bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin Control of Hepatic Glucose Production
Authors: Matsuura Nobuo , Cheng Jose S. , Kalant Norman
Published: December 2009

Insulin injected intravenously caused a rapid, marked decrease in hepatic glucose secretion in the rabbit, as determined by an isotope-dilution procedure. This was associated with a decrease in the concentrations of gluconeogenic intermediates from phosphoenolpyruvate to triose phosphates, inclusive, compatible with inhibition of gluconeogenesis at phosphoenolpyruvate carboxykinase. The concentration of glucose 6-phosphate was unaltered but that of hepatic glucose was reduced. The specific activities of the hexose phosphates, relative to that of liver glucose, were the same in control and insulin-treated animals. These observations can be explained by a decrease in the activity of glucose-6-phosphatase. It is concluded that this enzyme is a control point for hepatic glucose production and is inhibited by insulin.In the rat, insulin produced a rapid fall in blood sugar. The hepatic glucose output remained normal despite a fall in hepatic glucose 6-phosphate concentration during the initial period of insulin action. This suggests that glucose-6-phosphatase activity was increased. Subsequently the concentration of glucose 6-phosphate returned to normal with no change in the rate of glucose production. The data suggest that in the rat, insulin produces a transient increase in glucose-6-phosphatase activity.
3-Methyl-2-butenal: An Enzymatic Degradation Product of the Cytokinin, N6-(Δ2-Isopentenyl)adenine
Authors: Brownlee Brian G. , Hall Ross H. , Whitty C. Dennis
Published: December 2009

An enzyme preparation from immature corn kernels catalyzes cleavage of N6-(Δ2-isopentenyl) adenine to give the aldehyde, 3-methyl-2-butenal, as the major side-chain derived product. This product, in the form of the semicarbazone, was identical with an authentic product by several criteria: chromatographic behavior, mass and ultraviolet spectra.
Identification and Distribution of m-Tyramine in the Rat
Authors: Philips S. R. , Davis B. A. , Durden D. A. , Boulton Alan A.
Published: December 2009

A procedure for the quantitative evaluation of m-tyramine in mammalian tissues is described. It involves isolation of the amine by ion-exchange chromatography, followed by conversion to the dansyl derivative, chromatographic separation, and quantitation by the mass spectrometric integrated ion current technique using an isotopically labelled internal standard. The concentrations of m-tyramine in some tissues of male Wistar rats were (mean ± S.D., nanograms per gram): brain 0.32 ± 0.03, heart 0.44 ± 0.13. kidney 12.6 ± 3.4, liver 0.27 ± 0.04, lung 0.33 ± 0.11, spleen 0.25 ± 0.07, and blood 0.15 ± 0.04.
An Improved Method for the Isolation of Surface Membranes from Cells in Monolayer Culture
Authors: Carlsen S. A. , Till J. E.
Published: December 2009

A modification of the procedure reported by Scher and Barland (Biochim. Biophys. Acta 255 (1972) 580–588) for the isolation of the surface membranes of cells grown in monolayer culture is described. The modification involves addition of a purification step utilizing the two-phase polymer system described by Brunette and Till (J. Membr. Biol. 5 (1971) 215–224). The modified method permits the isolation of surface membranes from cells not yet at confluence, when they may easily become detached from the substratum.
Subunit Structure and some Properties of Pyruvate Kinase of Neurospora
Authors: Kapoor M.
Published: December 2009

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000–52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS–polyacrylamide gels. The enzyme has an isoelectric point of 6.35–6.41. Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg2+, is required for activity. At low concentrations, Ca2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.
Effect of Triphenylmethane Derivatives on Cell-Free Macromolecular Synthesis. II. mRNA–Ribosome Binding
Authors: Igarashi S. J. , Zmean J. A.
Published: December 2009

The specific inhibition by aurintricarboxylic acid (ATA) of mRNA binding to ribosomes was re-examined. Among triphenylmethane compounds, all of which are potent inhibitors for phenylalanyl-tRNA synthetase, ATA is the most potent inhibitor. Two other dyes, Aurine and Azure Blue B, inhibit the reaction, but the concentration for 50% inhibition is far higher than that for ATA. It appears that the presence of the carboxyl group on the skeletal-triphenylmethane structure enhances the inhibition of the mRNA-binding reaction. This feature is slightly different from that of the phenylalanyl-tRNA synthetase reaction, in which the skeletal structure of the dye itself is essential to its inhibitory action.
Mammalian Fatty Acid Synthetase II. Modification of Purified Human Liver Complex Activity
Authors: Roncari Daniel A. K.
Published: December 2009

Highly purified human-liver fatty acid synthetase complex was used to study the effect of several potential modifiers. Adenosine 3′,5′-phosphate did not alter the activity of either purified synthetase or of multienzyme present in 700 × g supernates. Its dibutyryl derivative was also ineffective when incubated with liver slices. Fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6-phosphate stimulated significantly the activity of the purified enzyme. Fructose 1,6-diphosphate, which was most effective, decreased the Km of the synthetase for NADPH. Phosphoenolpyruvate, rac-glycero-3 -phosphate and potassium phosphate were ineffective. All long-chain fatty acyl-CoA thioesters tested were inhibitory, but this effect was not observed until the regions of their critical micellar concentrations were reached. Free myristate, palmitate, and stearate did not inhibit synthetase activity up to the highest concentration tested (1 mM). An enzyme preparation derived from livers of fasted rats inactivated purified rat-liver 4′-phospho[14C]pantetheine-fatty acid synthetase by releasing its prosthetic group. It also decreased the activity of the purified human-liver complex.
Lysolecithin–Cholenterol Interaction. A Spin-Resonance and Electron-Micrographic Study
Authors: Purdon A. D. , Hsia J. C. , Pinteric L. , Tinker D. O. , Rand R. P.
Published: December 2009

Another publication (Rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O. (1975) Can. J. Biochem. 53, 189–195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r.) and electron microscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol% cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol% cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-doxylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol%. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.
Protein Kinases in Rat Testes: Evidence for Different Fractions of the Enzyme
Authors: Bernard Elena A. , Wassermann G. F.
Published: December 2009

Protein kinase activity of rat testis homogenate was separated into five fractions by means of pH 4.8 acidification and DEAE-cellulose chromatography. The five fractions showed a peculiar pattern of activity and cAMP dependency with the substrates used: casein, protamine, histone mixture, arginine-rich histone, lysine-rich histone, and phosvitin. The casein–sepharose substrate affinity column separated two fractions from the pH 4.8 precipitate. Peak number one phosphorylates histone preferently and is cAMP-dependent, while peak number two has a strong affinity toward casein as substrate and is not cAMP-dependent.
Dehydroepiandrosterone and Dehydroepiandrosterone Sulfate Dynamics in Obesity
Authors: Fehér T. , Halmy L.
Published: December 2009

Dehydroepiandrosterone (D) and dehydroepiandrosterone sulfate (DS) dynamics were studied in three obese female subjects following a single injection of [4-14C]D and [7α-3H]-DS tracers. Dynamic parameters were calculated simultaneously by both the urinary and blood method of compartmentalization. Estimates for the urinary secretion and production rates of D were found to be high, and those of DS varied within normal range. Calculation of the conversion factors, ρDDS and ρDSD, by the urinary method revealed a normal extra-glandular DS→ D conversion, while that for D → DS appeared deficient in obese female subjects. Estimates of inner and outer pool distribution volumes were extremely increased for free D; in contrast to this, moderately increased inner and decreased outer pool volumes of DS were observed. The metabolic clearance rates of D were normal or decreased and those for DS were greater than normal. The blood production rates of both D and DS were higher in obese female subjects than those estimated for normal women in our previous study.These observations suggest a considerable uptake of unconjugated D by adipose tissue, an overall poor D → DS conversion and an accelerated DS metabolism in obese female subjects.
Development of Pyridoxal Kinase Activity in the Cerebral Hemispheres of the Chick Embryo and Young Chick
Authors: Wright S. D. Wain
Published: December 2009

Low levels of pyridoxal kinase activity were found in the cerebral hemispheres of the 10-day chick embryo. Specific activity rose eightfold by the 10th day post-hatching and thereafter remained constant to the 30th day.
Acid Proteases from Species of Mucor. II. Partial Characterization of the Acid Protease Produced by a Strain of Mucor miehei Isolated in Cuba
Authors: Rickert W. S. , McBride-Warren P. A.
Published: December 2009

The acid protease produced by a strain of Mucor miehei isolated in Cuba was purified by column electrofocusing and partially characterized as to amino-acid composition, molecular weight, helical content, total carbohydrate content, and approximate isoelectric point. A detailed comparison of these results with those reported previously for Mucor miehei protease (Ottesen, M. &Rickert, W. S. (1970) C.R. Trav. Lab. Carlsberg 37, 301) suggested that the two enzymes are similar but not identical. This conclusion was reinforced by an analysis of circular-dichroism spectra.
Wheat-Embryo Ribonucleates. IV. Factors That Influence the Formation and Stability of a Complex Between 5S rRNA and 18S rRNA
Authors: Azad A. A. , Lane B. G.
Published: December 2009

Under the conditions used in this study, wheat-embryo 5S rRNA complexes with its homologous 18S rRNA from wheat embryos and with heterologous 18S rRNA from other eukaryotic source materials such as yeast, L cells, and HeLa cells, but it does not complex with heterologous 16S rRNA from a prokaryote such as Escherichia coli or with homologous or heterologous 26S(23S) rRNA of either eukaryotic or prokaryotic origin.If a solution of wheat-embryo rRNA is simply made 0.3 M with respect to NaCl and then heated at 60 °C for 3 min before quick cooling to room temperature (ca. 20 °C), there is both preferential and efficient complex formation between 5S and 18S rRNA and between 5.8S and 26S rRNA.The laboratory-prepared' complex between wheat-embryo 5S rRNA and its homologous 18S rRNA is more thermostable in 0.1 M NaCl solution than is the 'natural' complex between wheat-embryo 5.8S rRNA and its homologous 26S rRNA, and both complexes 'melt' over a narrow range of temperature.The possible physicochemical and physiological importance of both homologous and heterologous rRNA complexes is the subject of a brief discussion.
Studies of Folic Acid Compounds in Nature. IV. Folic Acid Compounds in Soybeans and Cow Milk
Authors: Shin Y. S. , Kim E. S. , Watson J. E. , Stokstad E. L. R.
Published: December 2009

Folate compounds in soybean and cow's milk were identified by Sephadex chromatography and differential microbiological assay. Soybean contained mainly monoglutamates (ca. 52%), some diglutamates (ca. 16%), and polyglutamates (pentaglutamates representing the major portion). 5-CHO-H4PteGlun(1–6) constituted 65–70% of total folate activity. Cow's milk contained monoglutamates (60%) and polyglutamates (ranging from di- to hepta-conjugates). In contrast to soybean, 90–95% of milk folate was in the 5-CH3-H4PteGlun(1–7) form.
The Orientation of Cytochrome c Oxidase in Coupled Phospholipid Membrane Vesicles—A Spin-Label Study
Authors: Kornblatt J. A. , Chen W. L. , Hsia J. C. , Williams G. R.
Published: December 2009

Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. We have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-labeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim &Racker ((1972) J. Biol. Chem. 247, 1338–1339) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles. NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with [Formula: see text] of 1 min at 22 °C. The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.
Effects of Hormones and Drugs on Phosphoribosyl Pyrophosphate Concentrations in Mouse Liver
Authors: Lalanne Marcel , Henderson J. Frank
Published: December 2009

Concentrations of phosphoribosyl pyrophosphate (PP-ribose-P) in mouse liver were studied as a function of time of day and following injection of a number of metabolites and drugs. A twofold diurnal variation in liver PP-ribose-P concentration was observed. Liver PP-ribose-P concentrations were elevated following injection of glucagon, insulin, epinephrine, and ethylaminothiadiazole.
The Conformational States of Oxidized and Oxygenated Beef-Heart Cytochrome c Oxidase
Authors: Kornblatt Jack A. , Kells D. I. C. , Williams G. R.
Published: December 2009

1. The "oxygenated" form of cytochrome oxidase has been generated by treatment of the enzyme with ascorbic acid.2. "Oxygenated oxidase" so generated is stable over long periods (24 h).3. Sedimentation velocity experiments have shown the "oxygenated oxidase to be a less compact molecule than the oxidized.
The Conformational States of Cytochrome Oxidase in the Mitochondrion
Authors: Kornblatt Jack A. , Williams G. R.
Published: December 2009

The Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity, the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.
Effects of Lubrol Detergents on Adenylate Cyclases
Authors: Bär H. P. , Kulshrestha S.
Published: December 2009

The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects on basal and stimulated adenylate cyclases. Above concentrations of 0.001–0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibited about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer certain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.
Cell-Free Protein Synthesis in the Rabbit Liver Ribosomal System. II. Large Scale Preparation of Purified 80S Ribosomes
Authors: Dufresne M. J. , Igarashi S. J.
Published: December 2009

A procedure for the preparation of a large quantity of biologically active, highly purified ribosomes from rabbit liver is described. The method employs polyethylene glycol – dextran sulfate partition and DEAE-cellulose chromatography to overcome the limitations encountered in conventional procedures. The entire process takes only 48 h to obtain 10 000 A260 units of ribosomes. The ribosomes thus obtained are predominantly 78 S particles with a constant protein–RNA ratio of 0.95. The ribosomes are free from RNase, amino-acyl-tRNA synthetase, and amino-acyl-tRNA:protein transferase activity. The protein synthesizing activity is dependent on added mRNA and protein factors. These ribosomes are stable for prolonged periods of storage in a liquid nitrogen refrigerator.
DNA Polymerase Activity Associated with Endogenous Template: Release by Ribonuclease Treatment
Authors: Moranelli Francesco , Penner P. E.
Published: December 2009

A ribonuclease-sensitive DNA polymerase, which uses an endogenous template, is detectable in the 39 000 g supernatant of a rat thymus homogenate, and appears as a single peak of activity in the void volume after Sephadex G 150 or G 200 gel filtration chromatography. Native and "activated" DNA-dependent DNA polymerase activities coincide with the endogenous-templated polymerase activity. Treatment of the thymus extract with ribonuclease(s) prior to gel filtration chromatography yields two other peaks of activity in addition to the void volume peak. The appearance of the two lower molecular weight peaks of activity is accompanied by a concomitant decrease in the endogenous-templated activity. The effect of ribonuclease is specific and cannot be reproduced by a similar deoxyribonuclease treatment.
Identification of Ornithine and Arginine Conjugates of Cholic Acid by Mass Spectrometry
Authors: Myher J. J. , Marai L. , Kuksis A. , Yousef I. M. , Fisher M. M.
Published: December 2009

Nα-Cholylornithine, -arginine, and -histidine were prepared according to a method previously employed for the chemical synthesis of the monoamino acid conjugates of bile acids. The products were shown to involve the α amino group of the dibasic amino acids by examination of the mass spectra of the original compounds, their lactams, their methyl esters and the methyl ester acetates. Only the methyl ester acetates gave detectable amounts of molecular ion. The free acids and the methyl esters of Nα-cholylornithine and -arginine gave identical lactams upon sublimation from the direct insertion probe. The synthetic Nα-cholylarginine was shown to yield a mass spectrum identical to that of an arginocholic acid recovered from the bile of an isolated perfused rat liver.
Membrane Protein Synthesis in Micrococcus lysodeikticus and Selective Effect of Chloramphenicol
Authors: Mendoza C. Garcia , Ledieu M. Novaes
Published: December 2009

Micrococcus lysodeikticus cytoplasmic membranes labeled with [14C]arginine plus [14C]-threonine were prepared and subjected to mild washing treatments to fractionate membrane proteins. Polyacrylamide gel electrophoresis of total membranes, in the presence of sodium dodecyl sulfate, results in the separation of 28–30 bands of labeled protein. Three peaks of protein show higher specific radioactivity than the others. Chloramphenicol at 100 μg/ml inhibits the incorporation of labeled precursors into membrane proteins by 45–70%, some of them being more affected by the antibiotic. From all available results, we suggest that the partial inhibitory effect shown by this antibiotic could be due to the existence of specific biosynthetic sites for some membrane proteins, which are differently affected by chloramphenicol.
Titration Study of Guaiarcol Oxidation by Horseradish Peroxidase
Authors: Santimone Marius
Published: December 2009

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.
The Functional Groups of the Mg–Ca ATPase from Escherichia coli
Authors: Ahlers Jan , Kabisch Doris , Günther Theodor
Published: December 2009

The influence of hydrogen ion concentration on binding and conversion of MgATP and CaATP by membrane bound and solubilized ATPase from Escherichia coli has been investigated. The reaction of enzyme (E), hydrogen ion (H+), and substrate (S) proceeds according to the following scheme, where Me is the metal ion and P is the product(s).[Formula: see text]Within experimental error, the results obtained with membrane-bound and solubilized ATPase are identical. Changing the concentration of Mg2+ ions or replacement of Mg2+ by Ca2+ ions alters the dissociation constants Kb, KHMeATP, and [Formula: see text]. The kinetics and experiments with group-specific inhibitors suggest that integrity for amino, imidazole, tyrosyl, carboxyl, and arginyl residues is required for activity of membrane-bound and solubilized E. coli ATPase.
Specificity of γ-Glutamyl Cyclotransferase
Authors: Szewczuk Apolinary , Connell George E.
Published: December 2009

Using the phthaloyl method, 18 γ-L-glutamyl peptides labelled with 14C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified γ-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that γ-glutamyl cyclotransferase acts only on the L-form of γ-glutamyl substrates; the enzyme could, therefore, be used for preparation of γ-D-glutamyl peptides from their racemic mixtures. The specificity of γ-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as γ-L-glutamyl—NH—CHR—COOH. The amino acid linked to the γ-glutamyl group must be in the L configuration.
Identification and Turnover of Glycosaminoglycans in Rat Kidneys
Authors: Barry D. N. , Bowness J. M.
Published: December 2009

The turnover of sulfate label in crude glycosaminoglycan fractions from rat kidney cortex, medulla, and papilla has been determined. Heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronate have been separated electrophoretically and their specific activities determined after injection of labeled sulfate or glucose. The half-lives of the sulfated glycosaminoglycans are within the ranges found for other organs and tissues, but hyaluronate has a somewhat faster turnover in the kidney than elsewhere.
The Effect of a Myelin Apoprotein on Lipid Structure: A Spin Probe Study
Authors: Butler K. W.
Published: December 2009

Spin probes, stable free radical derivatives of stearic acid and cholestanone, were used to observe the effects of the "Folch–Lees" protein isolated from the white matter of bovine brain on the organization and motion of lipid molecules. The incorporation of the organic solvent soluble form of this protein decreased the tendency of a variety of lipid molecules with zero, positive or negative net charges to arrange themselves close to the normal to the lipid bilayer. The aqueous form of the protein also had a profound chaotropic effect on the molecular geometry of the lipid, but only if the lipids had a net negative charge (the protein has a net positive charge in the pH range investigated). Examination of the ESR spectra indicated that this protein altered the geometry of the lipid structure without causing major changes in the mobility of the individual lipid molecules.
In Vitro Synthesis of Estrogen Glucuronides and Sulfates by Human Renal Tissue
Authors: Mellor Joyce D. , Hobkirk R.
Published: December 2009

17β-[6,7-3H]Estradiol (E2) was incubated with slices and homogenates of adult human renal tissue. The metabolites formed were identified by chromatography on DEAE-Sephadex, thin layer chromatography and crystallization with carrier steroids or steroid derivatives. The major metabolites formed by slices were estradiol-17-glucuronide (E217G), estrone sulfate and estradiol-3-sulfate. This is the first report of in vitro synthesis of estrogen sulfates by adult renal tissue. Minor quantities of the 3-glucuronides of estrone and estradiol were also found. An oxygen atmosphere appeared to stimulate the production of E217G. A time study with tissue slices showed similarities between the in vitro pattern of glucuronide synthesis and the excretion pattern of these compounds seen in earlier in vivo studies. Homogenates fortified with uridine diphosphoglucuronic acid formed the same pattern of glucuronide products but in lesser amounts. No sulfates were formed under these conditions. Testosterone did not act as a substrate in the experimental conditions used.
Partial Characterization of a Histone Acetyltransferase from Trout Testis
Authors: Candido E. P. M.
Published: December 2009

Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts, With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin.Treatment of nuclei with 0,4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates.
Studies on the Fatty Acid Binding Proteins in Cytosol of Rat Liver
Authors: Suzue Ginzaburo , Marcel Yves L.
Published: December 2009

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing.Peak I (mol. wt. 60 000, pI 5.0) was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10 000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated.These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.
Carboxyl-Terminal Sequences of Procaryotic Ribosomal Proteins from Escherichia coli, Bacillus stearothermophilus, and Halobacterium cutirubrum
Authors: Duggleby Ronald G. , Kaplan Harvey , Visentin Louis Peter
Published: December 2009

The carboxyl-terminal amino acid sequences of two ribosomal proteins, Escherichia coli L12 and E. coli S4, and the proteins believed to be their equivalents from Bacillus stearothermophilus and Halobacterium cutirubrum, were determined. These proteins are known to be required for peptide chain termination (L12) and in ribosome assembly (S4). The carboxyl-terminal sequences obtained suggest that the E. coli and B. stearothermophilus proteins have retained structural homology in this region, whereas the H. cutirubrum proteins have not.
Phosphoenolpyruvate Carboxylase of Thiobacillus thioparus. I. General Properties
Authors: Hoban Daryl J. , Lyric Ronald M.
Published: December 2009

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxylase (phosphorylating), EC 4.1.1.31) was purified 19-fold from the obligate chemoautotroph, Thiobacillus thioparus.Michaelis constants for the substrates were found to be 0.44 mM for phosphoenolpyruvate, 0.89 mM for bicarbonate, and 0.37 mM for magnesium, using Tris–HCl, pH 7.3.1-Aspartate, 1-malate, and orthophosphate were found to be inhibitors of enzyme activity, while acetyl CoA, FDP, GTP, and CDP had no effect. Dioxane greatly stimulated enzyme activity.
Nuclear Demethylation and C-24 Alkylation During Ergosterol Biosynthesis in Saccharomyces cerevisiae
Authors: Fryberg M. , Avruch L. , Oehlschlager A. C. , Unrau A. M.
Published: December 2009

The role of 4,4-dimethylzymosterol (3), 4,4-dimethylfecosterol (4) and 31-norlanosterol (5) in the biosynthesis of ergosterol in Saccharomyces cerevisiae has been investigated. The synthesis of 4 and 5 coupled with the availability of 3 facilitated a search for these sterols in commercial yeast sterol concentrates, fresh laboratory grown yeast and fresh brewery grown yeast. Sterol 4 was not detected in any of these mixtures whereas 5 was found in the first and last and 3 was present in all three sources investigated. Investigation of incorporation of [2-3H]lanosterol into 3, 4 and 5 revealed significant incorporation into 3 but neither 4 nor 5. This observation suggests the principle pathway for ergosterol biosynthesis initially involved 1 → 3 → 7.Incubation of a mixture of [2,4-3H]zymosterol and [26,27-14C]lanosterol with S. cerevisiae revealed that during the initial phases of aerobic growth the major route from 7 to ergosterol involves zymosterol (11) but as 11 accumulates 4α-methyl-24-methylenezymosterol (8) assumes equal importance.
The Immunoglobulin M Molecule: Isomeric Forms of the Monomer Subunit
Authors: Percy M. E. , Percy J. R.
Published: December 2009

We have devised a novel technique which uses a simple theoretical model to simulate the reduction of immunoglobulin M (IgM) molecules. By fitting the results of the simulated depolymerization to experimental data, we have obtained statistical evidence which suggests that two major isomeric forms of covalently-bonded IgM monomer are liberated when human 19S IgM is reduced with dithiothreitol. The two heavy chains of isomer 1 are linked by two dilsulfide bridges, one in the segment designated the "hinge" region (position 337), the other penultimate to the COOH terminus of the chains (position 575). The half-cystines at position 414, which are free in isomer 1, form an inter-heavy chain bridge in isomer 2. Theoretically, the relative proportions of the two forms of monomer liberated in the reduction depend upon the dithiothreitol concentration, with isomer 2 predominating at higher dithiothreitol concentrations. Although in this paper we have assumed the conventional structure of the IgM molecule, the liberation of isomers depends only upon a symmetrical arrangement of the three types of inter-heavy chain bonds in the cyclic 19S pentamer.
Amino Acid Transport in a Water-mould: The Possible Regulatory Roles of Calcium and N6-(Δ2-isopentenyl)adenine
Authors: Singh Danny P. , LéJohn Hérb. B.
Published: December 2009

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different transport systems elucidated are: (i) methionine, (ii) cysteine, (iii) proline, (iv) serine–threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine–tyrosine–tryptophan and leucine–isoleucine–valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.Calcium played more than a secondary role in the transport of the amino acids. When bound to the membrane-localized glycopeptide, it permits concentrative transport to take place. However, excess calcium can inhibit transport which can be overcome by chelating with citrate. Calculations show that the concentration of free citrate is most important. At low citrate concentrations (less than 1 mM) in the absence of exogenously supplied calcium, enhancement of amino acid transport occurs. At high concentrations (greater than 5 mM), citrate inhibits but this effect can be reversed by titrating with calcium. Evidently, the glycopeptide acts as a calcium sink to regulate the concentration of calcium made available to the cell for its membrane activities.N6-(Δ2-isopentenyl) adenine (a plant growth 'hormone') and analogues mimic the inhibitory effect of citrate and bind to the glycopeptide as well. Replot data for citrate and N6-(Δ2-isopentyl) adenine inhibition indicate that both agents have no more than one binding constant. These results implicate calcium, glycopeptide, and energy-dependent transport of solutes in some, as yet undefinable, way.
Nuclear Magnetic Resonance Studies of Phenylthiohydantoin Amino Acids
Authors: Tsai C. S. , Fraser N. L. , Avdovich H. , Farant J. P.
Published: December 2009

Proton magnetic spectra of 3-phenyl-2-thiohydantoin derivatives of common amino acids in deuterated dimethyl sulfoxide were recorded. Spectral data pertaining to characteristic protons for diagnostic purpose were compiled. Their application to the N-terminal amino acid analysis of peptide by Edman degradation was examined.
Pentaglutamate Derivatives of Folate as Substrates for Rat Liver Tetrahydropteroylglutamate Methyltransferase and 5,10-Methylenetetrahydrofolate Reductase
Authors: Cheng F. W. , Shane B. , Stokstad E. L. R.
Published: December 2009

Tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13) and 5,10-methylenetetrahydrofolate reductase (EC 1.1.1.68) were purified more than 100-fold from rat liver. The specificity of each enzyme for 5-methyl-H4PteGlu and 5-methyl-H4PteGlu5 did not change throughout the purification procedures.A comparison of enzyme properties as well as kinetic analysis showed that pteroylmono- and pentaglutamates were binding to the same enzyme in each case. There was no evidence of any pteroylpentaglutamate specific methyltransferase or reductase in rat liver.(+)-5-Methyl-H4PteGlu5 was a more effective substrate for the methyltransferase (Km ~ 4 μM) and reductase (Km ~ 3 μM), at low substrate concentrations, than (+)-5-methyl-H4PteGlu (Km ~ 13 μM and ~ 23 μM, respectively), although V values were lower. High levels of the pentaglutamate substrate inhibited the reductase reaction (Ki ~ 40 μM). The unnatural (−)-5-methyl-H4PteGlu1,5 diastereoisomers were not inhibitors of either enzyme.The results suggest that any 'methyl trap' operating at the monoglutamate level under conditions of vitamin B12 deprivation would be at least as effective at the pentaglutamate level.
The Action of β-Galactosidase (Escherichia coli) on Allolactose
Authors: Huber R. E. , Wallenfels K. , Kurz G.
Published: December 2009

The parameters involved in the action of β-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of β-galactosidase as lactose. The pH optimum with allolactose (7.8–7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The α-anomer of allolactose was hydrolyzed about two times as rapidly as was the β-anomer.
The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes
Authors: Jothy Serge , Bilodeau Jean-Louis , Simpkins Henry
Published: December 2009

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.
Anti-Inflammatory Steroids, Lysosomal Stabilization and Parachor
Authors: Ahmad Parvez , Fyfe Colin A. , Mellors Alan
Published: December 2009

Parachor is an additive constitutive property of a molecule and is related to the molar volume and the surface tension. The parachor of a steroid can be calculated from its constituent atoms and bonds. The parachor of a biologically active molecule is related to the ability of that molecule to permeate hydrophobic regions of cells, especially cellular membranes. An examination of the parachor values of a large number of steroids shows that these values are correlated with a number of different biological activities, from independent sources. The ability of steroids to release lysosomal enzymes from isolated lysosomes in vitro is inversely related to the parachor of the steroid. A similar relationship holds for the release of lysosomal β-glucuronidase (EC 3.2.1.31) from isolated lysosomes of rat preputial gland following in vivo administration of steroids. The relative anti-inflammatory potencies of steroids by several assay methods are directly proportional to their parachors. The relative ability of corticosteroids to uncouple oxidative phosphorylation and to swell isolated mitochondria in vitro show a direct proportionality with the steroidal parachor. The percutaneous absorptions of steroids show good correlation with parachors, stratum corneum – water partition coefficients and amylcaproate–water partition coefficients; but not with hexadecane–water partition coefficients. The application of parachor as a structure–activity correlation parameter in drug design is likely to yield useful information. The advantages and limitations of the calculated parachor method are discussed.
Flow Kinetics of β-Galactosidase Chemically Attached to Nylon Tubing
Authors: Narinesingh D. , Ngo T. T. , Laidler K. J.
Published: December 2009

β-Galactosidase (EC 3.2.1.23) has been attached covalently to the inner surface of nylon tubing. An experimental study has been made of the flow kinetics for the hydrolysis of o-nitrophenylgalactose, the substrate concentration and flow rate being varied. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, three different methods of analysis being employed. It is concluded that at the lower substrate concentrations and flow rates employed, the reactions are largely diffusion controlled; with increase in flow rate and substrate concentration the width of the Nernst diffusion layer decreases, and there is found to be less diffusion control. The values of Km(app) vary with flow rate VF, being linear in VF−1/3, and the value extrapolated to very high flow rate agrees well with the Km value for β-galactosidase in free solution. The theory and results are shown to provide guidelines for the design of open tubular heterogeneous enzyme reactors for industrial, biomedical, and analytical applications.
Identification of Radioactive 17β-Estradiol-17-β-D-glucuronide and 17α-Estradiol-17-β-D-glocuronide in the Urine of the Domestic Fowl after Intramuscular Injection of [4-14C]Estrone
Authors: Robinson Arthur Robin , Baker Susan Jane , Common Robert Haddon
Published: December 2009

Two previously uncharacterized radioactive estrogen conjugates, 17β-estradiol-17-β-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17β-yl-β-D-glucopyranosiduronate) and 17α-estradiol-17-β-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17α-yl-β-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.
Cysteine Biosynthesis in Salmonella typhimurium: the Presence of ATP-Sulfurylase and APS-Kinase in Various Cysteine-Requiring Mutants
Authors: Collins James M. , Monty Kenneth J.
Published: December 2009

Enzymatic tests were performed on a series of cysteine-requiring mutants for the presence of the sulfate activating enzymes, ATP-sulfurylase (sulfate adenylyltransferase EC 2.7.7.4) and APS-kinase (adenylylsulfate kinase EC 2.7.1.25). The enzymatic products adenosine 5′-[35S]sulfatophosphate and adenosine 3′-phosphate 5′-[35S]sulfatophosphate were identified by paper electrophoresis and measured quantitatively without elution from the paper. Cys mutants mapping in cistrons, A, H, I, J, G, and Ea contain both enzymes. Mutation in the D cistron leads to the loss of ATP-sulfurylase. Mutants mapping in the C cistron lack APS-kinase. Ba, Bb, and Bc mutants lack both enzymes. The control of the synthesis of these enzymes by cysteine was examined. Both enzymes are missing when cells are grown on cysteine.
Effect of Nε-Alkylation of the Substrate on the Hydrolysis of Benzoylglycyl-L-Lysine by Carboxypeptidase B
Authors: Moore Graham J. , Benoiton N. Leo
Published: December 2009

The action of carboxypeptidase B (EC 3.4.12.3) on the benzoylglycyl dipeptides Bz-Gly-Lys(X) where X = methyl, ethyl, propyl, formyl, dimethyl, isopropyl, trimethyl, and benzyl has been investigated. All were hydrolyzed, at a rate decreasing in the order indicated, except where X = trimethyl and benzyl. Compounds where X = dimethyl, formyl, and isopropyl were hydrolyzed very slowly, and did not inhibit the hydrolysis of Bz-Gly-Lys by the enzyme. The kinetic parameters kcat and Km were determined for compounds with X = methyl and ethyl. The observed decrease in rate of hydrolysis of substrates with increasing size of X is consistent with increasing steric hindrance effects arising from the interaction of the Nε-alkyl group with residues of the protein in the cleft which accommodates the substrate side-chain, and resulting in weaker binding of the substrate.Bz-Gly-Lys(Me2) was prepared by reductive methylation of Bz-Gly-Lys. Bz-Gly-Lys(Me3) was prepared by the reaction of Bz-Gly-Lys(Me2) with diazomethane in aqueous solution. Bz-Gly-Lys(Me) and Bz-Gly-Lys(Et) were synthesized by classical coupling procedures from the appropriately protected lysine derivatives.
Diacylglycerol Biosynthesis in Everted Sacs of Rat Intestinal Mucosa
Authors: Breckenridge W. C. , Kuksis A.
Published: December 2009

The molecular specificity in the biosynthesis of diacylglycerols by rat intestinal mucosa was examined by means of radioactive markers, 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 diacylglycerols were isolated by solvent extraction and thin-layer chromatography. Stereospecific analyses of the X-1,2-diacylglycerols labeled from 2-monoacylgiycerols showed that the sn-1,2-isomers (45–55%) were slightly in excess of the sn-2,3-isomers (34–45%) with the X-1,3-diacylglycerols accounting for the rest of the radioactivity (5–10%). This suggests that racemic diacylglycerols may be intermediates in the resynthesis of dietary fat in rat intestinal mucosa. Detailed analyses of the molecular species of the sn-1,2-diacylglycerols labeled from free fatty acids revealed that 10–45% of the total did not contain the acid present in the 2-monoacylglycerol supplied, and therefore had originated from the phosphatidic acid pathway. These findings are at variance with those obtained in isolated microsomes, which have suggested an inhibition of the phosphatidic acid pathway by monoacylglycerols as well as have given evidence of an exclusive synthesis of sn-1,2-diacylglycerols from 2-monoacylglycerols.