the enzyme had been activated by a similar mechanism in the intact cell. The physiological role of this high level of cholesterol esterase activity in adipose tissue is unclear. Its relationship to hormone-sensitive triglyceride lipase, with which it extensively co-fractionates, and its possible involvement in fat mobilization remain to be determined.


Subunits of fatty acid synthetase complexes. Enzymatic activities and properties of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The separation of the half-molecular weight, nonidentical subunits (I and II) of the pigeon liver fatty acid synthetase complex has been achieved on a large (20 mg) scale by affinity chromatography on Sepharose epsilon-aminocaproyl pantetheine. This separation requires a careful control of temperature, ionic strength, pH, and column flow rate for success. The yield of subunit II is further improved by transacetylation (with acetyl-CoA) of the dissociated fatty acid synthetase prior to affinity chromatography. The separated subunit I (reductase) contains the 4'-phosphopantetheine (A2) acyl binding site, two NADPH binding sites, and beta-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contains the B1 (hydroxyl or loading) and B2 (cysteine) acyl binding sites, and acetyl- and malonyl-CoA: pantetheine transacylases. When subunit I is mixed in equimolar quantities with subunit II, an additional NADPH binding site is found even though subunit II alone shows no NADPH binding. Both subunits contain activities for the partial reactions, beta-hydroxybutyryl thioester dehydrase (crotonase) and palmityl-CoA deacylase. Subunit I has 8 sulfhydryl groups per mol whereas subunit II has 60. Reconstitution of fatty acid synthetase activity to 75% of the control level is achieved on reassociation of subunits I and II.


Crystallization and properties of L-arginine deiminase of Pseudomonas putida.

Crystalline L-arginine deiminase of Pseudomonas putida was prepared by the following steps: sonic disruption, ammonium sulfate fractionation, protamine sulfate treatment, DEAE-cellulose column chromatography, and L-arginine-Sepharose 6B chromatography followed by crystallization. This procedure yields a crystalline pure enzyme with a 45% recovery of the activity in crude cell-free extracts. The yield is significantly higher than that reported for this enzyme. The purified enzyme appears to be homogeneous in ultracentrifugation (s-o20, w equals 10.2 S) and isoelectric focusing (pI equals 6.13). The purified enzyme showed two bands on disc gel electrophoresis, both carrying out the deimination of L-arginine. Electrophoresis in the presence of beta-mercaptoethanol plus Na dodecyl-SO4 gave a single band (Mr, 54,000). Specific activity of this enzyme was 58.8 mumol of L-citrulline formed per min per mg of protein at 37 degrees. The optimum pH of the purified enzyme was 6.0 and maximal activity was obtained at 50 degrees. The molecular weight of the native protein was 130,000 by gel filtration and 120,000 by sedimentation-equilibrium measurements. The spectrum of the pure enzyme showed absorption maximum at 280 nm and the value of E-1%-1 CM AT 280 NM WAS 10.48 IN 0.05 M potassium phosphate buffer (pH 7.0). The crystalline enzyme hydrolyzed several L-arginine analogues. L-Homoarginine, L-alpha-amino-gamma-guanidinobutyric acid, and L-alpha-amino-beta-guanidinopropionic acid competitively inhibited the hydrolysis of L-arginine with Ki values of 25.7, 7.5, and 4.0 times 10- minus 3 M, respectively. p-Chloromercuribenzoate, Ag-+, and Hg-2+, and several metal ions inhibited the enzyme.


Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.


The extended active site of guinea pig liver transglutaminase.

The catalytic activities of guinea pig liver transglutaminase toward glutamine-containing peptide derivatives of three series have been studied. These series include: (a) formylheptapeptides of the basic structure, HCO-GLY3-L-Gln-Gly3. A single L-leucine residue was systematically substituted for glycine at a different position in each peptide; (b) formyltripeptides of the basic structure, HCO-Gly-L-Gln-Gly. L-Leucine was substituted for glycine in each position and in both positions; (c) various N-acyl derivatives of the dipeptide, L-Gln-Gly. Comparison of the values of the kinetic constants for methylamine incorporation and for hydroxylamine incorporation with the peptide derivatives shows that the length of the peptide chain has a pronounced influence on catalysis, as does the position of the leucine residue in the longer chain peptide derivatives. The kcat/Km(app) values for each substrate calculated from data for methylamine incorporation and from those for hydroxylamine incorporation were found to be in good agreement. However, both the observed maximum velocity and the apparent Michaelis constant for each peptide derivative were significantly larger for hydroxylamine incorporation than for methylamine incorporation. Interpretation of these findings as evidence for a normal catalytic mechanism for each amine incorporation reaction and for the limiting nature of deacylation to methylamine is discussed. Two observations caution against such an interpretation. These are the significantly higher inhibitor constants found fo formylhexaglycine and for several other competitive inhibitors in the hydroxylamine incorporation reaction, and earlier findings of higher turnover values with hyroxylamine in cases were acylation appears to be limiting for methylamine incorporation. Methods of preparation, supporting analytical data and properties of the peptide intermediates, the peptides, and their derivatives used in this study are presented in the miniprint supplement immediately following this paper.


Glucose-sensitive adenylate cyclase in toluene-treated cells of Escherichia coli B.

Toluene treatment of Escherichia coli B makes it possible to measure adenylate cyclase activity directly using [alpha-32-P]-ATP as substrate. In contrast to French press extracts, the activity of adenylate cyclase in toluene-treated cells shows many of the characteristics of the enzyme seen in the intact cell. In both toluene-treated and intact cells the activity of adenylate cyclase is inhibited at least 85% by glucose, while in French press extracts the enzyme activity is much lower and is not sensitive to inhibition by glucose. In toluene-treated cells, glucose inhibits at 10 muM, and the effect is rapid in onset and readily reversible. The activity is not inhibited by glucose 6-phosphate suggesting that glucose is responsible for the inhibition. The measurement of the activity and sensitivity to glucose of adenylate cyclase in toluene-treated cells requires the presence of potassium phosphate in the assay medium. Since it does not increase the activity or sensitivity of the enzyme in the French press extract, it is suggested that potassium phosphate is required for the maintenance of cellular integrity necessary for the activity and sensitivity of adenylate cyclase.


Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.


The in vitro biosynthesis of dhurrin, the cyanogenic glycoside of Sorghum bicolor.

A microsomal fraction from seedlings of Sorghum bicolor (Linn) Moench has been shown to catalyze the conversion of L-tyrosine to p-hydroxymandelonitrile via p-hydroxyphenylacetaldoxime. This transformation is consistent with the general pathway for cyanogenic glycoside biosynthesis proposed on the basis of in vivo experiments. When the microsomal fraction was combined with a protein fraction from the soluble portion of the cell and uridine diphosphate glucose, it was possible to demonstrate the synthesis of the cyanogenic glycoside dhurrin from L-tyrosine.


Characterization of a polyriboadenylate polymerase from vaccinia virions.

A poly(A) polymerase with a molecular weight of approximately 80,000 containing 51,000 and 35,000 molecular weight subunits, was purified by affinity chromatography from vaccinia virus cores. The enzyme had a pH optimum of about 8.6, was dependent on divalent cations, and had considerably more activity with Mn-2+ than Mg-2+. At equimolar concentrations, other ribonucleoside triphosphates inhibited poly(A) polymerase activity by less than 10%; NaCl was extremely inhibitory at concentrations above 0.1 M. Under standard assay conditions, poly(A) polymerase activity was stimulated more than 10-fold by poly(C), but to small extent or not at all by other homopolyribonucleotides or natural RNA species unless they were first subjected to partial hydrolysis and alkaline phosphatase treatment. The ineffectiveness of most long polyribonucleotides was attributed to enzyme binding to internal regions. Short poly- or oligoribonucleotides prepared from natural or synthetic RNAs, except poly(G), exhibited similar priming abilities, and isotope transfer experiments indicated the covalent attachment of poly(A) to cytidylate, uridylate, and inosinate residues. Experiments with a series of uridylate oligomers indicated that the minimum effective primer length was four to six nucleotides. Partially digested DNA and short poly- and oligodeoxyribonucleotides of dT, dC, and dI, but not of dA and dG, also acted as effective primers for the poly(A) polymerase.


Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.


Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.


Beta-Ketoacyl-acyl carrier protein synthetase.  Characterization of the acyl-enzyme intermediate.

Beta-Ketoacyl-acyl carrier protein (ACP) synthetase catalyzes the condensation reaction of fatty acid synthesis in Escherichia coli. The homogeneous enzyme reacts with hexanoyl-CoA to form hexanoyl-enzyme which was isolated and characterized. Hexanoyl-enzyme contains 2 mol of hexanoate/mol of enzyme (molecular weight 66,000); it is liable at alkaline pH, and it reacts with neutral hydroxylamine to form hexanoyl hydroxamic acid. Hexanoate was cleaved from the enzyme when hexanoyl-enzyme was subjected to performic acid oxidation. These properties indicate that hexanoyl-enzyme is a thioester. Studies of the circular dichroism spectra of fully acylated and nonacylated forms of the enzyme indicated that the secondary structure of the enzyme is relatively unperturbed by the presence of the hexanoyl groups. An alpha helical content of 65% was estimated for the enzyme from the circular dichroism spectrum. Hexanoyl-enzyme is active in both partial reactions that comprise the beta-ketoacyl-ACP synthetase reaction; it reacts with ACP to form hexanoyl-ACP and with malonyl-ACP to form beta-ketooctanoyl-ACP. Although the hexanoate of hexanoyl-enzyme is transferred very rapidly to ACP, the physiological acceptor in this reaction, it is also transferred very slowly to CoA, dithiothreitol, and 2-mercaptoethanol, indicating that the enzyme can react nonspecifically with a number of unrelated mercaptans.


Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli.

Two forms of beta-ketoacyl-acyl carrier protein (ACP) synthetase (designated I and II) have been identified in extracts of Escherichia coli. Synthetase I corresponds to the condensing enzyme that was studied earlier (GREENSPAN, M.D., ALBERTS, A.W., and VAGELOS, P.R. (1969) J. Biol. Chem. 244, 6477-6485); synthetase II represents a new form of the enzyme. Synthetase II was isolated as a homogeneous protein. It differs from synthetase I in having a higher molecular weight (76,999 versus 66,000), a lower pH optimum (5.5 to 6.1 versus 7.2), and a greater resistance to denaturation by heat. Synthetase II is similar to synthetase I in that both are inactivated by iodoacetamide, and prior incubation of the enzymes with fatty acyl thioesters prevents the inhibitory effect of iodoacetamide. Both also react with a fatty acyl thioester to form an acyl-enzyme intermediate, and the latter reacts with malonyl-ACP to form a beta-ketoacyl thioester. Specificity studies indicated that synthetase II, like synthetase I, has similar affinities with saturated and cis unsaturated fatty acyl thioesters of ACP that are intermediates in the synthesis of saturated and unsaturated fatty acids, respectively. The two synthetases differ only with respect to reactivity with palmitoleyl thioesters: synthetase II has a lower Km and higher Vmax than synthetase I with palmitoleyl-ACP. This finding suggests that synthetase II functions specifically in the elongation of palmitoleyl-ACP to form cis-vaccenyl-ACP. An investigation of synthetases I and II in two classes of unsaturated fatty acid auxotrophs revealed that synthetase I is absent in one class, fabB. Addition of wild type synthetase I to fabB fatty acid synthetase, which synthesizes only saturated fatty acids, permitted this fatty acid synthetase to synthesize unsaturated fatty acids. These experiments indicate that synthetase I plays a critical role in the synthesis of unsaturated fatty acids.


Intermediates and enzymes between alpha-ketoarginine and gamma-guanidinobutyrate in the L-arginine catabolic pathway of Pseudomonas putida.

In Pseudomonas putida P2 grown on L-arginine as the sole source of carbon and nitrogen, catabolism of L-arginine forms of alpha-ketoarginine, gamma-guanidinobutyrate, and gamma-aminobutyrate. A previously undetected intermediate, gamma-guanidinobutyraldehyde, is identified as the product of alpha-ketoarginine decarboxylase. An 86-fold purification of this enzyme is described. Activity is thiamine pyrophosphate-dependent and cofactor reassociation is facilitated by divalent cations. The order of effectiveness is Mn-2+ greater than Mg-2+, Co-2+ greater than Ca-2+ greater than Ni-2+ greater than Zn-2+. An inducible enzyme that catalyzes conversion of gamma-guanidinobutyraldehyde to gamma-guanidinobutyrate has been studied in cell-free extracts. NAD-+, but no other cofactors, is required. By differential nutritional growth experiments, 4 regulatory units for the L-arginine pathway are proposed and inducers of 2 units are identified.


Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi.  Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+.  ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport.  Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone.  These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.


Reactions of pyrzdoxal 5'-phosphate, 6-aminocaproic acid, cysteine, and penicilamine. Models for reactions of Schiff base linkages in pyridoxal 5'-phosphate-requiring enymes.

Schiff base formation, transaldimination, and reaction with aminothiols are important reactions which occur on the surface of pyridoxal-P-requiring enzymes. As a first step in assessing the role of the protein in these reactions, models for these reactions were studied in the absence of enzyme at 25 degrees, gamma/2 0.28. The reaction of 6-aminocaproic acid with pyridoxal-P to form the Schiff base N6-(P-pyridoxylidene)-aminocaproic acid was studied as a model for formation of Schiff base on the holoenzyme...


The binding of CO2 to human hemoglobin.

CO2-dissociation curves of concentrated human deoxy- and carbonmonoxyhemoglobin at 37 degrees, pH 7.6 to 7.0, PCO2 equal to 10 to 160 mm Hg, have been obtained by a rapid mixing and ion exchange technique.  The CO2-dissociation curves for deoxyhemogloblin can only be fitted by assuming two classes of binding sites for carbon dioxide.  The simplest way to account for the experimental data is to assume that the alpha-amino groups of the alpha and beta chains react with carbon dioxide with affinities that differ by at least a factor of 3.  No difference in reactivity with CO2 was found among the four terminal alpha-amino groups of carbonmonoxyhemoglobin.


Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.


Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied  by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.


Chemical modification of bovine heart mitochondrial malate dehydrogenase.  Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide.  NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site.  N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue.  The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea.  NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation.  This enzyme has a subunit weight of 33,000 and is a dimer.  The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.


Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%.  The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer.  The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol.  The reduced flavin is instantaneously reoxidized by addition of GSSG.  The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158).  This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+.  However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism.  NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates.  The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.


Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex.

The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5.  Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E. coli cells.  The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38).  Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates.  The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen.  The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA).


Characterization of the deoxyribonucleic acid polymerase associated with Kilham rat virus.

Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.


Nuclear and cytoplasmic deoxyribonucleic acid polymerases from rat nephroma cells.

We have examined the DNA polymerases found in a rat nephroma cell line. Using DEAE- and DNA-cellulose chromatography, we have found two major cytoplasmic DNA polymerases and one major and three minor DNA polymerases from the nucleus. The enzymes were all purified, characterized, and distinguished from each other by several criteria. The enzyme require, for maximal activity, a natural or synthetic double-stranded DNA, four deoxynucleoside, triphosphates, and magnesium. They are inhibited to varying degrees by sodium pyrophosphate, ethidium bromide, and rho-chloromercuribenzoate.


A unique reactive residue in adenosine triphosphate phosphoribosyltransferase sensitive to five conformation and dissociation states.

Adenosine triphosphate phosphoribosyltransferase is inactivated rapidly by bulky alkylating reagents in a biphasic reaction.  The initial inactivation rate is dependent upon an optimal concentration of histidine and is more rapid at low enzyme concentrations and low ionic strength.  A histidine-free dimer form of the enzyme is the proposed reactive species.  The dimer is shown by ultraviolet difference spectroscopy to bind histidine about 1 order of magnitude more weakly than the hexameric form of the enzyme.  Alkylated enzyme is similar to native enzyme in dissociation and histidine-binding properties.  Native enzyme must exist at significant levels in at least five different conformational and dissociative states to account for the inactivation behavior.


The effects of ionic conditions, temperature, and chemical modification on the fluorescence of myosin during the steady state of ATP hydrolysis.  A comparison of the fluorescnece and electron spin resonance spectra of the spin-labeled enzyme.

The ATP-induced enhancement of the intrinsic fluorescence of myosin and heavy meromyosin (HMM) that persists during the steady state of hydrolysis has been investigated. To compare the substrate-induced changes in fluorescence with those in the electron spin resonance spectrum of the spin-labeled enzyme, we studied the influence of temperature, pH, and ionic strength, as well as the effect of chemical modification (spin labeling) of the SH-1 sulfhydryl groups. Changing the pH between 6 and 9 does not affect the enhancement of fluorescence of myosin or HMM; changing the ionic strength, which could be studied only with HMM, also has no effect; and decreasing the temperature from 20 to 5 degrees slightly diminishes the enhancement with both myosin and HMM. Chemical modification with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, which blocks the SH-1 thiol groups, reduces the enhancement of fluorescence, induces a strong dependence on ionic strength and pH, and substantially increases the dependence on temperature. The enhancement with labeled myosin or labeled HMM increases with increasing pH, ionic strength, and temperature, closely paralleling the effects of these parameters on the electron spin resonance spectrum of spin-labeled myosin (SEIDEL, J.C. and GERGELY, J. (1973) Arch. Biochem. Biophys. 158, 853), suggesting that the same molecular change, induced by ATP and associated with formation of the MADP-P1 complex, underlies both the change in fluorescence and the change in ESR spectrum. Those analogues of ATP that produce the maximal enhancement of fluorescence (WERBER, M., SZENT-GYORGYL, A.G., and FASMAN, G. (1972) Biochemistry 11, 2872) also produce the maximal change in the ESR spectra. Both an amino group at position 6 of the substrate and an unmodified triphosphate chain are required for maximal change in either fluorescence or ESR spectra. The smaller enhancement of fluorescence produced by spin labeling the SH-1 groups persists after the nitroxide has been chemically changed to a diamagnetic species. Thus the small enhancement cannot be attributed to paramagnetic quenching of tryptophan fluorescence by the spin label. An initial burst of phosphate liberation accompanies the hydrolysis of ATP, cytidine 5'-triphosphate, uridine 5'-triphosphate, guanosine 5'-tryphosphate, iosine 5'-triphosphate, 2'-deoxyadenosine 5'-tryphosphate, adenosine 5'-tetraphosphate, and tripolyphosphate. The presence or absence of the burst does not correlate with the extent of the spectral change.


Gram-negative osteomyelitis following puncture wounds of the foot.

Seven cases of gram-negative osteomyelitis following puncture wounds of the foot are presented. Regardless of age (range, five to thirty-two years) all patients had a similar, typical clinical picture of one to two weeks of improvement after the initial post-traumatic inflammation followed by worsening of the local signs. Two patients were successfully treated with antibiotics alone and five, with antibiotics in combination with curettage.


Effect of ploidy on spontaneous mutation rate to asparagine non-requirement in cultured cells.

Variations in ploidy do not affect the spontaneous mutation rate to asparagine non-requirement in Jensen rat sarcoma cells cultivated in vitro.


Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts.

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate. Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells. The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2--16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.


Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B.

After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50 mum S minus 1, but but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2nlevels, free Ca2nat 10 minus 7 M or less and was inhibited by high levels of Clminus and by pH's on either side of 7.0. The reactivated movements were rapidly and completely inhibited by 25mug ml minus 1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.


Chromatographic analysis of the neutral and phosphatide glyceryl ethers from various biological sources.

A method of analysis is described which permits the facile evaluation of the neutral glyceryl ether lipids (mild alkaline hydrolysis) and total glyceryl ether lipid (Vitride reduction) thereby allowing an assessment of the phosphatide contribution. The alkyl and alk-enyl glyceryl ethers are chromatographically resolvable on Gelman glass-fiber type SG and Whatman SG-81 and detected by the periodic acid-Schiff and plasmal reaction, respectively. The attending problems and interpretation are discussed with examples from a number of unicellular organisms and animal tissues.


Ion-exclusion chromatography of compounds of biological interest.

The principle of ion exclusion was examined as a method for the separation of small ionic compounds. The systems employed consisted of very porous column packings, substituted with fixed charges, which were eluted by buffer solutions of low ionic strength. DEAE-Sephadex A-50 was principally employed, and it was shown that there was a linear relationship between the net charge on a cation and its partition coefficient into the gel phase. A similar relationship existed in the chromatography of amino acids on various columns bearing fixed negative charges. It was concluded that this was an efficient form of chromatography, which gave results directly related to the ionic charge of the sample being examined. The charge characteristics of biologically active compound could be determined by this method.


Identification of glucagon in the gastrointestinal tract.

Gel filtration studies on Bio-Gel P-10 columns of a 50-fold purified porcine duodenal extract revealed a main peak of glucagon-like immunoreactivity (GLI) in the 2,900 mol wt zone and a smaller peak in the 3,500 mol wt zone, the same zone as the pancreatic glucagon marker. Like pancreatic glucagon, samples of 3,500 mol wt material gave essentially identical measurements in radioimmunoassays employing the pancreatic glucagon-specific antiserum 30K and the GLI crossreacting antiserum 78J, whereas the 2,900 mol wt peptide gave 60-fold higher readings in the 78J assay. On disk gel electrophoresis, the 3,500 mol wt fraction, like pancreatic glucagon, migrated at pH 8.3, whereas the 2,900 mol wt peptide remained at the origin; at pH 4.7, the 2,900 mol wt peptide migrated while the 3,500 mol wt immunoreactive peptide and glucagon remained at the origin. Isoelectric focusing revealed the 3,500 mol wt moiety to have an isoelectric point (pI) of 6.2, the same as pancreatic glucagon, whereas the 2,900 mol wt peptide had an pI greater than 10. The glycogenolytic activity of the 3,500 mol wt peptide in the perfused rat liver did not differ significantly from glucagon, and its adenylate cyclase stimulating activity in partially purified liver cell membranes was comparable to that of glucagon; the 2,900 mol wt peptide had less than 20% of these activities. In samples of 3,500 mol wt material subjected to isoelectric focusing, adenylate cyclase-stimulating activity was confirmed to fractions containing 30K immunoreactivity with a pI of 6.2. In samples of 2,900 mol wt material subjected to isoelectric focusing, adenylate cyclase-stimulating activity was confined to fractions containing 78J immunoreactivity with an pI greater than 10. Displacement of [125-I]glucagon from the membranes was limited to these two biologically active fractions. However, the affinity of both pancreatic glucagon and the 3,500 mol wt peptide was an order of magnitude greater than of the 2,900 mol wt peptide. Thus, by all of several biologic, physiocochemical, and immunometric techniques, the 3,500 mol wt gut immunoreactive peptide could not be distinguished from pancreatic glucagon, while the 2,900 mol wt peptide was readily differentiated by all these techniques. "True" A-cells, ultrastructurally indistinguishable from pancreatic A-cells but differing from the A-like cells of the lower bowel, were identified in the gastric fundus of dogs. Their distribution corresponded to that of the 3,500 mol wt immunoreactivity resembling pancreatic glucagon, while the distribution of "A-like cells" in the lower small intestine corresponded to that of GLI.


The acetylation of hemoglobin by aspirin.  In vitro and in vivo.

The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.


An alternative pathway for fibrinolysis.  I. The cleavage of fibrinogen by leukocyte proteases at physiologic pH.

An alternative fibrinolytic system, active at physiological pH, is present in peripheral blood leukocytes. The fibrinolytic proteases localized predominantly in the leukocyte granules are capable of degrading both fibrinogen and fibrin, and plasmin activity does not contribute significantly to this proteolytic event. The specificity of the alternative fibrinolytic proteases for fibrinogen and the characteristics of the derivative cleavage fragments are clearly distinguishable from the classical plasmin system. The high molecular weight derivatives of fibrinogen, generated by the alternative system, under physiological conditions, are larger than the plasmin-generated X fragment, exhibit immunoelectrophoretic mobility comparable to native fibrinogen, and are not coagulable by thrombin. Analysis of the constituent polypeptide chains of the fragments reveals cleavage of the Aalpha, Bbeta, and gamma chains of fibrinogen. The lower molecular weight derivatives of fibrinogen, generated by the alternative system, are structurally distinct from previously described fibrinogen degradation products and exhibit potent anticoagulant activity. This anticoagulant activity can be attributed to interference with normal fibrin polymerization. The proteases of the alternative fibrinolytic systems are actively secreted by leukocytes when stimulated to undergo a nonlytic release reaction. These results provide direct evidence for a fibrinolytic system resident in leukocyte granules that is associated with the leukocyte release reaction and is capable of generating unique fibrinogen cleavage fragments.


Tributyrate as a substrate for the determination of lipase activity in milk.

A procedure is described for the determination of the lipolytic activity in milk using tributyrate as substrate. The method is based on the titration of liberated butyric acid in a diethyl ether: light petroleum (2-75:1, v/v) extract of the incubation mixture. Some of the fundamental factors involved in the procedure are discussed.


Lipases in bovine milk and the relationship between the lipoprotein lipase and tributyrate hydrolysing activities in cream and skim-milk.

The lipoprotein lipase and tributyrate hydrolysing activities were found to be similarly distributed in the fractions obtained when whole milk was separated into skim-milk and cream, and when the cream was washed and freed from lipid. These enzyme activities in skim-milks and in extracts of lipid-free cream could not be separated by affinity chromatography on heparin-Sepharose. The enzymes were inactivated to the same degree when incubated at 37 degrees C in the presence of 1-5 M-NaCl, pH 8-5,  and both showed marked decrease in stability at 4 degrees C in UV-light caused the same decrease in both lipoprotein lipase and tributyrate hydrolysing activities. An antiserum against a highly purified skim-milk lipoprotein lipase caused total inhibition of the lipoprotein lipase and tributyrate hydrolysing activities in skim-milk and in extracts of lipid-free cream. It is suggested that in bovine milk there is only one major lipase and that it is identical to lipoprotein lipase.


The precipitation of proteins by carboxymethyl cellulose.

Carboxymethyl cellulose (CMC) formed insoluble complexes with beta-lactoglobulin, bovine serum albumin and Na caseinate. Maximum precipitation of the beta-lactoglobulin-CMC complex occurred at pH 3-2, whereas maximum precipitation of the bovine serum albumin. The percentage of protein precipitated by CMC decreased with increasing ionic strength of the solution, the rate of decrease being least for bovine serum albumin. At a given ionic strength, more protein was precipitated by CMC of high degree of substitution than by CMC of low degree of substitution. The change in pH (delta pH) occurring on mixing CMC and unbuffered protein solutions, each initially at the same pH, was measured. delta pH was negative for beta-lactoglobulin-CMC mixtures over the pH range 7--2 (minimum at pH 5-5). For bovine serum albumin--Cmc and Na caseinate--CMC mixtures, delta pH was positive between pH 7 and 3-2 (maximum at pH 4-5), zero at pH 3-2 and negative between pH 3-2 and 2-0 (minimum at pH 2-8).


Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.


Electrophoresis of cottage cheese whey proteins and their polymers.

Cottage cheese whey solutions containing sodium dodecyl sulfate were resolved electrophoretically on 5% sodium dodecyl sulfate polyacrylamide gels. Major bands were identified by comparing their electrophoretic mobilities to those of known whey components. Other components were identified principally from molecular weights determined from a calibration correlating mobility and molecular weight. Conditions which affect polymerization of proteins were studied in whey and solutions of purified beta-lactoglobulin. Formation of a number of polymers was induced by concentrating the whey samples, lowering the temperature, adjusting pH, or adding salts. The dimer, trimer, tetramer, and octamer of beta-lactoglobulin, the dimer and trimer of bovine serum albumin, and several unidentified components in the 100,000 to 300,000 molecular weight range were observed. The octamer state of beta-lactoglobulin was observed in whey at pH values between 5.1 and 8.0, at temperatures below 10 C, and with .2M addition of potassium thiocyanate, potassium iodide, calcium chloride, or sodium acetate. Similar polymer formation, and temperature and pH effects, were observed with solutions of purified beta-lactoglobulin, which contained dimers, trimers, and tetramers. The beta-lactoglobulin octamer in whey samples could be dissociated by the addition of acid. The bovine serum albumin dimer appeared in whey at pH above 6.2 and at -minus 1 C.


Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.


Heat stability and reactivation of mare milk lysozyme.

Mare milk and aqueous solution of mare milk lysozyme were incubated for variable times between 30 C and 100 C at pH 3, 6, or 9. Lysozyme activity was stable at acid and neutral pH and labile at alkaline pH. Some of the results show the existence of a reactivation process in mare's milk and in aqueous solution. reaching 30 to 40% after incubation of the aqueous solution at 4 C for 20 days at pH 3 or 6.


Chemistry of milk proteins in food processing.

This paper analyzes the current knowledge of mild protein chemistry to explain the reactions and their control for the major processes utilized by the modern milk processing industry. The compositon and chemical properties of casein micelles and whey proteins are summarized. The effect of processing upon denaturation, aggregration, and destabilization of milk proteins is updated. The role of milk proteins in the gelation of sterile milk concentrates, destabilization of frozen milk, rennet-clotting of milk, and stabilization of the fat emulsion in milk is also described.


Enamel demineralization by snack foods.

The amount of enamel destroyed by salivary fermentation of snack foods and confections was not dependent on their sugar content; starch, flavoring agents, and other components also played a part. Most enamel destruction was produced by fruit-flavored candies in which the inherent acid or high sugar concentration or both inhibited bacterial fermentation.


Formation of Ca HPO4-2H2O from enamel mineral and its relationship to caries mechanism.

Tooth enamel, when treated with dilute H3P94 solutions, dissolved incongruently with formation of large CaHPO4-2HO2O crystals. Equilibrated solutions were saturated with respect to Ca5(PO4)3OH, probably an impure, defective apatite. The CaHPO4-2H2O crystals formed at considerably higher pH values than expected becuase of enhanced solubility of the apatitic phase in enamel. Pyolysis of carious enamel revealed the presence of acidic calcium phosphate presumed to be CaHPO4-2H2O.


The physician's assistant.

A physician's assistant is trained to act in the place of the physician in the diagnostic and therapeutic management of patients. Tasks that can be performed by assistants, legislation related to their responsibilities, the question of physician supervision, educational programs and certification, and factors in employment are matters that are being dealt with by our colleagues in medicine. The acceptance of this new category of health care personnel has implications for the status of expanded duty dental auxiliaries in dentistry.


Observations on the psychopharmacology of the aged.

Psychotropic drugs are discussed under the descriptive categories of antipsychotic, antidepressive, antimanic, anti-anxiety, and cognitive acting. In the antipsychotic classification, special attention is given to side effects (extrapyramidal motor signs, tardive dyskinesias, akathisis) and to dosage for the elderly. The section on congenitive acting drugs includes some pertinent observations on cognitive ability in the aging, the importance of correct diagnosis, and therapeutic strategy. The aged are a population at risk for not only disease, but iatrongenic illness due to direct and indirect drug action. Rational therapy involves understanding the underlying dynamics of the disease and thus the selection of the most effective treatment. Psychotropic drugs in an appropriate program are valuable therapeutic assets.


A survey of the prescribing and administration of drugs in a long-term care institution for the elderly.

The prescription and administration of drugs (especially of the neuroactive class) was observed in 131 patients in an extended care facility. The average number of neuroactive drugs prescribed (2.1) was distinctly different from the average number administered (1.3) because of the large number of pro re nata (prn) prescriptions. More neuroactive substances were prescribed for patients with superior mentation and minimal physical disability; the difference between low and high groups was 1.7 (mentation) and 2.8 (physical status). The most common neuroactive drugs prescribed were: 1) analgesics, 2) major tranquilizers, and 3) hypnotics. Questionable prescribing practices were demonstrated by the fact that 30 patients had prescriptions for 38 "not-recommended"drugs; 23 of these prescriptions were for propoxyphene compound. After requiring physicians to rewrite drug orders every thirty days, a survey made ten months later showed that there was a decline (0.8) in the number of drugs prescribed per patient and a slight increase (0.45) in the number of drugs administered. Professional drug surveillance is crucial for improving the therapeutic process. At least two modifications of current prescribing practices are recommended: 1) a record should always be made of the precise condition(s) under which a drug prescribed "prn" is to be administered; and 2) a strong effort should be made to reduce the total number of drug prescriptions. The results of this survey suggest that certain procedural matters necessitating change are not in themselves the most substantive factors in improvement. Present "third party" review mechanisms likely will not ameliorate the current situation. It will be necessary to implement complex organizational changes in most extended care facilities.


Human epidermal transamidase.

The possible presence of epsilon-(gamma-glutamyl) lysine covalent bonds in human epidermal proteins prompted a study of transamidase activity in human hair-free epidermis. Callus contains an enzyme which catalyzes the incorporation of radioactive putrescine into alpha-casein. The enzyme is active without prior treatment with exogenous proteolytic enzymes. The putrescine incorporation is calcium dependent and inhibited by iodoacetamide. The enzyme was partially purified (50-fold over starting material), and has an apparent molecular weight between 50,000 daltons and 55,000 daltons by agarose 0.5m gel filtration. The apparent molecular weight is unaltered by chromatography in the presence of 11 mMCaCl2, a condition known to dissociate plasma transglutaminase (Factor XIII) into its ultimate subunits. The enzyme is active over a wide pH range up to pH 10.4 The Km for putrescine varies by 1-fold over the pH range 6.0 to 10.2, although enzyme activity increases at least 20-fold over the same pH range. The human epidermal transamidase is similar to the guinea-pig hair follicle transglutaminase and cow snout transamidase in its ability to cross-link fibrin.


Cyclic 3',5'-nucleotide phosphodiesterase in rat skin. II. Biochemical characterization.

The biochemical characteristics of cyclic 3',5'-nucleotide phosphodiesterase were studied in homogenates of male albino rat skin using preparations which were predominantly epidermal. Enzymatic activity was detected in both the particulate and soluble fractions of these skin homogenates. Two kinetically distinct phosphodiesterase (PDE) activities were detected in the soluble fraction (100,000 times g supernatant). This 100,000 times g supernatant contains at least two distinct protein bands that hydrolyze cyclic AMP as demonstrated by gel electrophoresis. Divalent cations (Mg-++ or Mn-++) and 2-mercaptoethanol were required for maximal enzymatic activity. Epinephrine, dibutyryl cyclic AMP, and methylxanthines inhibited while imidazole and histamine phosphate stimulated the cyclic AMP phosphodiesterase activity at high and low cyclic AMP concentrations. Cyclic GMP competitively inhibited hydrolysis of low, but not high, concentrations of cyclic AMP. Hydrocortisone phosphate in pharmacologic concentrations blocked PDE denaturation by heat. These studies indicate that there are complex interrelationships between cyclic nucleotides and PDE in rat skin.


Quantitative microbiology of the scalp in non-dandruff, dandruff, and seborrheic dermatitis.

The composition of the scalp microflora was assessed quantitatively in normal individuals and in patients with dandruff and seborrheic dermatitis, disorders characterized by increasing scaling. Three organisms were constantly found: (1) Pityrosporum, (2) aerobic cocci, and (3) Corynebacterium acnes. Pityrosporum (mainly Pityrosporum ovale) made up 46% of the total microflora in normals, 74% in dandruff, and 83% in seborvheic dermatitis. The geometric mean number of organisms per cm-2 in non-dandruff subjects was 5.04 times 10-5; 9.22 times 10-5 in dandruff subjects; and 6.45 times 10-5 in those with seborrheic dermatitis. The cocci were dominantly Baird-Parker type SII and no quantitative or qualitative change occurred in the scaling disorders. C. acnes comprised 26% of the flora on the normal scalp, 6% in dandruff, and only 1% in seborrheic dermatitis. These results differ significantly from previous reports which describe a much more complex microflora and suggest an etiologic role for microorganisms in dandruff.


Analysis of lipid composition of isolated human sebaceous gland homogenates after incubation with cutaneous bacteria. Thin-layer chromatography.

The effects of specific species of skin bacteria on human sebaceous gland lipids in vitro were analyzed. Isolated dissected sebaceous glands were pooled, homogenized, and sterilized, then incorporated into peptone-yeast extract medium and used as substrate for growth of Propionibacterium acnes, P. granulosum, and Staphylococcus epidermidis subgroup II. The sebaceous lipids were analyzed by thin-layer chromatography before and after bacterial growth. The most striking effect of bacteria on sebaceous gland lipid composition was the hydrolysis of sebaceous triglycerides. The degree of hydrolysis varied with bacterial strain but was most complete with P. acnes and P. granulosum. Staphylococci were not effective in hydrolyzing sebaceous triglycerides at pH 4.5 although, when the pH of the medium was raised to pH 6.4, some strains of staphylococci were as effective as the propionibacteria in hydrolyzing sebaceous triglycerides to free fatty acids. Thus minor changes in acidity may play asignificant role in controlling the lipolytic activity of staphylococci on skin. Another effect of bacterial action on sebaceous gland lipids was the esterification of sebaceous cholesterol to cholesteryl esters. Thus, bacterial action must be taken into account in evaluating studies of alterations in cutaneous cholesterol and cholesteryl esters in skin surface lipids in normal and disease states.


Human necrotizing vasculitis: immunoglobulins and complement in vessel walls of cutaneous lesions and normal skin.

Immunoglobulins and C3 were detected by immunofluorescence in the blood vessel walls of biopsies of clinically normal skin in patients with active necrotizing vasculitis. Of the 13 patients studied, 9 had C3 and 6 of these had IgM or IgA in biopsies of lesions of vasculitis. In adjacent clinically normal skin, 7 patients had C3 and 3 of these also had IgM or IgA. These findings support the hypothesis that immunoglobulins and complement are present in vessels of some patients prior to chemotaxis of polymorphonuclear leukocytes and the resulting inflammatory purpuric lesions so characteristic of necrotizing vasculitis.


In vivo hepatic and intestinal toxicity of sodium cyanate in rats: cyanate-induced alterations in hepatic glycogen metabolism.

To determine the hepatic and intestinal toxicity of sodium cyanate, this compound was administered to rats by orogastric tube (PO) or intraperitoneal injection (IP). At low dosage (50 mg. per kilogram per day PO for 8 weeks), the animals showed no clinical effects other than mild lethargy. They had normal intestinal absorption studies, but demonstrated decreased liver G6PD activity and a slight increase in hepatic glycogen. At higher dose levels (200 mg. per kilogram per day PO for 10 days, 400 mg. per kilogram per day PO for 3 days, and 100 mg. per kilogram per day IP for 10 days), the animals became very lethargic and developed hind-limb paralysis; many animals died during the period of dosing. The severity and rate of onset of symptoms increased proportionally with the dose level. Liver sections from rats receiving these higher doses showed striking increases in glycogen deposition. Activities of hepatic enzymes involved in glycogen synthesis and degradation were measured in rats receiving 200 mg. per kilogram per day PO  or 100 mg. per kilogram per day IP. Significant decreases were noted in the activities of glucose-6-phosphatase and G6PD in PO-dosed rats. The activities of phosphorylase, UDPG-pyrophosphorylase, glycogen synthetase, phosphoglucomutase, and debrancher did not differ from control rats. In IP-dosed rats, significant decreases were observed in the activities of glucose-6-phosphatase, G6PD, phosphorylase, and UDPG-pyrophosphorylase, but not in the other glycogen-related enzymes. Our data suggest that sodium cyanate affects several enzymes of hepatic glycogen metabolism but that the enzymes vary in their susceptibility (glucose-6-phosphatase and G6PD greater than phosphorylase and UDPG pyrophosphorylase.


Catecholamine content of serum and adrenals in protracted anaphylactic shock of guinea pigs.

In ovalbumin-sensitized guinea pigs, the serum catecholamines were significantly increased 20 or 120 min after antigen injection. The highest serum cathecholamine values were obtained in animals which were selected during a severe state of protracted shock, and in those which died in protracted shock. Antihistamine pretreatment reduced the serum noradrenaline, but not the adrenaline values in anaphylaxis. In urethane anesthesia, the anaphylactic serum catecholamine levels (especially noradrenaline) were lower than in nonanesthetized animals. The adrenaline and noradrenaline content of the adrenals in protracted and in acute anaphylactic shock did not differ from control values.


Release of corticotrophin releasing factor and other hypophysiotropic substances from isolated nerve-endings (synaptosomes).

Synaptosomes isolated from the sheep and the rat hypothalamus contain corticotrophin releasing factor, vasopressin, prolactin-release inhibiting factor and probably all of the substances which participate in the regulation of adenohypophysial function. Electrical field stimulation or depolarizing concentrations (55 mmol/l) of potassium ions cause a release of these factors from the incubated nerve-endings in a calcium-dependent manner. It is suggested that synaptosomes may provide a valuable approach to the study of mechanisms involved in hypothalamic neurosecretion in vitro.


Phosphatase active antigens in sea urchin eggs and embryos. I. Substrate specificity, pH-optima and inhibitors.

Phosphatase activity in sea urchin embryonic antigens was investigated by histochemical staining of immunoprecipitates separated by two-dimensional (crossed) immunoelectrophoresis. Unfertilized eggs were homogenized in a hypotonic medium which solubilized cytoplasmic antigens. Antigens integrated in membranes or enclosed in particles were solubilized by detergent treatment of the residual pellet. Two different phosphatase activities were discerned in the unfertilized eggs, nucleoside diphosphatase (EC 3.6.1.6.) and acid phosphatase (EC 3.1.3.2.). Nucleoside diphosphatase activity was obtained in both the water soluble and detergent extracted protein fractions. This activity was confined to one antigen. Acid phosphatase acitivity on the other hand was almost exclusively obtained in the detergent extracted fraction and about ten distinct antigens displayed this activity. The nucleoside diphosphatase active antigen preferentially hydrolyzed purine nucleoside diphosphates and to a lesser degree triphosphates of these nucleosides. The acid phosphatase active antigens had a broader substrate specificity and hydrolyzed equally well beta-glycerophosphate and nucleotides. Both activities were essentially inactive at neutral or alkaline pH values. The activities were inhibited by p-choloromercuribenzoate and accordingly stimulated by cysteine. Tartrate and sodium fluoride, however, inhibited the acid phosphatase activity while nucleoside diphosphatase activity was either stimulated or not affected at all by these agents.


Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.


Beta-lactamases from Yersinia enterocolitica.

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.


Temperature-sensitive mutants of foot-and-mouth disease virus: the isolation of mutants and observations on their properties and genetic recombination.

A number of temperature-sensitive mutants were isolated from two strains of foot-and-mouth disease virus (FMDV). Various properties of the mutants were examined including comparative growth curves at permissive and restrictive temperatures, cut-off temperatures, thermal lability and pH sensitivity. Recombination was observed between various pairs of mutants of FMDV strain Pacheco. It occurred early in the growth cycle and the proportion of recombinants remained constant thereafter. Maximum recombination was achieved if the input multiplicity of each virus was 6 p.f.u./cell or greater, provided the ratio of the input multiplicities did not vary by more than a factor of two. Day-to-day variations could be substantially reduced by normalizing recombination frequencies in terms of a standard cross. The results suggested that genetic mapping was possible with two-or three-factor crosses.


Sites of organic acid production and pattern of digesta movement in the gastrointestinal tract of swine.

Twelve swine were used to assess the movement of fluid and particulate digesta through their gastrointestinal tracts and to determine the diurnal variations in organic acid levels for various segments of the tract. Animals were fed twice daily at 12-hour intervals. Fluid (polyethylene glycol and chromium-labeled ethylenediamine-tetraacetic acid) and particulate markers (2 mm OD, and 2 mm and 1 and 2 cm long) were administration of markers. The gastrointestinal tract was divided into 12 segments for measurements of markers, pH, volatile fatty acids (VFA), and lactic acid (LA) contents. The data indicated a rapid evacuation of the fluid and the smaller particles from the stomach and their relatively rapid passage through the small intestine and cecum. There was, however, prolonged retention of both fluid and particulate markers first in the ascending and then in the descending colon. Larger particles (2 cm) were retained in the stomach throughout much of the 60-hour experimental period. LA levels were observed 8 hours postfeeding. The highest levels of VFA in gastric contents averaged 20 mmoles/liter. Gastrointestinal pH values showed significant changes with time postfeeding only within the stomach, where they did not reflect the changes in LA of VFA concentrations. VFA constituted 92% of the organic acids present in the large intestine. Their concentrations varied markedly with time (150-230 mmoles/liter), but the VFA at all times constituted the major anions in the large intestinal contents. The results demonstrated that digesta can be retained for prolonged periods of time in that swine stomach and colon. The high concentrations of organic acids also indicated that substantial degrees of microbial digestion of carbohydrates occurred at both sites.


Effects of dietary calcium level, acid stress, and age on renal, serum, and bone responses of rats.

The effects of age and acid stress on renal, serum and bone responses in 13- and 25-month-old rats, which were fed two levels of dietary calcium, 100 and 500 mg/100 g of diet, for 9 months, with and without dietary ammonium chloride (2%), were investigated. Acid-stressed animals showed significant decreases in urinary pH and significant increases in urinary total acid, calcium and phosphorus excretions, kidney weights, and phosphate-dependent glutaminase activities. Renal responses were affected by the level of calcium in the diet and the age of the animal. Acid stress tended to decrease serum calcium and phosphorus. Serum phosphorus was decreased in old animals, while serum calcium was unaffected by age. Tibia ash weights of old animals were significantly less and their fat content was significantly higher than that of young animals. However, neither acid stress nor the level of calcium in the diet significantly affected bone analysis in either age group.


Changes in fatty acid synthesis and lipogenic enzymes in adipose tissue from fasted and fasted-refed steers.

Controls of fatty acid synthesis in bovine adipose tissue were investigated. Six Brown Swiss steers were fasted for 8 days and then refed for 56 days. Biopsy samples of backfat adipose tissue were taken during the fasting and refeeding periods. Rates of acetate incorporation into fatty acids (FAS), activities of acetyl CoA carboxylase (CBX), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP:isocitrate dehydrogenase, and plasma free fatty acids (FFA) and plasma acetate were determined. FAS decreased 60% after 1 day of fasting and 99% after 8 days. FAS did not increase until day 3 of refeeding when energy intake was above maintenance, then returned to normal by 14 days. CBX followed a pattern similar to FAS, except its activity did rise above the control rate during refeeding. Plasma FFA increased 350% and acetate decreased 67% during fasting. After 4 days of refeeding, FFA returned to normal, and acetate increased to 156% of initial concentration, then returned to normal by 21 days. These data suggest that CBX limits FAS in adipose tissue of cattle.


The influence of cosolvents and substrate substituents on the sorption of benzoic acid derivatives by polyamides.

The sorption of some substituted benzoic acid derivatives by polyamides (nylons) from aqueous solution has been examined and the influence of their nature together with those of the cosolvent and polymer have been assessed. In all cases the sorption isotherms were linear and could be expressed by a simple distribution law, enabling the influence of cosolvent concentration to be predicted. The sorption of a series of p-substituted benzoic acids and some of their esters is related to their solubility except where the p-substituent is capable of hydrogen-bonding with the polymer. The extent of the interaction is also dependent upon the amide frequency of the polymer.


Non-ionic surfactants and membrane transport of thioridazine in goldfish.

Polysorbate 80, which has been widely used in studies of the effects of surfactants on drug absorption, increases the rate of absorption of some drugs at concentrations near its critical micelle concentration (cmc). To determine whether all non-ionic surfactants were capable of inducing this effect, the effects of six commercial non-ionic surfactants on thioridazine absorption in goldfish have been compared with the effect of polysorbate 80. The reciprocal death time (T-1) determined when the fish were immersed in the solution under study was the index of absoprtion rate used. Not all surfactants tested increased T-1. Cremophor EL (polyoxyethylated castor oil), Atlas G1295 (a polyoxyethylene fatty glyceride), Atlas G1300 (a polyoxyethylene glyceride ester) had no effect below their cmc's. Those surfactants that did increase T-1 [polysorbate 80 (a polyoxyethylene lanolin derivative), G2162 (a polyoxyethylene oxypropylene monosterate) and Renex 650 (a polyoxyethylene alkyl aryl ether)] display the concentration-dependent behaviour reported previously - a decrease in absorption rate when the surfactant concentration is increased above its cmc. The factor determining whether or not the surfactant will increas absorption rate appears to be the configuration of the surfactant molecule rather than its hydrophile-lipophile balance or its surface activity.


Surface tension and cloud point changes of polyoxyethylenic non-ionic surfactants during autoxidation.

Changes in the surface tension-concentration curves of the non-ionic surfactant cetomacrogol, a polyoxyethylenic (POE) hexadecyl ether containing about 24 ethylene oxide (EO) groups, have been examined during autoxidation of aqueous solutions. The curves exhibit decreases in cmc values and changes in the slopes below and above the cmc which lead to the loss of the sharp break characteristic of micelle-formation. There is also a progressive decrease in the cloud point during autoxidation. Surface tensions and cloud points of a series of known POE hexadecyl ethers containing from 10 to 60 EO units have been measured and related to the number of EO units present. From these data, it is apparent that autoxidation of cetomacrogol is accompanied by degradation of POE chain rather than the hydrocarbon chain, the number of EO units lost up to the time at which the solution becomes turbid being about 14. The significance of such measurements is discussed in relation to the detection of decomposition and pending rapid decomposition in synthesized and commercial surfactants.


Isolation, characterization and quantitation of chloral hydrate as a transient metabolite of trichloroethylene in man using electron capture gas chromatography and mass fragmentography.

A method for the quantitative determination of plasma concentrations of chloral hydrate by electron capture gas chromatography is described. Characterization by multiple ion monitoring confirms, for the first time, that chloral hydrate exists as a transient metabolite of trichloroethylene in man.


Quantitative determination of morphine in biological samples by gas-liquid chromatography and electron-capture detection.

A highly sensitive method for the determination of morphine in plasma and brain samples down to 500 pg ml-1 in plasma and 100 pg in 30 mg of brain tissue is described. This sensitivity is in the range of that described for radioactive and radioimmunological tehcniques. The samples (50-500 mu-l) are extracted with toluene-butanol (9:1) at pH 8.9 and then re-extracted into 0.1 N sulphuric acid and back extracted to a toluene-butanol phase at pH 8.9. The pentafluoropropionic anhydride derivatives of morphine and the internal standard nalorphine were quantitatively determined by gas-liquid chromatography with a 3-H electron capture detector.


Dextromethorphan inhibits 5-hydroxytryptamine uptake by human blood platelets and decreases 5-hydroxyindoleacetic acid content in rat brain.

The effect of dextromethorphan on the uptake and metabolism of 5-hydroxytryptamine (5-HT) was studied in human blood platelets and in rat brain. In the concentration of 120 nM dextromethorphan inhibited the uptake of 5-HT (1 mu-M) into platelets by 50%. The corresponding concentrations of imipramine and methadone under similar conditions were 22 and 590 nM, respectively. Dextromethorphan (20 to 40 mg kg-1) decreased the concentration of brain 5-hydroxyindoleacetic acid (5-HIAA) and the probenecid-induced accumulation of 5-HIAA time- and dose-dependently. However, dextromethorphan did not alter the pargyline-induced changes in brain 5-HT metabolism. Dextromethorphan-induced changes in brain 5-HT metabolism could arise from the inhibition of the re-uptake of 5-HT into neurons.


Evidence for a central alpha-sympathomimetic action of clonidine in the rat.

The antihypertensive effects of clonidine (0.15 mg kg-1, i.p.) were studied in conscious DOCA/saline hypertensive rats having chronically implanted arterial cannulae. The response to clonidine was markedly reduced by simultaneously administered desipramine (3 mg kg-1, i.p.), antagonized dose-dependently by piperoxan (2-10 mg kg-1, i.v.) and prevented by pretreatment with phentolamine (0.2 mg, i.c.v.). Pretreatment with 6-hydroxydopamine (3 x 250 mu-g, i.c.v.), haloperidol (1 mg kg-1, i.p.), p-chloro-N-methylamphetamine (3.5 mg kg-1, i.p.) or 5,6-dihydroxytryptamine (50 mu-g and 25 mu-g, i.c.v.) did not significantly modify the antihypertensive response. It is concluded that the antihypertensive response to clonidine is mediated via stimulation of central alpha-adrenoceptors and is independent of central dopaminergic receptors and intact central serotoninergic neurons. The necessity for intact central noradrenergic neurons remains uncertain.


Comparison of the time course of the anorectic effect of fenfluramine and amphetamine with drug levels in blood.

Time courses of the suppressive effects on food intake of (+)-amphetamine and (plus or minus)-fenfluramine in deprived rats were found to be different. Amphetamine displayed a potent initial action which rapidly decayed, and this behavioural effect was consistent with the measured blood concentration of amphetamine which showed a peak at 1 h followed by rapid clearance. For fenfluramine, the initial suppression of eating was maintained over several hours and was, for the first hour, related to the blood concentration of fenfluramine but later to an active metabolite, norfenfluramine. The study shows how drug-induced changes in feeding behaviour fluctuate over time and illustrate how single measures of food intake may overlook information about the effectiveness of anorectic drugs.


How does angiotensin II increase cardiac dopamine-beta-hydroxylation?

The potent accelerating effect of angiotensin II (Ang II) on caridac dopamine beta-hydroxylation was studied on slices of rat heart. Ang II did not affect the kinetics of beta-hydroxylation but it increased the axonal uptake of dopamine, and, concomitant with the acceleration of biosynthesis, it enhanced the accumulation of dopamine into tissue. Puromycin, in contrast to actinomycin D, antagonized the stimulation of dopamine beta-hydroxylation by Ang II, but did not suppress the rise in cardiac dopamine. Therefore, to promote the acceleration of dopamine beta-hydroxylation, (i) the rise in tissue dopamine available for conversion appeared to be insufficient, (ii) the formation of new proteins by activation of traduction seemed to constitute the basic mechanism of Ang II action.


Fluorescence characteristics of benzodiazepines in strong acid.

The fluorescence characteristics of 10 substituted 1,4-benzodiazepines in strong acid solution were investigated. The compounds that fluoresce in the Hammett acidity region possess, or can form by enolization, an azomethine linkage in the 1,2- or 4,5-position. All benzodiazepines that fluoresce in strong acid show increases in fluorescence intensity with corresponding blue shifts as acidity increases. Two pKa's in the Hammett acidity region were observed for both the fluorometric and absorptiometric titrations of the benzodiazepines possessing a carbonyl in the 2-position. No evidence of excited state prototropism was observed.


Determination of estrogens in dosage forms by fluorescence using dansyl chloride.

A sensitive, reproducible, fluorometric procedure for the determination of estrogens in pharmaceutical preparations is presented. The estrogens are determined fluorometrically following their reaction with dansyl chloride. The optimum conditions for the reaction such as pH, reaction solvent composition variations, and speed of reaction are discussed. In addition, a linearity study of the relationship between concentration and fluorescence intensity for estrone, estradiol, and ethinyl estradiol is reported. Solvent extraction procedures based on acid-base behavior or column chromatography are used when necessary to isolate the estrogen prior to reaction with dansyl chloride and fluorometric measurement. The recovery of estrogens from spiked samples indicated that the proposed method is efficient and reproducible. Comparison of the dansyl procedure with the official NF method in the analysis of estrone aqueous suspension showed the proposed method to be accurate and more precise than the NF assay. Estrone aqueous suspension, estradiol in sesame oil, estradiol valerate in castor oil and in sesame oil (in the latter case, in the presence of testosterone), estradiol benzoate in sesame oil, and ethinyl estradiol tablets (0.5 mg/tablet) from commercial sources were satisfactorily analyzed, with average results within compendial limits and no coefficient of variation greater than 2%.


In vitro antibacterial activity of drugs against human intestinal anaerobic bacteria.

The in vitro antibacterial activity, against the major groups of anaerobic fecal bacteria, of a series of drugs was examined by an agar diffusion test and the minimum inhibitory concentration (MIC) test. Only the phenothiazines and amitriptyline showed any marked antibacterial activity, the MIC's being in the 40-640-mu-g/ml range. It is suggested that the detergent nature of the molecules of these drugs in aqueous solution is responsible for the observed antibacterial activity.


Effect of beta receptor blockade on the initiation and perpetuation of ouabain-induced ventricular tachyarrhythmia.

The role of the cardiac beta receptor in the genesis and perpetuation of ouabain-induced ventricular tachyarrhythmias (VT) was investigated in the chloralose (20 mg/kg)-urethane (200 mg/kg) anesthetized dog. This was achieved by determining: 1) the effect of oxprenolol (0.3 mg/kg), a potent beta receptor blocking agent possessing no significant direct membrane effects on the myocardium, on the dose of ouabain necessary to cause VT (i.e., the toxic dose); and 2) whether oxprenolol had antirrhythmic efficacy against preexisting VT. The toxic dose was determined by giving a bolus injection of ouabain (similar to 36 mug/kg) plus a subsequent ouabain infusion (similar to 0.40 mug/kg min-minus 1) until VT occurred. VT was defined as at least one-third of the ventricular beats being of ectopic ventricular origin. The existence of an effective beta receptor blockade was verified by showing that 0.75 mug/kg of isoproterenol (an ED50 dose) injected intravenously had no detectable cardiovascular effects. The results were: 1) oxprenolol (0.3 mg/kg) did not alter the toxic dose significantly (P greater than .5 for both paired and group comparisons) [63.9 plus or minus 2.3 (S.E.) mug/kg vs. 64.4 plus or minus 2.9 (S.E.) mug/kg]; and 2) attempts to convert VT to sinus rhythm with oxprenolol (0.3 mg/kg) failed in 20 of 21 trials. Oxprenolol (1.0 mg/kg) effected only a temporary [10.6 plus or minus 6.4 (S.D.) minutes] reversion to sinus rhythm in 10 of 21 attempts. These results indicate that an activation of the cardiac beta receptor is not prerequisite for either the initiation or the perpetuation of ouabain-induced ventricular tachyarrhythmia in this particular model. The results cannot provide support for the notion that an increase in cardiac sympathetic nerve activity is a principal factor in VT in the dog. However, the results are consistent with the hypothesis that the direct membrane actions of ouabain are a principal cause of ouabain-induced ventricular tachyarrhythmia.


Antihypertensive drugs and catecholamine metabolism: effects of reserpine and hydralazine on tyrosine hydroxylase activity and norepinephrine concentrations in the spontaneously hypertensive rat.

The antihypertensive drugs, reserpine and hydralazine, produce different effects on tyrosine hydroxylase activity and norepinephrine levels in blood vessels and other tissues of the spontaneously hypertensive rat at doses which cause an equivalent reduction in blood pressure. Reserpine administration is associated with increased tyrosine hydroxylase activity in the mesenteric artery, mesenteric vein and adrenal, but the vasculature appears more sensitive to the effects of reserpine than the adrenal. This increase in tyrosine hydroxylase activity can be related to catecholamine depletion in the mesenteric artery, mesenteric vein and adrenal. Since chlorisondamine, a ganglionic blocking agent, diminished the ability of reserpine to increase tyrosine hydroxylase activity in the mesenteric artery and adrenal, it is likely that increased nerve activity is involved in regulation of the enzyme in both tissues. Hydralazine neither alters tyrosine hydroxylase activity in arteries or veins, nor depletes catecholamine levels in these tissues. In the adrenal, hydralazine increases tyrosine hydroxylase activity independently of any change in catecholamine levels. It would appear that changes in tyrosine hydroxylase activity produced by antihypertensive drugs are organ dependent and may involve both neuronal activity and amine depletion.


Mechanical response in the wall of ovarian follicles mediated by adrenergic receptors.

The identity of the adrenergic receptors involved in the contractility of the follicular wall was studied in bovine ovaries. Strips were prepared mainly from the protruding part of large Graafian follicles and mounted in an organ bath for recording of mechanical activity. Some intact ovaries and strips from the bottom of follicles were also studied. The latter two preparations showed spontaneous rhythmic contractions, and the tension was increased by norepinephrine and reduced by terbutaline. Norepinephrine, epinephrine, phenylephrine, isoproterenol and terbutaline alone or in combination with various concentrations of the inhibitors, phenoxybenzamine, piperoxan and propranolol, were used to analyze adrenoceptors in strips from the protruding portion of the follicle. Such strips showed no appreciable spontaneous contractions. A contractile response was found to be mediated by alpha receptors. The KA value for norepinephrine (determined in combination with phenoxybenzamine) was found to be 2.26 X 10- minus 6 M and KB for piperoxan (determined in combination with norepinephrine) was 1.02 X 10-minus 8 M (pA2 = 8.19). Adrenergic beta receptors mediated relaxation of the strips, and the KB value for propranolol (determined in combination with terbutaline) was 7.28 X 10-minus 9 M (pA2 = 8.17).