bluuebunny/crossref_metadata_2025 huggingface dataset:
Some Distinctive Characteristics of the Alkaline Phosphatase of Serratia marcescens
Authors: Bhatti A. R.
Published: December 2009

A mutant strain of Serratia marcescens produces a constitutive enzyme (phosphatase F) which differs from the alkaline phosphatase of Escherichia coli in the following characteristics: one enzyme species with higher mobility on electrophoresis, less heat stability, no rapid reactivation following exposure to high hydrogen ion concentrations, no hybridization with E. coli enzyme in vitro, little activation at increased ionic strength, greater sensitivity to EDTA inhibition, and no cross reaction of rabbit anti-serum with the E. coli enzyme.
Cell Surface Sialoglycopeptide Metabolism and Surface Glycosyl Transferase Activity in the Ehrlich Ascites Cell
Authors: Irwin D. , Anastassiades T. P.
Published: December 2009

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained.2. However, when intact cells were incubated with labelled uridine 5′-diphosphate – N-acetyl glucosamine or cytidine 5′-monophosphate (CMP) – sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity at the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP – sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP – sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.
In Vitro Metabolism of 17β-Estradiol by Human Liver Tissue
Authors: Hobkirk R. , Mellor Joyce D. , Nilsen Mona
Published: December 2009

17β-[6,7-3H]Estradiol was incubated with adult human liver slices in Krebs–Ringer phosphate buffer containing glucose. Of the identified 3H recovered, 51–76% consisted of estrone-3-sulfate (E13S) and 17β-estradiol-3-sulfate (E23S). E13S was the main metabolite and was found in both tissue and medium. E23S was present only in the medium. Minor amounts of estrogen glucuronides were formed. When a human liver homogenate was incubated with [3H]E2 in a medium fortified with excess uridine diphosphate glucuronic acid only some 4% of conjugation with glucuronic acid was observed. It is suggested that human liver favors sulfurylation as the conjugating mechanism for E2 and E1.
Essential Sulfhydryl Groups of Rat Liver Monoamine Oxidase
Authors: Symes Aston L. , Sourkes Theodore L.
Published: December 2009

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 °C are 5.80 × 10−4M and 4.35 × 10−5 M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO.The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 ± 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 total sulfhydryl groups (n = 2) on denaturation with 8 M urea.
Ammoniagenesis: D-Glutamyltransferase as a Source of Ammonia in the Rat Kidney
Authors: Wadoux P. , Welbourne T. C.
Published: December 2009

The contribution of D-glutamyltransferase (D-GT) (EC 2.3.2.1) to total renal ammonia production was determined by employing DL-methionine-DL-sulfoximine (MSO) as an inhibitor of D-GT. Rat kidney homogenates were assayed for NH3-liberating activity under optimal D-GT or γ-glutamyltranspeptidase (γ-GTP) (EC 2.3.2.2) conditions. MSO inhibits only D-GT activity. The contribution of D-GT to total renal ammonia production was then evaluated in the isolated perfused rat kidney employing identical substrate (5 mML-glutamine) and inhibitor (15 mM MSO) concentrations as employed in the homogenate study. Under these conditions, MSO inhibits 70% of the total ammonia production by the normal kidney; in addition, the ratio of ammonia produced per glutamine taken up rose from 1.0 to 1.8. In kidneys from chronically acidotic rats, MSO reduced total ammonia production only 35% while the NH3/glutamine ratio rose from 1.0 to 1.8. D-GT appears to be the predominant source of NH3 production in the normal rat kidney; γ-GTP does not contribute significantly. The rise in the NH3/glutamine ratio after D-GT inhibition is consistent with glutamine utilization via the activated mitochondrial glutaminase (EC 3.5.1.2) – glutamate dehydrogenase (EC 1.4.1.2) pathway.
A Comparison of Lactate Dehydrogenases from an Ectothermic and an Endothermic Animal
Authors: Borgmann Uwe , Moon Thomas W.
Published: December 2009

Adaptation to environmental temperature is examined in beef heart, beef muscle, and flounder muscle lactate dehydrogenases (EC 1.1.1.27). Low temperature adaptation in the ectothermic (flounder) enzyme is indicated by a reduced enthalpy of activation for kcat (enzyme turnover number, s−1) and increased catalytic efficiency. Also, the reaction rate at low substrate concentrations has a maximum at a lower temperature than in the endothermic enzymes. This is a result of altered bonding in the enzyme–substrate complexes. Adaptation to higher temperatures in the endothermic (beef) enzymes is suggested by a decreased sensitivity to heat denaturation, especially in the presence of substrates. A direct correlation is found between the degree of bonding in the enzyme–substrate complexes and the decrease in rate of heat denaturation caused by addition of substrates.
Glycerolipid Biosynthesis in Isolated Rat Intestinal Epithelial Cells
Authors: O'Doherty P. J. A. , Kuksis A.
Published: December 2009

Intestinal epithelial cells were prepared from fasted rats by dispersion with collagenase (EC 3.4.24.3). The structural and metabolic integrity of the cells was verified by electron microscopy, a high percentage of Trypan Blue exclusion, a low degree of release of lactate dehydrogenase (EC 1.1.1.27) in the medium, and by the retention of sensitivity to agents known to modify metabolic and transport activity in everted sacs of intestinal mucosa. The isolated intestinal epithelial cells were used to study glycerolipid biosynthesis from glucose, glycerol, 2-monoacylglycerol, and free fatty acids. The cells actively incorporated the labeled precursors into glycerolipids without specific cofactor requirements. Addition of fatty acids stimulated the incorporation of both glucose and glycerol into triacylglycerols and glycerophospholipids, the greatest effect being observed with palmitate. The stimulation of monoacylglycerol acylation appeared to depend on both the nature of the monoacylglycerol and fatty acid supplied. Stereospecific analyses of the diacylglycerols formed from 2-monoacylglycerols and free fatty acids showed that 1,2-diacyl-sn-glycerols (62–70%) were the major and that 2,3-diacyl-sn-glycerols (30–38%) the minor intermediates in triacylglycerol biosynthesis. The data indicate that isolated intestinal epithelial cells exhibit a total capacity of glycerolipid synthesis and a stereochemical course of reaction which is comparable to that observed for triacylglycerol formation in everted sacs of intestinal mucosa, but much less specific than that seen in microsomal preparations of intestinal mucosa.
Mechanism of Action of Mg2+ and Zn2+ on Rat Placental Alkaline Phosphatase. I. Studies on the Soluble Zn2+ and Mg2+ Alkaline Phosphatases
Authors: PetitClerc Claude , Delisle Monique , Martel Marc , Fecteau Claude , Brière Normand
Published: December 2009

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135 000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH, but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate.Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site.A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.
Effect of Sample Volume and Column Length on Transcortin Determination by Sephadex G-25 Gel Filtration
Authors: Dubé J. Y. , Ho-Kim M. A. , Tremblay R. R.
Published: December 2009

When Sephadex G-25 columns are used to separate transcortin-bound corticosterone from free corticosterone, there is a non-linear increase of total binding with increasing plasma volume filtered. However, the specific binding (total binding — unspecific binding) shows a linear relationship with the plasma volume filtered. Furthermore, the specific binding decreases with increasing Sephadex column length and absolute binding values are found by extrapolating to a zero length column.
The Hydrolysis of Some N-Acylaspartic and N-Acylglutamic Monoamides in Dilute Mineral Acid
Authors: Ali M. , Capindale J. B.
Published: December 2009

The release of ammonia by hydrolysis of N-benzoyl-L-asparagine, glycyl-DL-asparagine, L-asparagine, and succinamic acid, and of aniline from N-benzoyl-L-glutamic-α-anilide, N-benzoyl-L-aspartic-α-anilide, L-aspartic-α-anilide, and the monoanilides of succinic and glutaric acids is first-order with respect to substrate in dilute (0.4–0.03 M) aqueous hydrochloric acid at 100 °C. The first-order rate constants (kobs) for these reactions can be expressed as kobs = kintra + k2[H+]. The above hydrolyses are used as models for developing a tentative mechanism to account for the selective release of aspartic acid from proteins under these conditions. The data are also used to suggest reasons why glutamic acid is not released with equal facility.
Charge Properties of Mitochondrial Matrix Proteins
Authors: Strasberg P. M. , Freeman K. B.
Published: December 2009

Proteins of the rat liver mitochondrial matrix have been separated into anionic (acidic), cationic (basic), and neutral groups by electrophoresis. These groups represent 69, 8, and 23% of the total matrix protein, respectively, compared to 69, 21, and 10% for the cytosol protein. The acidic nature of the mitochondrial matrix proteins has been confirmed by cellulose ion-exchange chromatography, isoelectric focusing in sucrose gradients, and amino acid analysis. The anionic, cationic, and neutral matrix proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis into 18, 6, and 5 bands, respectively, compared to 22 bands for the total fraction. The significance of the charge properties of these proteins in terms of mitochondrial biogenesis is discussed.
The Histones of Rainbow Trout Erythrocytes Include an Erythrocyte-Specific Histone
Authors: Miki B. L. A. , Neelin J. M.
Published: December 2009

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erytbrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation.To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to establish identities of corresponding histone fractions.An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.
A Nuclear Magnetic Resonance Study of the Metal Binding Sites in Bacitracin
Authors: Wasylishen Roderick E. , Graham Moira R.
Published: December 2009

Carbon-13 nuclear magnetic resonance spectroscopy has been used to identify sites in bacitracin which bind Cu2+ and Mn2+. Results are presented which implicate the free carboxyl groups of the aspartic and glutamic acid residues and the imidazole ring of the histidine residue as metal complexation sites between pH 6 and 8. Evidence is presented which also indicates that the thiazoline ring of bacitracin binds Mn2+. Bacitracin does not bind Cu2+ or Mn2+ at pH values of 2.5 or less.
Sterol and Fatty Acid Composition of Polyene Macrolide Antibiotic Resistant Torulopsis glabrata
Authors: Lomb M. , Fryberg M. , Oehlschlager A. C. , Unrau A. M.
Published: December 2009

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystatin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa.Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquisition of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.
Purification and Properties of Phenylalanyl-tRNA Synthetase from Baker's Yeast
Authors: Hossain Afzal
Published: December 2009

In an effort to avoid proteolytic fragmentation of enzymes extracted from yeast cells, the (L-phenylalanine:tRNAPhe ligase (AMP-forming) phenylalanyl-tRNA synthetase (EC 6.1.1.20)) has been isolated from toluene lysates of baker's yeast in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride. The procedure includes ammonium sulfate fractionation and chromatography on DEAE-cellulose and hydroxyl-apatite columns. Acrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate indicates a single subunit of 75 000; other isolations have led to two subunits of 75 000 and 63 000, respectively, in agreement with other workers. Steady state kinetic analysis of the enzyme has also been carried out. The apparent kinetic patterns resulting from application of Cleland's procedure, in which the substrates are varied pair-wise in the presence of saturating concentrations of the third component, suggest a reaction mechanism in which ATP and phenylalanine enter the reaction in an obligatory ordered fashion but do not completely eliminate a random mechanism.
Nonpolar lipids of a halotolerant species of Staphylococcus epidermidis
Authors: Kushwaha S. C. , Kates M.
Published: December 2009

The nonpolar lipids of a halotolerant Staphylococcus epidermidis, isolated in pure culture from a growth medium for extreme halophiles containing 25% sodium chloride, were found to contain squalene, menaquinone-7, free fatty acids (mainly anteiso-15:0 and anteiso-17:0), undecaprenol, nonaprenol with predominately cis-isoprene residues, heptaprenol, with predominately trans-isoprene residues, and 1,2- and 1,3-diglycerides containing anteiso-15:0 and anteiso-17:0 branched chain fatty acid residues. The above compounds were isolated in pure form by column and thin-layer chromatography and were characterized by ultraviolet, proton magnetic resonance, and mass spectra. Fatty acid moieities were characterized by gas–liquid chromatographic retention times of their methyl esters.
In vivo and in vitro phosphorylation of nuclear proteins in rat liver
Authors: Thomson Judith A. , Brade W. P. , Chiu Jen-Fu , Hnilica L. S. , McGill M.
Published: December 2009

The incorporation of 32P into nuclear nonhistone proteins was compared in rat liver in vivo, in liver slices incubated in vitro, and in isolated nuclei incubated with γ-[32P]ATP. The highest specific activities of nuclear phosphoproteins were obtained by incubating isolated nuclei. However, the radioactivity profiles of polyacrylamide gel electrophoretograms of these proteins differed from those obtained in vivo or in liver slice experiments. A group of low molecular weight nonhistone proteins exhibited a very high incorporation of labelled phosphate. These proteins could be obtained from the interface when the phosphoproteins were isolated by the buffered phenol extraction procedure. Phosphorylated proteins were also obtained from three cytoplasmic fractions (mitochondria, microsomes, and cytosol). The specific activities of these proteins were much lower than of the nuclear phosphoproteins.
Characterization of tryptic peptides from porcine haptoglobin light chain and an amino acid sequence
Authors: Chung W. P. , Smith David B.
Published: December 2009

The tryptic peptides derived from porcine haptoglobin light chains have been separated and characterized by composition, chromatography, electrophoretic mobility, and partial sequencing. Depending on homology with the corresponding human polypeptide, the amino acid sequence of the chain is proposed. Twenty differences from the human chain are indicated in the total of 84 residues.
Uptake and binding of disaccharides in human erythrocytes
Authors: Beneš Ivan , Kotyk Arnošt
Published: December 2009

Disaccharides (sucrose, lactose, melibiose, cellobiose, trehalose, maltose, and isomaltose) are not transported across the human erythrocyte membrane. Maltose alone is bound in appreciable amounts to the intact cell as well as ghost membranes and competes mutually for uptake with D-glucose. In (NH4)2SO4-precipitated membrane preparations, maltose binds more strongly than other disaccharides (KD = 1.3 × 10−5 M; maximum binding capacity, 71 pmol/mg protein) and again competes mutually with D-glucose. Phloretin inhibits the binding of glucose much more than that of maltose.
The metabolism of higher chlorinated benzene isomers
Authors: Kohli J. , Jones D. , Safe S.
Published: December 2009

The metabolism of the isomeric tri- and tetrachlorobenzene isomers, penta- and hexachlorobenzene was investigated in the rabbit. The major urinary metabolites of the isomeric tri- and tetrachlorobenzenes were identified as the corresponding tri- and tetrachlorophenols whose structures were confirmed by chromatographic analyses. The genesis of the formation of metabolites is discussed in terms of their possible arene oxide intermediates in which the NIH shift of a chlorine atom is observed in the oxidation of many of the isomers. Pentachlorobenzene is metabolized to give both pentachlorophenol and a dechlorination–hydroxylation product which was identified as 2,3,4,5-tetrachlorophenol. The hexachlorobenzene substrate did not give any phenolic metabolites.
Effects of diet on catabolism and excretion of [26-14C]cholesterol in rats
Authors: Kim Jae Jin , Hamilton R. M. G. , Carroll K. K.
Published: December 2009

The catabolism and excretion of [26-14C]cholesterol was studied in rats on semisynthetic and commercial diets low in fat or containing 15% butter or corn oil. Rats on the low fat commercial diet oxidized the labeled cholesterol to 14CO2 at more than twice the rate of those on the semisynthetic diet. Fecal excretion of labeled lipid was also somewhat higher with the commercial diet. The added fats had little effect on rate of oxidation of cholesterol but dietary corn oil stimulated fecal excretion of labeled lipid. The rate of loss of labeled cholesterol through oxidation and excretion showed a positive correlation with cholesterol biosynthesis, as measured previously by acetate incorporation into cholesterol in rats on the same kinds of diet. A simple method for efficient trapping and counting of 14CO2 was developed, which facilitated measurement of low levels of 14CO2 in expired air. Estimation of bile acid production from the rate of oxidation of [26-14C]cholesterol to expired 14CO2 and the specific activity of plasma cholesterol gave somewhat higher values than those obtained by other methods. Possible reasons for this difference are discussed.
The purification from Escherichia coli of a protein relaxing superhelical DNA
Authors: Burrington M. G. , Morgan A. R.
Published: December 2009

The Escherichia coli omega protein was first described by Wang (Wang, J.C.: J. Mol. Biol. 55, 523–533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, α. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an αβ-ype subunit protein with a molecular weight of the intact protein of about 80 000 (determined by gel filtration) and of the individual subunits of 56 000 and 31 000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.
Some properties of acetylcholinesterase from rat retina
Authors: Tunnicliff G. , Tsukada Y. , Barbeau A.
Published: December 2009

Acetylcholinesterase (AChE, EC 3.1.1.7) of rat retina was studied with respect to its kinetic and other properties, and a comparison was made with the enzyme from brain. The subcellular distribution of the retinal AChE showed that the enzyme was concentrated in the synaptosomal–mitochondrial fraction although in the brain the AChE was distributed more evenly between the fractions studied. The enzyme from both retina and brain was easily solubilised and exhibited a Km of the order of 10−4 M. The pH optimum was 8.3–8.6 for the AChE from both tissues for both the soluble and particulate enzyme.
Evidence of a novel role for monovalent cations in pyruvate kinase catalysis
Authors: Robinson James L.
Published: December 2009

In the pyruvate kinase (EC 2.7. 1.40) reaction, the complete enzyme–products complex consists of enzyme, pyruvate, ATP, a divalent cation, and a monovalent cation, usually K+. The dissociation of this, complex can be slow permitting reversible enolization of pyruvate in this complex during the course of the forward reaction. High concentrations of each component in the enzyme–products complex inhibits the forward reaction, but only elevated concentrations of the monovalent cation decrease the net rate of product dissociation relative to that of pyruvate enolization. This result indicates that the monovalent cation can be the first component released from the enzyme–products complex and that the presence of an inhibiting monovalent cation concentration leads to re-formation of the complete complex, which is necessary for pyruvate enolization. The evidence suggests that the monovalent cation can bind and release with each turnover of the enzyme. While the data do not permit the conclusion that first release of monovalent cation is the exclusive pathway for dissociation of the enzyme–products complex, no other component once released can reassociate rapidly enough to form a complete complex during the forward reaction. Inhibition by these components must be attributed to the formation of abortive complexes.
Studies related to antitumor antibiotics. Part VIII. Cleavage of DNA by streptonigrin analogues and the relationship to antineoplastic activity
Authors: Lown J. William , Sim Soo-Khoon
Published: December 2009

A group of substituted 5,8-quinolinequinones which exhibit antineoplastic activity and which are structurally related to the antitumor antibiotic streptonigrin induce single strand cleavage of PM2 covalently-closed circular-DNA (ccc-DNA) when reductively activated. The cleavage which is detected by an ethidium fluorescence assay is specifically enhanced by cuprous and ferrous ion and is selectively inhibited by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) and by free radical scavengers. Independent generation of the superoxide ion by xanthine – xanthine oxidase (EC 1.2.3.2) also cleaves PM2 DNA and therefore a chemical mechanism for the scission process induced by the streptonigrin analogues is formulated. A correlation between rate of PM2 ccc-DNA cleavage and inhibition of Walker carcinosarcoma 256 is observed.
The interaction of ligands with chemically modified phosphorylase b
Authors: Brooks D. , Busby S. J. W. , Griffiths J. R. , Radda G. K. , Avramovic-Zikic O.
Published: December 2009

Phosphorylase b which had been inactivated with 5-diazo-1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein.Phosphorylase b which had been inactivated by carbodiimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose- 1-phosphate.The actions of 5-diazo-1H-tetrazole and carbodiimide on phosphorylase are discussed in the light of the above observations.
The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver
Authors: Nestruck A. Christine , Rubinstein David
Published: December 2009

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor–product relationship. Analysis of the apoproteins of the Golgi VLDL by polyacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF3, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1 showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent.The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.
Mechanisms of suppression in Drosophila. IV. Specificity and properties of tyrosyl-tRNA synthetase
Authors: Warner Cynthia K. , Jacobson K. Bruce
Published: December 2009

Tyrosyl-tRNA synthetase from wild type Drosophila can distinguish tRNATyr of wild type from that of the suppressor mutant, su(s)2. The method of fractionation of the enzyme on DEAE-cellulose can produce three different forms of the enzyme. One of these forms is characterized in this study. We describe the apparent Km for K+, Mg2+, ATP, tyrosine and tRNA, as well as its heat stability and molecular weight. Spermine does not replace Mg2+ but is inhibitory; a Ki of 1 mM was obtained. Inhibition by p-chloromercuribenzoate results in a Ki of 1 μM. The properties of tyrosyl-tRNA synthetase of Drosophila are compared to the properties of this enzyme from E. coli, B. subtilis and yeast.
Assembly of three major subclasses of mouse immunoglobulin G: a theoretical model for covalent assembly in vivo
Authors: Percy J. R. , Percy M. E. , Baumal R.
Published: December 2009

A mathematical model, based on second-order reaction kinetics, has been used to describe the covalent assembly of immunoglobulin G (IgG) in vitro from its heavy (H) and light (L) chains (Percy, M. E., Baumal, R., Dorrington, K. J. &Percy, J. (1976) Can. J. Biochem. 54, 675–687). In the present paper, the same model has now been applied to the steady-state assembly of IgG in vivo. This mathematical approach permits a quantitative comparison of the pathways of covalent assembly used by given immunoglobulins in vivo and in vitro. The assumptions in the model are: the species L, H, HL, HH, HHL and LHHL belong to a common pool; incompleted IgG intermediates may freely assemble to form HL, HH, HHL and LHHL; the reaction rate for covalent linkage between any two reacting species is proportional to the products of the number densities of the reactants and to a parameter P which takes the value PHH if the reaction joins two H chains, and PHL if it joins an H and L chain. In vivo values of PHH/PHL were determined for the 18 mouse myeloma tumours and cell lines studied by Baumal et al. (Baumal, R., Potter, M. &Scharff, M. (1971) J. Exp. Med. 134, 1316–1334). From these analyses, we have arrived at the following conclusions: (1) the three major IgG subclasses have distinctive values of PHH/PHL (mean value 53 for IgG1, 12 for IgG2a and 2.8 for IgG2b); (2) for IgGs of the same subclass, the values of PHH/PHL are similar; (3) the mean in vivo values of PHH/PHL are very close to those determined from in vitro assembly experiments. Finally, the individual values of PHH/PHL have been used to simulate pulse-chase experiments in the various tumours and cell lines. Considering the sources and magnitude of experimental error, the theoretical pathways of assembly agree with those determined qualitatively from the pulse-chase experiments.
Human intestinal goblet cell mucin
Authors: Jabbal I. , Kells D. I. C. , Forstner G. , Forstner J.
Published: December 2009

Goblet cell mucin (GCM) has been purified for the first time from mucosal scrapings of human small intestine. Proteolytic enzymes and organic solvents were avoided during the isolation procedure. The mucin was purified by Sepharose 4B and 2B column chromatography of high-speed supernatant fractions. The most purified fraction was compared with rat intestinal GCM. The two were similar with respect to chemical composition, antigenic features, and polyacrylamide disc gel electrophoresis. The major chemical differences included a higher hexosamine–fucose and hexosamine – sialic acid ratio in human mucin. The two mucins showed strong concentration dependence in sedimentation velocity studies. Human mucin at a concentration of 0.2 to 1.5 mg protein per millilitre gave multiple associated peaks with variable s0 values (10.8–36.6). Rat mucin, in contrast, gave a constant (although polydisperse) pattern with s0 = 15.15. To explore these differences both mucins were stained with periodic acid – Schiff reagent and subjected to band ultracentrifugation at concentrations of 0.6–1.9 μg protein per millilitre. At this low concentration, rat mucin did not change in its sedimentation characteristics. In contrast, human GCM produced a single peak with s0 = 37.9. Thus dilution abolished polydispersity in the human but not the rat mucin, suggesting that intermolecular bonding forces in the human mucin are weaker.
Quantitative effects of unsaturated fatty acids in microbial mutants. VI. Selective growth responses of yeast and bacteria to cis-octadecenoate isomers
Authors: Ohlrogge John B. , Barber Eugene D. , Lands William E. M. , Gunstone F. D. , Ismail I. A.
Published: December 2009

The full series of positional isomers of cis-octadecenoate were tested for their suitability in meeting the nutritional requirement for unsaturated fatty acids by mutants of Escherichia coli and Saccharomyces cerevisiae that were unable to synthesize unsaturated fatty acids.Quantitative comparisons of the efficiencies of the various isomers showed a range from 0–48 cells per femtomole for the prokaryotic cells and 0–5 for eukaryotic cells. The Δ5 isomer was much more effective than the Δ6 isomer with the bacterial cells whereas the reverse was true with the yeast cells. In general, isomers containing a cis ethylenic bond between carbons 7 and 12 were able to support extensive growth of either type of mutant.Since all of the various isomers were incorporated into cellular lipids by both types of microorganism, the different efficiencies observed in supporting growth were not a simple reflection of the inability of an acid to be esterified. The differences may reflect the suitability of the resultant esterified product to function as a normal membrane lipid.The contents of various fatty acids in the cellular phospholipids when growth ceases may have a linearly cumulative relationship to the degree of expansion of the acyl chains.
Purification and partial chemical characterization of human pituitary lipolytic hormone
Authors: Chrétien M. , Gilardeau C. , Seidah N. , Lis M.
Published: December 2009

Frozen human pituitary glands contain a lipotropic hormone which is similar to but not identical with ovine, bovine, and porcine beta-lipotropin. Six of the first seven residues from the N-terminus are [Formula: see text] The C-terminal amino acid is a leucine residue.
Comparative biochemistry of the phytopathogenic fungus Helminthosporium. XVI. The production of victoxinine by H. sativum and H. victoriae
Authors: Pringle RossB.
Published: December 2009

The toxic metabolite victoxinine (C17H29NO), considered previously to be a unique product of Helminthosporium victoriae, has been found consistently in culture filtrates of H. sativum strains isolated from a wide variety of sources. This indicates a close relationship between the two phytopathogenic fungi and adds another toxin to be considered in the etiology of diseases incited by H. sativum.
Isolation and carbohydrate composition of glycopeptides of human apo low-density lipoprotein from normal and type II hyperlipoproteinemic subjects
Authors: Lee P. , Breckenridge W. Carl
Published: December 2009

Glycopeptides were prepared from the delipidized protein of low-density lipoprotein (LDL, d = 1.019–1.063) of three normal and three familial heterozygous type II hyperlipoproteinemic (HLP) subjects. The glycopeptides of all subjects were resolved into three groups by gel filtration on Bio-Gel P6 following papain (EC 3.4.22.2) digestion and initial purification on Bio-Gel P2. In normal individuals the component of largest molecular weight (F-1) contained mannose (Man), N-acetyl glucosamine (GlcNAc), galactose (Gal), and N-acetyl neuraminic acid (NANA) in the respective amounts of 45.9 ± 6.7, 37.3 ± 5.9, 28.6 ± 3.4, and 27.0 ± 3.9 nmol/mg original apoprotein. The group of smallest molecular weight (F-3) contained essentially only Man (25.8 ± 1.5 nmol/mg protein) and GlcNac (3.0 ± 0.4 nmol/mg protein) with traces of Gal and NANA. A group of intermediate molecular weight (F-2) exhibited considerable heterogeneity and contained Man, GlcNAc, Gal, and NANA in the amounts of45.9 ± 5.1, 18.3 ± 1.7, 11.0 ± 1.7, and 7.7 ± 1.2 nmol/mg protein. While the major portion of NANA (78%), Gal (71%), and GlcNAc (64%) was present in F-1, approximately 22% of the total Man was in F-3. No major differences were detected in the carbohydrate composition of the three glycopeptide fractions of LDL apoprotein from normal and Type II subjects.
Evidence against the involvement of adenosine 3′,5′-cyclic monophosphate in glucose inhibition of β-galactosidase induction in Bacillus megaterium
Authors: Yeung Kwok-Him , Chaloner-Larssgn Gillian , Yamazaki Hiroshi
Published: December 2009

When Bacillus megaterium cells are grown on D-galactose as the sole carbon source, the cells actively synthesize β-galactosidase (β-D-galactoside galactohydroIase, EC 3.2.1.23). However, D-galactose, when added to a glucose-grown culture, did not induce β-galactosidase, apparently because of the glucose inhibition of the transport of galactose. On the other hand, when glucose was added to a galactose-grown culture, the transport of galactose continued at a reduced but significant rate, whereas further synthesis of β-galactosidase was halted. Adenosine 3′,5′-cyclic monophosphate (cAMP) or guanosine 3′,5′-cyclic monophosphate (cGMP) did not relieve the glucose inhibition of β-galactosidase synthesis in the preinduced culture. A method which gave a reproducible assay of c[32P]AMP in Escherichia coli did not detect cAMP or cGMP in a B. megaterium culture undergoing β-galactosidase induction, but revealed the extracellular accumulation of two unknown phosphorylated compounds. Cell-free extracts prepared from galactose-grown cells did not catalyze the degradation of cAMP or cGMP.
Amino acid sequence of penicillopepsin. II. Isolation and characterization of thermolytic peptides
Authors: Rao Leticia , Hofmann Theo
Published: December 2009

The determination of the amino acid sequences of 70 peptides obtained from a thermoiytic digest of penicillopepsin (EC 3.4.23.7) is described. Fifty-six unique sequences ranging from 2 to 13 amino acids were compiled. Among these was a heptapeptide whose sequence is nearly identical with that of the epoxide-reactive active site peptide of porcine pepsin (EC 3.4.23.1). Considering unrecognized overlaps, a minimum of 272 and a maximum of 293 unique amino acids have been obtained. They account for about 90% of the amino acids of the enzyme.
Binding constants for tetramethylammonium ion determined with irreversible inhibitors of acetylcholinesterase
Authors: Iverson F.
Published: December 2009

The reversible binding constant (Ki) for tetramethylammonium ion (TMA) was determined from the decrease in the bimolecular rate constant (ki) observed with each of 21 organophosphate or carbamate inhibitors of acetylcholinesterase (EC 3.1.1.7). The Ki values obtained were reasonably constant (5.8 × 10−4 ± 0.38 M), and this is consistent with reports indicating that TMA binds to a single site on the enzyme.
Adenosine triphosphate - dependent calcium binding and accumulation by guinea pig cardiac sarcolemma
Authors: St. Louis Patrick J. , Sulakhe Prakash V.
Published: December 2009

Sarcolemma isolated from guinea pig heart ventricles possessed ATP-dependent Ca2+ binding and accumulation (+ oxalate) activities which were not inhibited by sodium azide, oligomycin, or ruthenium red. Ca2+ binding and accumulation by sarcolemma were sensitive to pH, the optimum being about pH 6.8. The concentrations of ATP required for half-maximal binding and accumulation were 94.3 and 172 μM, respectively. Mg2+ up to 5 mM significantly enhanced both activities but was inhibitory at higher concentrations (> 10 mM). Sarcolemmal Ca2+ binding and accumulation were stimulated 100% by K+, half-maximal enhancement occurring at 5–10 mM K+. Ca2+ binding and accumulation were both saturable processes and the respective apparent Km values for Ca2+ were 16.4 and 14.3 μM. Ca2+ binding by sarcolemma was a rapid process and the bound Ca2+ was released upon depletion of ATP in the medium. It is suggested that the sarcolemmal Ca2+ transport system may well be of significance in regulation of the contraction–relaxation cycle of cardiac muscle.
The effect of p,p′-dichlorodiphenyltrichloroethane on levels of guanosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyciie monophosphate in two species of insects
Authors: Bodnaryk Robert P.
Published: December 2009

Within 1 h after topical application of a convulsive dose (4 μg per fly, 47 mg/kg) of p,p′-dichlorodiphenyltrichloroethane (DDT) to the adult male of Sarcophaga bullata Parker, guanosine 3′,5′-cyclic monophosphate (cyclic GMP) levels rose by 71.5% (P < 0.05) in the head, 159.5% (P < 0.01) in the thorax, and 23.4% (P > 0.05) in the abdomen compared to controls. Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) levels were not significantly affected by the DDT treatment. A convulsive dose (100 μg per larva, 250 mg/kg) of DDT applied to larvae of Mamestra configurata Wlk. caused the whole body level of cyclic GMP to rise by 81.6% (P < 0.01) after 1 h, and by 95.9% (P < 0.01) after 3 h. Levels of cyclic AMP were not affected.A hypothesis is advanced suggesting that an abnormally high rate of discharge of acetylcholine (and in the later stages of poisoning, its actual accumulation) at central cholinergic synapses causes cyclic GMP levels to rise, perhaps in post-synaptic cells. The elevated cyclic GMP – cyclic AMP ratio found in DDT-poisoned insects may be of fundamental importance in the complex sequence of events leading to tremor, hyperexcitability, paralysis, and death.
Cholera toxin and adenylate cyclases properties of the activated enzyme in liver plasma membranes
Authors: Franks Douglas J.
Published: December 2009

Adenylate cyclase (EC 4.6.1.1) activity in mouse liver plasma membranes is increased fivefold when animals are pretreated with cholera toxin. The increase in activity is detectable within 20 min of an intravenous injection of the toxin. The response of the control and cholera-toxin-activated adenylate cyclase to hormones, GTP, and NaF is complex. GTP causes the same fold stimulation of control and toxin-activated cyclase, but glucagon and NaF remain the most potent activators of liver adenylate cyclase irrespective of whether the enzyme is activated by cholera toxin.Determination of kinetic parameters of adenylate cyclase indicates that cholera toxin, hormones, and NaF do not change the affinity of the enzyme for ATP–Mg nor do they alter the Ka for free Mg2+. High concentrations of Mg2+ inhibit adenylate cyclase that is stimulated by either cholera toxin, glucagon, or NaF. These same Mg2+ concentrations have no effect on the basal activity of the enzyme or its activity in the presence of GTP.
Some observations on the interaction of dopamine-β-hydroxylase and its natural inhibitors in the rat brain
Authors: Yu P. H. , Boulton A. A. , Wu P. H.
Published: December 2009

The difficulty of assaying dopamine-β-hydroxylase (DβH) (EC 1.14.17.1) has been circumvented by including N-ethylmaleimide (5 × 10−2 M) in the incubation mixture along with a small amount of Cu2+ (20–60 μM). Such a procedure ensures that the maximum DβH activity is assessed in all extracts. As a result, the absolute levels of DβH in various regions of the rat brain have been established. The DβH values obtained are higher by 40% in the hypothalamus and 20% in the cerebral cortex, spinal cord, and caudate nucleus than those previously reported. The mechanism of the synergic effect seen when N-ethylmaleimide and Cu2+ are combined is not presently understood.
The preparation of human hemoglobin α-subunit and a study of its monomer–dimer association
Authors: Terpstra Frank A. , Smith David B.
Published: December 2009

An improved procedure for the isolation of the α-subunit of human hemoglobin is described. The monomer–dimer equilibrium in α-subunit solutions has been studied by boundary analysis in gel filtration, sedimentation velocity, sedimentation equilibrium, and cross-linking with dimethyl adipimidate. A dissociation constant has been determined from the sedimentation equilibrium data. The reaction with haptoglobin of cross-linked α-subunit showed that the dimer fraction would form a stable complex.
The hydrolytic activity of L-ascorbic acid
Authors: Kanfer J. N. , Spielvogel C. H.
Published: December 2009

The ability of L-ascorbic acid to catalyze the liberation of 4-methylumbelliferone from 4-methylumbelliferyl-β-D-N-acetylgiucosaminide, 4-methylumbelliferyl-β-D-glucuronide, 4-methlumbelliferyl-α-D-mannoside, and 4-methylumbelliferyl-β-D-galactoside is documented. There is an apparent metal and oxygen dependency. The cleavage of two lipids was shown in addition to the hydrolysis of these fluorogenic glycosides. Galactose was liberated from galactosyl-6-[3H]ceramide and oleic acid from cholesterol-[1-14C]oleate by L-ascorbic acid under conditions usually used for in vitro incubations. In common with most in vitro systems, only a small percentage of substrate was degraded.
Isoelectric points and molecular weights of salt-extractable ribosomal proteins
Authors: Saleem M , Atkinson Burr
Published: December 2009

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCl were characterized by acid urea–polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67 000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.