bluuebunny/crossref_metadata_2025 huggingface dataset:
Triacylglycerol Biosynthesis in Everted Sacs of Rat Intestinal Mucosa
Authors: Breckenridge W. C. , Kuksis A.
Published: December 2009

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas–liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled from free fatty acids showed that the phosphatide acid pathway contributed a minimum of 20–30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.
Kinetics and Thermodynamics of Lactate Dehydrogenases from Beef Heart, Beef Muscle, and Flounder Muscle
Authors: Borgmann Uwe , Laidler Keith J. , Moon Thomas W.
Published: December 2009

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC 1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 °C to 50 °C, and the corresponding activation parameters ΔG‡, ΔH‡, and ΔS‡ were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme–substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels for the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.
The Isolation and Characterization of the ATPase Inhibitory Protein (TN-I) from Bovine Cardiac Muscle
Authors: Burtnick L. D. , McCubbin W. D. , Kay C. M.
Published: December 2009

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated ATPase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 ± 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 ± 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.
Studies of the Metabolism of Asialotransferrins: the in Vivo Behavior of Baboon and Rhesus Asialotransferrins
Authors: Regoeczi E. , Hatton M. W. C. , Wong K.-L.
Published: December 2009

The catabolism and distribution of rhesus and baboon asialotransferrins relative to the corresponding parent proteins were studied in rabbits using a dual isotope tracer technique. Also a similar study with the baboon proteins in a baboon is reported.The metabolic data obtained in rabbits with both rhesus and baboon transferrins was close to the values established in a previous study for rabbit transferrin. Desialylation resulted in an average increase in the fractional catabolic rate of rhesus transferrin by 22.7%. This change is similar to that found earlier with asialotransferrins from several nonprimate mammals which are thought not to interact with the hepatic asialoglycoprotein receptor.Two kinetically distinct fractions were identified in baboon asialotransferrin. One of these, amounting to approximately one-third of the protein, was eliminated from the circulation very rapidly. The remaining two-thirds constituted a slowly catabolized fraction which behaved in vivo similarly to rhesus asialotransferrin. Unlike the rapidly cleared fraction, elimination of the slowly catabolized fraction in baboon asialotransferrin is probably not mediated by the hepatic asialoglycoprotein receptor. An amount comparable to the rapidly eliminated fraction in baboon asialotransferrin was recovered with the liver of rats in short-term experiments. In rats which were preinjected with chicken acid α1-glycoprotein the hepatic uptake of baboon asialotransferrin was markedly reduced. Data obtained in the baboon agreed with the findings in rabbits, although transferrin turnover was slower in the baboon.From its behavior in vivo as an asialoglycoprotein, baboon transferrin shows greater resemblance to human transferrin than rhesus transferrin. This conclusion is supported by carbohydrate analyses which show an intermediate position for baboon transferrin between man and a nonprimate mammal (rabbit), and a similarity between rhesus and rabbit transferrins.
The Effects of Substrate Concentration on the Mg-Adenosine Triphosphatase Activity of Myosin
Authors: Nihei T. , Filipenko C. A.
Published: December 2009

Using myosin, heavy meromyosin, and subfragment-1 the steady state rate of Mg-modified adenosine triphosphatase (Mg-ATPase) was determined over a range of substrate concentrations between 10−8 M and 5 × 10−3 M, at 0.5 M and 0.05 M KCl (pH 7.4 at 20 °C). At the substrate concentrations below 10−5 M, myosin Mg-ATPase was observed to show that two active sites interact, as suggested by the analysis of transient kinetic studies (Walz, F. G., Jr.: J. Theor. Biol. 41, 357–373 (1973)). The increase in the activity at Mg-ATP concentrations higher than 10−4 M corresponds to the binding of Mg-ATP to myosin sites not responsible for the catalytic action. With heavy meromyosin and subfragment-1, the activity was best expressed by the Michaelis equation. With heavy meromyosin, the activation at high ATP concentrations is detectable, though not as pronounced as with myosin, but not with subfragment-1.
Fractionation of nucleolar proteins by two-dimensional gel electrophoresis
Authors: Jackowski G. , Suria D. , Liew C. C.
Published: December 2009

Isolation of nucleolar proteins was obtained by dissociation in the presence of urea – guanidine hydrochloride, followed by high-speed centrifugation to remove nucleic acids. At least 31 fractions of nucleolar proteins were detected by isoelectrofocusing gel electrophoresis in the pH range 3.5–10. Following two-dimensional gel electrophoresis on sodium dodecyl sulfate – polyacrylamide slab gels, more than 100 components of nucleolar proteins were identified. Two-thirds of nucleolar proteins were located in the pH range 5–8 following isoelectrofocusing. The molecular weights of these classes of proteins were shown to be mostly 30 000 – 70 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis.
Effect of propylthiouracil-induced hypothyroidism on phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin synthesis in chick liver microsomes
Authors: Lyman E. M. , Dove J. L. , Sribney M.
Published: December 2009

Biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin was studied in liver endoplasmic reticulum obtained from newly hatched chicks which were made hypothyroid by feeding 0.2% propylthiouracil.In vitro measurements were made of the specific activities of phosphorylcholine-glyceride (cholinephosphotransferase (EC 2.7.8.2)), phosphorylethanolamine-glyceride (ethanolamine-phosphotransferase (EC 2.7.8.1)), and phosphorylcholine-ceramide (ceramide cholinephosphotransferase (EC 2.7.8.3)) transferases in control and hypothyroid chick liver for a period of 40 days. The specific activity of all three transferases began to decline after the chicks were on the propylthiouracil-containing diet for 5 days and steadily declined, reaching levels 10–15% of the controls after 15 days. These low levels were maintained for as long as the chicks were on this diet.Administration of L-thyroxine (15 μg/100 g of body weight) to the hypothyroid chicks caused a marked increase in the specific activities of all three transferases, reaching levels similar to those seen in the control chicks in 36–48 h. The specific activities then declined as the chicks were maintained on the diet of propylthiouracil, reaching the former low levels after 120 h.Administration of cycloheximide alone to the hypothyroid chicks caused a rise in the specific activities of the transferases after 24 h approximately equal to that caused by thyroxine alone, while thyroxine and cycloheximide together were no different than either alone.These studies indicate that in some manner circulating thyroxine controls the activities of enzymes involved in the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin in chick liver endoplasmic reticulum. There was no evidence that induction of hypothyroidism by propylthiouracil had any effect on the activities of these enzymes in the CNS.
On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli
Authors: Krebs Anita , Bridger William A.
Published: December 2009

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65 000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomelic structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding in the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzyme is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.
Enzymes of the orotate biosynthetic pathway in Crithidia fasciculata
Authors: Kidder G. W. , Dewey Virginia C. , Nolan Linda L.
Published: December 2009

A study of the enzymes of the orotate biosynthetic pathway in the kinetoplastid flagellate Crithidia fasciculata has revealed a number of differences between them and those of other organisms, either prokaryotic or eukaryotic. Carbamyl phosphate synthesis could not be demonstrated in cell-free extracts. However, the incorporation of both CO2 and the ureide carbon of citrulline into pyrimidines occurs in growing cells, the latter predominating over the former. The aspartate transcarbamylase of the flagellate has properties which are similar to those of this enzyme as it occurs in mammals rather than other microorganisms. Two enzymes, dihydroorotate synthetase and dihydroorotate hydrolase, are present, the former being responsible for the conversion of carbamylaspartate to dihydroorotate. Dihydroorotate hydroxylase, a soluble enzyme requiring a reduced pteridine as a cofactor, converts dihydroorotate to orotate. The hydroxylase is inhibited by orotate, but not by pyrimidine or purine ribonucleotides. Thus orotate serves to control its own biosynthesis.
Studies on the cell wall of Schizophyllum commune. Permethylation and enzymic hydrolysis
Authors: Siehr Donald J.
Published: December 2009

Two polysaccharide fractions S-glucan and R-glucan were isolated from the cell walls of Schizophyllum commune. S-Glucan is primarily alinear 1,3-α-glucan with occasional 1,6-αlinkages as shown by permethylation and partial acid hydrolysis. The glucan fraction also contains a small amount of xylose. The R-glucan fraction is a mixture of two polysaccharides, chitin and a highly branched glucan with linear 1,6-β and 1,3-β segments and 1,6-β branching. This conclusion is based on permethylation studies and enzymic hydrolyses.
Surface polypeptides of the Chinese hamster ovary cell; an immunochemical study
Authors: Behar-Bannelier M. , Juliano R. L.
Published: December 2009

Antibodies elicited by the injection of live Chinese hamster ovary cells (CHO) into rabbits precipitated four major components from detergent extracts of CHO membranes. The four components, of molecular weights 200 000, 125 000, 95 000 and 41 000 daltons, corresponded to cell surface components identified by the lactoperoxidase surface label technique.
Molecular weight of an extremely hydrophobic protein, zein, in dimethylformamide and in formamide
Authors: Danzer Laurence A. , Rees E. Douglas
Published: December 2009

Both alpha zein purified from a commercial preparation and beta zein prepared fresh from corn are soluble in the nonaqueous solvents formamide and dimethylformamide; in this regard zein resembles water soluble proteins such as insulin, ribonuclease, and lysozyme. On the basis of osmotic pressure measurements made in both formamide and dimethylformamide, alpha zein has a number average molecular weight of 21 000 – 24 000 daltons and shows no tendency to aggregate or dissociate. Beta zein exists in an aggregated state (dimer and higher forms) in dimethylformamide. Formamide dissociates the beta zein dimer into monomer units but aggregation to higher species occurs with increasing protein concentration.
The mechanism of the degradation of DNA by streptonigrin
Authors: Cone R. , Hasan S. K. , Lown J. W. , Morgan A. R.
Published: December 2009

The production of single strand cleavage in covalently-closed circular-DNA by the antitumour agent streptonigrin (reduced in situ by NADH) is demonstrated using the ethidium bromide fluorescence assay described previously. The degradation dependent on oxygen is completely inhibited by superoxide dismutase (EC 1.15.1.1) suggesting the intermediacy of the superoxide radical anion in the degradation. However similar complete inhibition of DNA strand breakage by catalase (EC 1.11.1.6) indicates that the hydroxyl radical (formed by interaction of superoxide with hydrogen peroxide) is the primary reactive species. Cupric ion stimulates the cleavage reaction and cobaltous ion has no effect in keeping with model studies using quinolinequinones.
Isotope-dilution studies of the effects of 5-fluorodeoxyuridine and hydroxyurea on the incorporation of deoxycytidine and thymidine by cultured thymus cells
Authors: Scott F. W. , Forsdyke D. R.
Published: December 2009

From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.
Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions
Authors: Bishop H. H. , Strickland K. P.
Published: December 2009

The enzyme, CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer–detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleoylphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60 mM for Mg2+, 6 mM for CTP and 250 μg per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90 000 × g fraction (microsomal) with small but significant activity in the 12 000 × g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses): 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl- (3.85), 1-stearoyl-2-arachidonoyl- (3.31), 1-arachidonoyl-2-stearoyl- (3.16), 1,2-diarachidonoyl- (0.72), 1,2-dicaproyl- (0.67), 1,2-dipalmitoyl- (0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.
Wheat embryo ribonucleates. VI. Comparison of the 3′-hydroxyl termini in 'rapidly labelled' RNA from metabolizing wheat embryos with the corresponding termini in ribosomal RNA from differentiating embryos of wheat, barley, corn and pea
Authors: Oakden K. M. , Lane B. G.
Published: December 2009

The NaCl-insoluble (2.5 M, 0 °C) fraction of wheat embryo RNA (iRNA) can be labelled when wheat embryos are subjected to either short-term (0.5 h) or long-term (24 h) imbibition in a medium that contains tritium-labelled adenosine, guanosine, cytidine and uridine. Electrophoretic analyses reveal that, after short-term labelling, there is a broadly heterodisperse distribution of radioactivity in 'rapidly labelled' i[3H]RNA, but after long-term labelling, there is an essentially trimodal distribution of radioactivity in i[3H]RNA. End-group analyses reveal that, after short-term labelling, adenosine is the principal 3′-hydroxyl terminus in all centrifugal subfractions of 'rapidly labelled' i[3H]RNA, whereas cytidine (in 5.8S rRNA), guanosine (in 18S rRNA) and uridine (in 26S rRNA) are the principal 3′-hydroxyl termini in centrifugal subfractions of wheat embryo i[3H]RNA. Guanosine is also the principal 3′-hydroxyl terminus in the 18S rRNA of differentiating embryos excized from both monocotyledonous (wheat, barley, corn) and dicotyledonous (pea) seedlings. The implications that the end-group measurements may have for current views about the possible biochemical involvements of 3′-hydroxyl terminal sequences in both mRNA and 18S rRNA are subjects of discussion. Incidental to the principal investigation, an existing technique for analyzing the RNA contents of cellular materials has been appropriately modified to circumvent interference from uv-absorbing pigments, which, when present, prevent application of the method to plant materials.
Persistent effects of ouabain treatment on human lymphocytes: synthesis of DNA, RNA and protein in stimulated and unstimulated cells
Authors: Dornand Jacques , Kaplan J. Gordin
Published: December 2009

Pretreatment of human lymphocytes for 2 days in 2 × 10−6 M ouabain caused irreversible loss of their subsequent capacity to stimulate in the mixed lymphocyte reaction (MLR). Pretreatment for the same period with 10−7 M ouabain resulted in an enhanced incorporation of thymidine into DNA of the responding cells in the MLR; this effect was also on the stimulating cells, as previously reported by Christen et al. (Cell. Immunol. 19, 137–142 (1975)). Pretreatment of stimulating lymphocytes with 10−7 M ouabain caused a persistent but reversible inhibition of the synthesis of RNA and protein in the MLR; peak incorporation of labelled uridine or alanine reached the same level as that of the control cultures, but 24 h later. Exactly the same persistent but reversible inhibition was found in the case of DNA synthesis of cells pretreated with 10−7 M ouabain and then stimulated by antigens (streptolysin-O and varidase) or by mitogens (phytohemagglutinin and concanavalin A); the same level of incorporation of labelled thymidine occurred but 24–48 h later than in the case of the controls. Pretreatment with the cardiotonic steroid under these conditions also resulted in a pronounced inhibition of the basal, unstimulated levels of RNA and protein synthesis in the case of both control lymphocytes and those which had been treated with mitomycin C. The effects of ouabain pretreatment on basal RNA and protein synthesis were identical for both 2 × 10−6 M and for 10−7 M; the effect of pretreatment of stimulating cells with these two concentrations was completely opposite: irreversible inhibition of the proliferative response of allogeneic responding cells at the former concentration and delayed activation at the latter.
Relationship between ppGpp levels and rates of protein and RNA synthesis in Escherichia coli
Authors: Chaloner-Larsson Gillian , Yamazaki Hiroshi
Published: December 2009

When amino acid starvation is ended in stringent (relA+) strains of Escherichia coli, the rates of RNA and protein synthesis as well as their accumulation return to normal more slowly in spoT− strains than in the spoT+ strains. The level of ppGpp accumulated declines more slowly in the spoT− strains than in the spoT+ strains. Thus, there is an inverse relationship between ppGpp levels and the rates of RNA and protein synthesis. The slow resumption of protein synthesis in the spoT−relA+ strains could therefore be explained in terms of the limited synthesis of mRNA species coding for the bulk of cellular proteins.
Studies on phosphoglyceromutase from chicken breast muscle: number and reactivity of sulfhydryl groups
Authors: Carne T. J. , McKay D. J. , Flynn T. G.
Published: December 2009

Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5′-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB. The total number of sulfhydryl groups determined by amino acid analysis of the performic acid oxidized and carboxymethylated enzyme was also found to be six. Disc gel electrophoresis in sodium dodecyl sulfate indicated that the enzyme is composed of subunits having the same molecular weights which suggests there are three sulfhydryl groups per subunit. Reaction with Nbs2 iodoacetate or iodoacetamide resulted in the modification of only two sulfhydryl groups. In each of the modifications, except that due to iodoacetamide both substrate and cofactor reduced the rate, but not the extent, of the reaction. Substrate was more effective than cofactor in reducing the rate of reaction with sulfhydryl reagents suggesting that a cysteine residue is involved in the binding of substrate. That the involvement is indirect is shown by the lack of protection offered by substrate and cofactor to reaction with iodoacetamide. Inactivation of PGM following titration with sulfhydryl reagents is not likely to be due to a conformational change since none of any significance was indicated by circular dichroism of the treated enzyme. This result was substantiated by the lack of a marked change in the fluorescence emission spectrum of the PMB treated enzyme.
Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors
Authors: Lin Kun-Tsan , Crawhall John C.
Published: December 2009

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.
Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase
Authors: Sulakhe Prakash V. , Leung Nicholas Ling-Kit , St. Louis Patrick J.
Published: December 2009

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmai calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.
Lipogenesis in muscle, liver, and adipose tissue of normal and dystrophic chickens
Authors: Vallyathan N. V.
Published: December 2009

The in vitro rate of oxidation and incorporation of [U-14C]glucose into lipids of muscle, liver, and adipose tissue of dystrophic and control chickens at 1 week, 1,6, and 12 months of age were determined. The muscle and liver from dystrophic birds showed a high rate of [U-14C]glucose oxidation and incorporation into lipids during 1 week, 6 and 12 months. Insulin did not have any consistent stimulatory effect in all the tissues on the rate of glucose oxidation and incorporation into lipids. The contributory role played by the liver in the accumulation of lipids in dystrophic muscle is discussed.
Utilization of dietary nucleic acid purines for nucleotide and nucleic acid synthesis in the mouse
Authors: Burridge Philip W. , Woods Robin A. , Henderson J. Frank
Published: December 2009

Three preparations of radioactive yeast nucleic acids were fed to mice. One was labeled predominantly in the guanine moiety, one was labeled predominantly in the adenine moiety, and in one adenine and guanine were labeled equally. Most of the nucleic acid purines produced by digestion were excreted in the urine. However, a small amount was utilized for nucleotide and nucleic acid synthesis in the mouse tissues. Small intestine, liver and skeletal muscle contained most of the purines that were retained in the tissues. Dietary nucleic acid adenine appeared to be utilized somewhat more efficiently than was dietary nucleic acid guanine.
The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin
Authors: Burtnick L. D. , McCubbin W. D. , Kay C. M.
Published: December 2009

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 ± 2 000, consistent with a value of 37 000 ± 1 000 determined by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent α-helical content of 43 ± 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.
Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholidate into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2′-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.
In vitro biosynthesis of γ-lipotropic hormone
Authors: Chrétien M. , Lis M. , Gilardeau C. , Benjannet S.
Published: December 2009

Sheep γ-lipotropic hormone (γ-LPH) is a pituitary polypeptide made of 58 amino acids and is formed of the first 58 residues of β-lipotropic hormone (β-LPH). The C-terminal portion (41–58) of γ-LPH is identical with the structure of β-melanophore-stimulating hormone (β-MSH). We hypothetized in 1967 that β-LPH could be the biological precursor of β-MSH and that γ-LPH could be an intermediate compound. We demonstrated in 1974 that β-LPH is actively synthesized in the bovine pituitaries. We now studied the biosynthesis of γ-LPH by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated γ-LPH from the other radioactive proteins with a method previously described. We characterized the radioactive proteins by ion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. Our results show that radioactive γ-LPH was actively synthesized. This γ-LPH has all the chemical characteristics of nonradioactive γ-LPH. However, in the conditions used, we were unable to demonstrate biosynthesis of β-MSH. These results suggest that γ-LPH is biosynthesized more slowly than β-LPH and that the conversion into β-MSH, if it exists, is a slow or subactive process in the species studied.
Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta
Authors: Baliga B. Suren , Hubert Christine , Murphy Alma , Meadow Felice , Dourmashkin Peter , Munro Hamish
Published: December 2009

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids.A fraction prepared from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5–7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA.These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.
Histone–histone interactions I. An electrophoretic study
Authors: Lewis Peter N.
Published: December 2009

Whole histone extracted from chromatin by either acid or protamine displacement was found by gel electrophoresis at pH 7 to contain only two histone complexes, H2A–H2B and H3–H4, and uncomplexed histone H1. Although both complexes are dissociated at low pH or with high urea concentrations, removal of the denaturant resulted in complete complex reformation within minutes at the most. A systematic investigation of binary, ternary and quaternary histone mixtures revealed that interactions also occur between histones H2B–H4 and H2A–H4. No evidence however was found for the formation of ternary and quaternary histone complexes.
The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs
Authors: Avruch L. , Fischer S. , Pierce Jr. H. , Oehlschlager A. C.
Published: December 2009

The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24,25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3β-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3β-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor.When S. cerevisiae was grown in the presence of 1.3 and 5 μM10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22, 24-tetraen-3β-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3β-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.
Characterization of the adenosinetriphosphatase and calsequestrin isolated from sarcoplasmic reticulum of normal and dystrophic chickens
Authors: Yap John L. , MacLennan David H.
Published: December 2009

The Ca2++Mg2+-dependent adenosinetriphosphatase (EC 3.6.1.3) and calsequestrin have been isolated from the sarcoplasmic reticulum of normal and dystrophic chicken muscle. The adenosinetriphosphatases, isolated from the two lines of chickens were identical in molecular weight, enzyme activity and in Ca2++Mg2+-dependence. Calsequestrins isolated from the two lines bound identical amounts of calcium. There were no differences in the Ca2+ transport functions of the sarcoplasmic reticulum membrane, isolated from the two lines of chickens. These results indicate that morphological differences in dystrophic chicken sarcoplasmic reticulum, described by Sabbadini et al. (Sabbadini, R., Scales, D. Inesi, G. FEBS Lett. 54, 8 (1975)), cannot be ascribed to qualitative differences in the adenosinetriphosphatase or calsequestrin.
Isolation of specific protease inhibitors from Neurospora crassa
Authors: Yu Peter H. , Kula Maria R. , Tsai Hsin
Published: December 2009

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.
Biosynthèse des glycoprotéines dans le parenchyme pulmonaire. II. Activité galactosyltransférase dans les fractions subcellulaires des pneumocytes
Authors: Levrat Christiane , Louisot Pierre
Published: December 2009

A soluble galactosyltransferase exists in the cell sap of the alveolar cells of the lung. The optimal conditions of galactosyltransferase are: pH 6,20 °C, 10 mM Mn2+, 1 mM Mg2+. Michaelis constant for substrate UDP-galactose is 390 nM. By electrofocusing on Ampholine column, galactosyltransferase appears in a unique peak, at pHi 4.8. The parameters of the purified enzyme are the same as that of the crude enzyme. UDP is a noncompetitive inhibitor for the purified enzyme.
GM1-Ganglioside and lactosylceramide β-galactosidase from rabbit brain: inhibitor and substrate competition studies
Authors: Callahan John W. , Gerrie Jacqualine
Published: December 2009

A rabbit brain β-galactosidase catalyzes the hydrolysis of synthetic substrates and the natural substrates GM1-ganglioside, lactosylceramide, and asialo-GM1-ganglioside. γ-D-Galactonolactone competitively inhibited hydrolysis of GM1-ganglioside, lactosylceramide, and MU-galactoside with Ki values of 0.26 mM, 0.13 mM, and 0.77 mM, respectively. From activity plots comparing the degree of inhibition to the inhibitor concentration, a single binding site for each substrate was found. NP-Galactoside inhibited the hydrolysis of GM1-ganglioside and lactosylceramide, whereas GM1-ganglioside inhibited lactosylceramide hydrolysis. At low substrate concentrations (<1 mM), GM1-ganglioside was hydrolyzed effectively in the presence of NP-galactoside, but at higher concentrations hydrolysis of the latter was preferred. Chloromercuriphenylsulfonic acid and iodoacetate were effective inhibitors of the enzyme, but N-ethylmaleimide was not. The degree of inhibition with chloromercuriphenyl sulfonic acid was different for each substrate.At 0.5 μM chloromercuriphenyl sulfonic acid, all activity towards NP-galactoside, 75% towards lactosylceramide, and 25% of the GM1-ganglioside activity was lost.Two possible models are presented to explain these results. The data favour the presence of multiple active sites in the enzyme.
A simple assay for acetyl-CoA holocarboxylase synthetase
Authors: Landman A. D. , Desjardins P. R. , Dakshinamurti K.
Published: December 2009

Biotin-mediated ATP-32PPi exchange has been used to assay acetyl-CoA holocarboxylase synthetase. The procedure is a potential alternative to the assay which uses the incorporation of biotin into acetyl-CoA apocarboxylase.
Effect of nicotine on biosynthesis of C50 carotenoids in Halobacterium cutirubrum
Authors: Kushwaha S. C. , Kates M.
Published: December 2009

The major carotenoids in pigmented extreme halophiles are the hydroxylated C50 red pigments bacterioruberin (major, 84%) and monoanhydrobacterioruberin (minor, 14%). When cells of Halobacterium cutirubrum were grown in the presence of nicotine they accumulated lycopene and bisanhydrobacterioruberin; maximal accumulation (80% lycopene, 20% bisanhydrobacterioruberin) occurred at 3 mM nicotine. Removal of nicotine resulted in the reformation of monoanhydrobacterioruberin and bacterioruberin at the expense of lycopene and bisanhydrobacterioruberin. Thus nicotine does not inhibit the addition of the two extra isoprene units plus two hydroxyl groups (at C1 and C1′) to a C40 carotenoid skeleton. It does, however, inhibit the hydration steps at C3″ and C3′″ that convert bisanhydrobacterioruberin to monoanhydrobacterioruberin and finally bacterioruberin. These results support the view that the C50 carotenoids can be formed from a C40 carotene, lycopene.
Amino acid sequence of penicillopepsin. IV. Myxobacter AL-1 protease II and Staphylococcus aureus protease fragments and homology with pig pepsin and chymosin
Authors: Cunningham Anne , Wang Hsin-Min , Jones Stephen R. , Kurosky Alexander , Rao Leticia , Harris C. Ian , Rhee Sung H. , Hofmann Theo
Published: December 2009

The digest of penicillopepsin (EC 3.4.23.7) with protease II from Myxobacter AL-1 gave five fragments which were separated on a Biogel P-100 column in 70% formic acid. The fragments were from 16 to 125 amino acids long. Two fragments were also isolated from a digest with a protease from Staphylococcus aureus. The analysis of these fragments by automatic sequencer gave a number of overlaps of the chymotryptic and thermolytic peptides. The available amino acid sequence data for penicillopepsin described in this paper and the accompanying papers (Kurosky, A. &Hofmann, T.: Can. J. Biochem. 54, 872 (1976); Rao, L. &Hofmann, T.: Can. J. Biochem. 54, 885 (1976); Harris, C. I., Rao, L., Shutsa, P., Kurosky, A. &Hofmann, T.: Can. J. Biochem. 54, 895 (1976)) have been combined and yield 15 fragments which range in lengths from 3 to 112 amino acid residues. These unique fragments account for virtually all the amino acids of the fungal protease. Four of the fragments with a total of 194 residues (about 60% of the molecule) have been aligned with corresponding sections of pig pepsin (EC 3.4.23.1) and with part of the N-terminal sequence available for calf chymosin (EC 3.4.23.4). In the alignments about 37% of the residues in the fungal enzyme are identical with at least one of the mammalian enzymes. An additional 20% are chemically similar. These results, together with previously reported active-site directed modifications, show conclusively that penicillopepsin is an evolutionary homologue of the mammalian acid proteases.
Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition
Authors: Roncari Daniel A. K. , Mack Esther Y. W.
Published: December 2009

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acid synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 μM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.
Histone–histone interactions. II. Structural stability of the histone H3–H4 complex
Authors: Lewis Peter N.
Published: December 2009

The stability of the histone H3–H4 complex toward urea, changes in pH and ionic strength, and certain chemical modifications have been examined by gel electrophoresis and circular dichroism. When uncomplexed, the two cysteine residues of histone H3 become rapidly oxidized, forming an intramolecular disulfide bridge which apparently blocks complex formation on return to complexing conditions. The complex was found to be unstable toward low values of pH and ionic strength, concentrations of urea exceeding 1M, modifications of the cysteine residues, and fragmentation in which the C terminal portions of either H3 or H4 are removed. A possible structure for this complex is proposed.
The purification of porcine haptoglobin by affinity chromatography with concanavalin A – Sepharose
Authors: Terpstra Frank A. , Smith David B.
Published: December 2009

Methods designed for the isolation of human haptoglobin (Hp) were found insufficient when applied to pig plasma due to the formation of a material tentatively identified as albumin dimer. Small scale separation is possible by preparative polyacrylamide gel electrophoresis. Larger scale purification from nonglycoprotein contaminants such as albumin dimer is achieved by affinity chromatography using immobilized concanavalin A. Porcine haptoglobin is microheterogeneous. More than 14 components, partially resolveable by gel filtration, were detected.