bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Requirements of D-Glucose for its Binding to Isolated Human Erythrocyte Membranes
Authors: Kahlenberg Arthur , Dolansky Donna
Published: December 2009

The inhibition by various derivatives of D-glucose of the stereospecific uptake of this sugar by isolated human erythrocyte membranes was determined. Single changes at each of the five hydroxyl groups of D-glucose, except for the 2-deoxy and 6-deoxy derivatives, resulted in a variable decrease in the binding affinity of the sugar which was dependent upon the nature and locus of the alteration. In addition, the presence of the pyranose ring oxygen is required for optimal binding. The largest decrease in affinity effected by a single change of any of the three hydroxyl groups of D-glucose which participate in the binding reaction (i.e. those at carbons 1, 3, and 4) was obtained by the removal or derivatization of the anomeric (C-1) hydroxyl. A similar decrease in binding affinity was obtained upon sulfur substitution of the ring oxygen. The results are interpreted as indicating that the binding of D-glucose to its erythrocyte membrane acceptor site is probably mediated by hydrogen bonds between this membrane component and the hydroxyl groups at carbons 1, 3, and 4 of D-glucose and the pyranose ring oxygen.
Inhibition of Rat Brain Protein and Nucleic Acid Synthesis by Cannabinoids In Vitro
Authors: Jakubovič Alexander , McGeer Patrick L.
Published: December 2009

The effect of Δ9-tetrahydrocannabinol (THC), cannabidiol, and cannabigerol on some metabolic processes in infant and adult rat brain cortex slices was studied in vitro. With L-leucine-U-14C as the tracer substrate, the incorporation of radioactivity into the protein and nucleic acid fractions was significantly inhibited by THC. The oxygen consumption of the slices, the uptake of L-leucine into the slices, and the evolution of 14CO2 were, however, unaffected by THC. Cannabidiol was comparable in activity to THC but cannabigerol was less active. The pattern of inhibition by THC was also observed when the rat brain cortex slices were stimulated by 100 mM K+ or 10 μM protoveratrine. THC also brought about a significant decrease in the incorporation of uridine-2-14C into the nucleic acid fraction of infant and adult rat brain cortex slices. There was a decreased formation of uridine nucleotides in the presence of THC and an increase in uridine and uracil in the low molecular weight fraction. Experiments with THC-2,4-14C established that there was rapid uptake and a concentration of radioactivity in the incubated brain tissue.
Comparison of Liver Glycogen Synthetase between Streptozotocin-Diabetic and Control Rats
Authors: Chang Albert Y.
Published: December 2009

Chronic diabetic rat, induced by streptozotocin, showed high blood glucose but low liver glycogen levels. The diabetic liver had subnormal amounts of glycogen synthetase and its I form when assayed in both 8000 × g supernatant and glycogen pellet suspensions. The pellet suspension of the control liver retained 68% synthetase I activity; the diabetic liver retained only 19.5%. The addition of deproteinized liver extract to the pellet suspension increased the activity of synthetase either in the presence or absence of glucose-6-PO4 for both groups of animals to different degrees. The deproteinized liver extract of the diabetic rat contained little ATP, less glucose-6-PO4, and slightly more inorganic phosphate than that of the control rat. The significance of these observations is discussed.
Isolation of the Bromouracil-Substituted Light Strand of Mouse Satellite DNA
Authors: Walker I. G. , Ewart D. F. , Chan A.
Published: December 2009

Mouse L-cells were grown in medium containing bromodeoxyuridine and fluorodeoxyuridine, and the DNA from these cells was centrifuged to equilibrium in alkaline cesium chloride. The bulk of the bromouracil-substituted DNA had a buoyant density lying between 1.82 and 1.85 g/cc. About 4–5% of bromouracil-substituted DNA was observed as a distinct peak with a buoyant density of 1.79 g/cc. After this DNA was purified by recentrifuging, nucleotide analysis revealed an asymmetric composition in which the base-pairing rules for double-stranded DNA were not obeyed. Comparison of the nucleotide composition with published data indicated that the bromouracil-substituted light strand of mouse satellite DNA had been isolated.
Cyanogen Bromide Fragments of Rabbit Skeletal Tropomyosin
Authors: Hodges R. S. , Smillie L. B.
Published: December 2009

Previous studies have demonstrated that rabbit skeletal tropomyosin consists of two or more chemically non-identical but highly homologous polypeptide chains. Attempts by a variety of techniques to prepare pure tropomyosin chains in amounts adequate for chemical characterization have been unsuccessful to date. To provide more extensive information for the purpose of elucidating the relationship between amino acid sequence and the coiled-coil structure of tropomyosin, a cyanogen bromide treatment of the S-carboxymethylated protein was carried out. The fragments were separated into small and large components by gel filtration on Sephadex G-50. The small fragments were fractionated by ion-exchange chromatography and electrophoresis on paper and their sequences elucidated by conventional methods. Coupled with previous data, these results indicate a minimum of seven unique methionine sequences and are consistent with a high degree of homology in the tropomyosin polypeptide chains. From the mixture of the larger cyanogen bromide polypeptides, a fragment was isolated by ion-exchange chromatography on QAE-Sephadex. In aqueous buffer it had a molecular weight of 35 000 and an α-helical content of about 60% as estimated by circular dichroism. In 8 M urea its molecular weight was reduced to 15 000, a value in reasonable agreement with a minimal molecular weight of 17 000 calculated from its amino acid composition. From its histidine content (two residues) and the known COOH-terminal amino acid sequence of the protein, the fragment was concluded to be derived from the COOH-terminal half of the molecule. These results are consistent with a degree of 'coiled-coil' structure in a fragment representing about one-half of the tropomyosin molecule.
Binding and Uptake of Amino Acids by Brain Tissue
Authors: Shiu P. C. , Elliott K. A. C.
Published: December 2009

(1) Of the endogenous glutamate, aspartate, alanine, glycine, and γ-aminobutyrate (GABA), 26–37% remains bound in particles when rat brain is homogenized in isosmotic salt-free sucrose solution; a smaller proportion, 16%, of the glutamine is bound. The amounts bound are increased if sodium chloride is present; the percentage increase is greatest in the case of GABA, followed by glutamate, and least with glutamine. When tracer amounts of radioactive amino acids are present in the solution in the absence of salt very little radioactivity appears in bound GABA or glutamine, but appreciable amounts are found in the other amino acids. In the presence of sodium chloride, the total amount of bound amino acid increases as does, to a lesser extent, the radioactivity bound. Ouabain and protoveratrine seem to cause some release of sodium-dependent binding of the amino acids; this is most marked with GABA.(2) Slices incubated in the presence of oxygen and glucose take up each of the amino acids when these are added to the incubation medium. The highest intracellular concentration and the greatest net uptake occur with GABA. The endogenous concentration of glutamate is higher than that of the other amino acids but the net uptake is the least. The highest ratios of uptake to endogenous content occur with alanine and glycine. Determinations of radioactivity indicate that, in the cases of GABA and glycine, the increase in radioactivity in the slices is almost completely accounted for by uptake from the medium with almost no exchange. Some exchange occurs with other amino acids. Protoveratrine inhibits uptake of all the amino acids and actually causes loss of glutamate and aspartate from slices. Ouabain inhibits in all cases; the uptakes of glutamate and aspartate are least affected. Tetrodotoxin, alone or with either of the other two drugs, tends to increase uptake of all the amino acids. When the net uptake is inhibited by drugs considerable exchange of endogenous amino acids with radioactive amino acids in the medium is observed.
Physical Reconstitution of 23 S RNA – 50 S Protein Complexes from Escherichia coli
Authors: Chu F. K. , Maeba P. Y.
Published: December 2009

The formation of ribosomal complexes in a system containing 50 S ribosomal proteins and ribosomal RNA (5, 16, and 23 S RNA's) is reported. Binding of 50 S proteins to 23 S RNA resulted in reconstituted particles that sedimented at 28, 32, 43, and 48 S. The process was highly influenced by temperature and indicated at least three temperature-dependent steps. The 28 S particle contained 14–20 proteins and formation of the largest particles was accompanied by the sequential binding of at least six to seven more proteins. The formation of 28 and 32 S complexes was independent of 5 and 16 S RNA's, as well as 30 S proteins or 30 S subunits. The failure to form functional 50 S subunits is discussed.
Purification of Superoxide Dismutase From Fungi and Characterization of the Reaction of the Enzyme with Catechols by Electron Spin Resonance Spectroscopy
Authors: Rapp Ursula , Adams Wyman C. , Miller Richard W.
Published: December 2009

Superoxide dismutase catalyzes the conversion of the single electron reduced species of molecular oxygen to hydrogen peroxide and oxygen. The widely distributed copper enzyme has been purified from two fungi. Identical chromatographic and electrophoretic behavior of the enzyme isolated from different sources indicates great similarity in the molecular properties of the enzyme from eucaryotic organisms. A photosensitized recording assay procedure for the enzyme was developed which elminates the use of a second enzyme system for generating the substrate, superoxide anion. Kinetic data indicate that the reaction between enzyme and superoxide anion shows a logarithmic dependence on concentration under the conditions of the method.The fungal enzyme contains 2 mol of zinc and 2 mol of copper per mole of holoenzyme. The reaction between the enzyme-bound copper and several catechol derivatives has been examined through the use of electron spin resonance spectroscopy. It was concluded from these studies that these compounds reduce a portion of the copper to the cuprous form and also may form a complex with enzyme copper. The substituted catechols are oxidized noncatalytically by the enzyme to the semiquinone forms in the presence of oxygen. Much higher concentrations of the semiquinones are formed in the presence of the enzyme than are possible with oxygen alone at pH 8. Catechols also react readily with superoxide anion. Because of the complex relationships between catechols, superoxide anion, and superoxide dismutase, it is difficult to assess the effect of catechols on the catalytic activity of the enzyme.
Estimation of p-Hydroxyphenylpyruvic Acid Hydroxylase Activity Based on 14CO2 Evolution from the 1-14C-labelled Substrate
Authors: Raheja M. C. , Dylewski I. , Crawhall J. C.
Published: December 2009

The rate of evolution of 14CO2 has been measured in a modified assay for the enzyme p-hydroxyphenylpyruvic acid oxidase, utilizing chemically synthesized p-hydroxyphenyl[1-14C]-pyruvic acid. The characteristics of the enzyme system with respect to substrate concentration, protein concentration, and the ionic environment have been studied. Short incubation periods and a short post-acidification shaking period for the collection of the 14CO2 into Hyamine gave optimum conditions for the assay, and reduced the amount of nonenzymic decarboxylation of the substrate to a minimum. This assay has an increased sensitivity when compared with previous analytical methods and has enabled the Km of the enzyme to be determined (2.3 × 10−5 M) with lower substrate concentrations than those that give rise to substrate inhibition (from 50 μM). The enzyme activity of liver and kidney of the human and rat have been determined. No enzyme activity could be observed in human leukocytes and cultured fibroblasts. o-Hydroxybenzaldehyde was found to be a powerful inhibitor of the enzyme.
Some Physicochemical and Chemical Properties of Light Chains Isolated from Rabbit Cardiac Myosin
Authors: Mani R. S. , Kay C. M.
Published: December 2009

Cardiac light chains released from myosin at alkaline pH were isolated by ammonium sulfate fractionation. The isolated light chains were separated on a diethylaminoethylcellulose column using a phosphate gradient. Sodium dodecyl sulfate acrylamide gel electrophoresis indicated the presence of two light chains, LC1 and LC2 of molecular weight 27 000 and 18 000, respectively. Densitometer traces of the gels gave a molar ratio of 2 to 1 for the two respective protein components. Molecular weights of 24 000 ± 1000 and 16 000 ± 500 were obtained for these two components by high-speed sedimentation equilibrium studies. Amino acid analyses of the two fractions revealed a high ratio of phenylalanine to tyrosine (4.0 and 6.7 for LC1 and LC2, respectively), which also dominates the aromatic circular dichroic (CD.) spectrum. The C.D. spectra of LC1 and LC2 exhibited ellipticity bands at 220 and 206.5 nm, positions typical of the α-helical conformation, but the low ellipticity values at these positions indicated that only a small portion of their polypeptide chains are in the α-helical conformation consistent with their large content of proline.
Structural Analysis of an Acidic Polysaccharide from Tremella mesenterica NRRL Y-6158
Authors: Fraser C. G. , Jennings H. J. , Moyna P.
Published: December 2009

An acidic polysaccharide has been isolated from the culture medium of T. mesenterica NRRL Y-6158. The heteropolymer contained D-xylose, D-mannose, D-glucuronic acid, and O-acetyl in the molar ratios of 7:5:1:0.7, respectively. Methylation analysis of the heteropolymer indicated that it was essentially a 1 → 3-α-linked mannopyranose backbone having approximately 80% of the backbone units substituted, thus forming a very highly branched structure. The substituents on the backbone were found to be D-glucopyranosyluronic acid end-group, β-linked to the O-2 positions of the mannopyranose units, and 2-O-β-D-linked xylopyranose side-chains, linked both to the O-2 and O-4 positions of the mannopyranose backbone. The methylation analysis suggests that these side-chains are probably two or three xylopyranose units long, although a limited variation in the length of the side-chains is a possibility.
Affinity Chromatography of Phenylalanine: tRNA Ligase
Authors: Forrester P. I. , Hancock R. L.
Published: December 2009

L-Phenylalanine: tRNA ligase from E. coli was successfully isolated by affinity chromatography on a column of Sepharose-phenylalanine. The phenylalanine was separated from the Sepharose backbone by two molecules of ε-aminocaproic acid to ensure that there was a minimum of steric interference between the enzyme and the solid matrix.
Effect of Relative Centrifugal Force on the Apparent Size of DNA from Escherichia coli Cells
Authors: Myers D. K. , Johnson L. D. , Chetty K. G.
Published: December 2009

Sedimentation coefficients were determined for the DNA from E. coli cells that had been lysed on top of alkaline sucrose gradients and some of the factors affecting the apparent size of the DNA were explored. Parallel experiments were carried out with the DNA from lysed T4 phage as a standard marker. The apparent size of single-stranded DNA from E. coli is strongly dependent on relative centrifugal force; regardless of the initial size, whether 50 or 140 S, all preparations sedimented at the same rate at high rotor speeds. The size of T4 phage DNA appeared to be too small to show an equally marked dependence of sedimentation coefficient on rotor speed under the conditions tested.Cells washed with EDTA solutions yielded DNA of a smaller size, while cells lysed on alkaline 25–40% sucrose gradients containing 1 M sodium ion yielded DNA with very high sedimentation coefficients.
Specific Immunosuppression of Antibody Response to Ox Insulin: Effect of Adjuvant and Dosage of Maleyl Insulin
Authors: Moloney Peter J. , Jackson Susan G.
Published: December 2009

Significant suppression of humoral antibody production to ox insulin has been induced in adult guinea pigs by the injection of multiple doses of maleyl insulin with incomplete Freund's adjuvant (IFA) in the dose range of 0.49–39.33 μg protein nitrogen (N), and by single doses in the dose range 10–200 μg N. The patterns of immunosuppression induced were similar whether multiple or single doses of maleyl insulin were used.Ox insulin with IFA in doses of 10 and 30 μg N did not induce immunosuppression.Immunosuppression was not induced by maleyl insulin with Al(OH)3 or by maleyl insulin or IFA alone.
Biosynthesis of Cerebral Phenolic Amines. II. In Vivo Regional Formation of p-Tyramine and Octopamine from Tyrosine and Dopamine
Authors: Boulton Alan A. , Wu P. H.
Published: December 2009

In rats pretreated with pargyline the highest cerebral concentration of 14C-labeled p-tyramine and octopamine following an intraventricular injection of tyrosine-14C was found in the hypothalamus. In the case of dopamine-14C the maximum formation was in the hypothalamus and brainstem. In the absence of pargyline only very small quantities of these amines were found in some regions. Following injection of p-tyramine-3H, β-hydroxylation to octopamine-3H occurred in all regions, the most active being the hypothalamus. Unchanged p-tyramine was most actively concentrated in the caudate nucleus. The formation and localization of p-tyramine and octopamine from their respective precursors tyrosine, dopamine, and p-tyramine itself are discussed with regard to the endogenous distribution of p-tyramine and certain of the enzymes involved in the synthesis and metabolism of these phenolic amines.
Carbon-13 Chemical Shifts Accompanying Helix Formation
Authors: Lyerla Jr. James R. , Barber Brian H. , Freedman Murray H.
Published: December 2009

The helix–coil transition in an aqueous solution of poly-L-glutamic acid has been observed using 13C nuclear magnetic resonance. The transition from coil to helix results in pronounced deshielding of the carbonyl and C-α carbons of the polymer and a less dramatic shielding effect on the C-β carbon. These results are contrasted with the titration behavior of L-glutamic acid to delineate those shifts directly attributed to the secondary-structure change. The biological significance of this observation is supported by the 13C chemical shift differences observed for the 13C-enriched phenylalanyl residue 8 in the free 1–15 amino-terminal peptide of ribonuclease (RNase) and the 1–15 peptide:RNase S-protein complex.
The Electron Paramagnetic Resonance Spectrum of Ferricytochrome c and Lysine-Modified Derivatives at Alkaline pH
Authors: Morton R. A.
Published: December 2009

The heme-associated, pH-induced transition of ferricytochrome c and two lysine-modified derivatives was investigated by absorption and electron paramagnetic resonance (E.P.R.) spectroscopy. The transition from type III to type IV ferricytochrome c (as defined by Theorell, H., and Åkesson, Å.: J. Am. Chem. Soc. 63, 1812 (1941)) produced a new E.P.R. spectrum in frozen solution measured at liquid nitrogen temperature. The measured g values were 3.2 and 2.1 (9.16 GHz). The third component of the expected set of three principal g values for low-spin type IV ferricytochrome c was not observed in this study. In two derivatives with modified lysyl residues, trifluoroacetylated and guanidinated ferricytochrome c, the type III to type IV transition was absent, and instead, at alkaline pH, a brown–red form was produced which had a strong absorption band at about 605 nm. The apparent pK's were 10.3 (trifluoroacetylated) and 9.4 (guanidinated). When an alkaline solution of the guanidinated ferricytochrome was frozen, the E.P.R. spectrum had a set of sharp lines (g values 2.79, 2.21, and 1.76) similar to those observed from low-spin type V ferricytochrome c (pH 14). The alkaline trifluoroacetylated protein gave a similar set of signals but in addition contained the signals observed at neutral pH. These results were interpreted by assuming that, at alkaline pH, a hydroxide ion displaced one of the normally coordinated iron ligands with a temperature-dependent equilibrium between high-spin and low-spin states. Freezing the solution to liquid nitrogen temperature shifted the equilibrium to the low-spin form, and, for the trifluoroacetylated derivative, led to partial recovery of the coordination structure present at about pH 7. The E.P.R. spectrum of the guanidinated cytochrome at neutral pH indicated that the iron electronic structure was essentially identical to type III ferricytochrome c. In contrast, the E.P.R. spectrum of the trifluoroacetylated protein was significantly different, but whether the coordination structure has been geometrically distorted or chemically changed remains to be determined.
The Degradation of Newly Synthesized RNA in Ehrlich Ascites Tumor Cells
Authors: von Tigerstrom Richard G.
Published: December 2009

The stability of rapidly labelled RNA in Ehrlich ascites tumor cells under in vitro conditions was investigated. [2-14C]Uridine was effectively incorporated into RNA and 90% of the acid-insoluble isotope was associated with the nucleus after 30 min labelling. When RNA synthesis was inhibited by actinomycin D or when [14C]uridine was chased with [12C]uridine at this time, a rapid degradation of approximately 50% of the labelled RNA could be observed. The rates of degradation, the extent of degradation under certain incubation conditions, and the classes of RNA from which the isotope was released were almost identical when the two chase techniques were compared. The concentration of uridine used during the chase did not inhibit RNA synthesis significantly as judged from incorporation of [8-14C]guanine. The results indicated that the degradation of the newly synthesized RNA in Ehrlich ascites cells occurred naturally and was not induced by actinomycin D.
The Amino Acid Sequence of the N-Terminal Region of Aldolase
Authors: Anderson P. J. , Gibson D.
Published: December 2009

The amino acid sequence of the N-terminal 39 residues of sturgeon muscle aldolase has been determined by a combination of automated and manual sequencing procedures. In addition, the sequence of the N-terminal 19 residues of rabbit muscle aldolase has been determined using a sequenator. It was apparent from these studies that aldolase primary structure is highly conserved in this region.
Adenosine Aminohydrolase from Halobacterium cutirubrum
Authors: Bauer Randy J. , Carlberg David M.
Published: December 2009

The extreme halophilic bacterium Halobacterium cutirubrum was examined for base, nucleoside, and nucleotide aminohydrolase activity on pyrimidine bases and their nucleosides and nucleotides. Only adenosine aminohydrolase activity was demonstrated. The enzyme was extracted from the cells and purified about 70-fold by liquid polymer two-phase fractionation, ammonium sulfate precipitation, and gel filtration. The enzyme has an apparent Km for adenosine of 4 × 10−5 M. The optimum pH is 7.5. The enzyme requires 4.0 M NaCl for maximal activity, but is irreversibly inactivated regardless of the concentration of NaCl when stored for extended periods of time.
Capacité de la méthionine, de la thiamine ou du pantothénate à renverser la toxicité, pour Escherichia coli, d'acides α-aminés artificiels homologues, dont la norleucine; mise en evidence du rôle probable de la méthionine dans la biosynthèse des deux vitamines
Authors: Planet G. , Abshire C. J.
Published: December 2009

We have previously made the chemical synthesis of artificial α-amino acids substituted by one or two alkylated linear groups on the α-carbon. Our present results indicate that they have a capacity to stop the growth of E. coli 9723. When they have toxicity, the inhibition is competitively reversed by L-methionine and noncompetitively by pantothenate or thiamine; these compounds act as methionine analogues. We have concluded that the mechanism of toxicity is due in part to the repression of biosynthetic enzymes of the methionine pathway and partly to the inhibition of homoserine-O-transsuccinylase, which is the first enzyme of this pathway. We think that the consequence is an intracellular deficiency of methionine which, in turn, causes a lack of pantothenate and thiamine. Our results, therefore, indicate that methionine is necessary for the biosynthesis of pantothenate and thiamine.
Studies on the Biosynthesis of Flavin Nucleotides from 2-14C-Riboflavin by Rat Liver and Kidney
Authors: Fazekas A. G. , Sandor T.
Published: December 2009

2-14C-Riboflavin was injected subcutaneously into young adult rats to study the biosynthesis of flavin mononucleotide (FMN) and flavin–adenine dinucleotide (FAD) in the liver and kidneys. Animals were sacrificed at different time intervals following the administration of labelled riboflavin (RF), and radioactive flavins were determined in their tissues by a newly devised method. Both tissues accumulated radioactive riboflavin rapidly and peak levels were obtained at 90 min after the injection, when over 80% of the total radioactivity of the liver was present in FAD. At this time the liver contained 17% of the injected dose of 2-14C-RF. The kidneys contained relatively high quantities of free RF due to the concentration and urinary excretion of the vitamin.Analysis of subcellular fractions of the liver of animals injected with 2-14C-RF revealed that most of the radioactivity was present in mitochondria and nuclei. The flavin nucleotides of rat liver cytosol became progressively associated with macromolecules in vivo. However, there was no significant binding of free RF by macromolecules in blood plasma or liver cytosol.Kinetic studies and incubations with liver slices indicated that RF freely diffuses into the liver cells, is rapidly converted into FAD, and becomes attached to apoenzymes. The tissue uptake of RF and FMN formation is considerably influenced by the concentration of RF present in the system, both in vivo and in vitro.
Rhizopus Acid Proteinases (Rhizopus-pepsins): Properties and Homology with Other Acid Proteinases
Authors: Graham J. E. S. , Šodek J. , Hofmann T.
Published: December 2009

Commercial acid proteinase from Rhizopus chinensis has been further purified by isoelectric focussing. Two forms of the enzyme (I and II) with very similar properties have been obtained. Like other fungal acid proteinases they activate trypsinogen at pH 3.4 and are inhibited by diazoacetyl norleucine methyl ester. Because of the lability of the label it has not been possible to isolate the active site peptide from a peptic digest. N-terminal amino acid sequences were determined for Rhizopus enzyme I (NH2∙Ala∙Gly∙Val∙Gly∙Thr∙Val∙Pro∙Asx∙Thr), for Rhizopus enzyme II (NH2∙Ala∙Gly∙Val∙Gly∙Thr∙Val∙Pro), and for penicillopepsin (NH2∙Ala∙Ala∙Ser∙Gly∙Val∙Ala∙Thr∙Asn∙Thr∙Pro∙Thr). The similarity in enzymic properties and in the sequence suggests that the Rhizopus enzymes are homologous with penicillopepsin and hence also with mammalian pepsins and calf chymosin.
Effects of Divalent Cations on 5′-Mononucleotidase of Bovine Cerebral Cortex
Authors: Tanaka Ryo , Teruya A. , Morita H.
Published: December 2009

A 5′-mononucleotidase preparation partially purified from bovine cerebral cortex was investigated with regard to its interaction with divalent metals. Magnesium, manganese, and zinc ions partly restored the activity lost by either acid or alkali treatment. Prolonged dialysis against an EDTA solution also eliminated the activity, which was restored by magnesium ions. The alkali-treated nucleotidase incorporated a larger amount of radioactive manganese than the untreated one did. The extra manganese incorporated by the treated nucleotidase roughly corresponded to the amount needed to bind to the enzyme molecule in a one-to-one ratio. The Km for magnesium (1.5 × 10−5 M) was independent of the type of substrate and was much lower than those for the substrates; the latter Km's were independent of the magnesium concentration. The facts suggest that the metal binds to the enzyme independently of the substrates. The inhibition patterns by nucleoside triphosphates were sigmoidal, and similar patterns were obtained with inorganic polyphosphates and chelators. The inhibition was a noncompetitive type, and the activity partially lowered by EDTA was not affected by ATP. The sigmoidal nature of the nucleotide inhibition, therefore, could be accounted for mainly by the removal of necessary metals for the enzyme activity instead of a cooperative mechanism.
Activity and Positional Specificity of a Rat Plasma Phospholipase on Phosphatidylethanolamine
Authors: Osmond D. H. , Ross L. J. , Holub B. J.
Published: December 2009

Lysophosphatidylethanolamine (LPE), rich in arachidonic acid, has been proposed by others as a physiologically important inhibitor of the renal enzyme renin that circulates in blood. Intravenously infused phosphatidylethanolamine (PE) has proposedly inhibited the acute pressor effect of injected renin in rats, suggesting a rapid conversion of PE to arachidonate-rich LPE by blood phospholipase activity. Therefore a definition of the activity was attempted. Incubation at 37 °C, pH 7.7–7.9, of a highly purified, 32P-labeled rat liver PE added to fresh rat plasma (heparin) in diethyl ether (0.2 ml/ml plasma) or sodium deoxycholate solution (5 mg/ml plasma) indicated that one phospholipase was dominant, and its activity greater when deoxycholate, rather than ether, was present. Lysophospholipase activity was negligible in either case. Two milliliters of plasma (with deoxycholate) deacylated 47% of 1.5 mg PE (approximately 2 μmol) during 24 min of incubation. Fresh serum had higher activity than corresponding plasma, suggesting activation associated with blood coagulation. C-2 specificity was demonstrated by selective removal of the label from 2-[1-C14]arachidonoyl rat liver PE, without concurrent cholesterol esterification, and by virtually identical compositions (gas–liquid chromatography) of fatty acids removed from PE by plasma or by C-2 specific snake venom phospholipase (Crotalus atrox). Our data appear to rule out C-1 specific post-heparin phospholipase and also C-2 specific lecithin:cholesterol acyltransferase (LCAT), and we present a plasma phospholipase A2 that is highly active against PE and creates an arachidonate-poor LPE, presumably not a renin inhibitor.
Chromatography of Drosophila tRNA on BD-cellolose
Authors: White Bradley N. , Tener G. M.
Published: December 2009

Conditions for the amino acylation of Drosophila melanogaster tRNA with the 20 14C-labelled amino acids were established. Crude tRNA prepared from adult flies of wild-type D. melanogaster was chromatographed on BD-cellulose columns, and the fractions were assayed for amino acid acceptor activity. The elution profiles of the tRNAs are presented. The crude tRNA was chromatographed on a Sephadex G-100 column to isolate the 5 S RNA, which constituted 9% of this material. The 5 S RNA was further chromatographed on BD-cellulose and was resolved into one major and two minor peaks. The nucleotide composition of these peaks was determined by RNase T2 digestion and two-dimensional thin-layer chromatography.
Effects of Elevated Intracellular ATP and GTP Concentrations on Purine Ribonucleotide Synthesis and Interconversion
Authors: Snyder Floyd F. , Henderson J. Frank
Published: December 2009

The effects of elevated intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and interconversion were examined in intact Ehrlich ascites tumor cells. Elevated ATP and GTP concentrations inhibit nucleotide synthesis from purine bases up to 50% and elevated concentrations of GTP severely inhibit IMP dehydrogenase (60%). In contrast, adenylate deaminase activity was inhibited 16% at the highest concentration of GTP studied and elevated ATP concentrations had only a slight inhibitory effect on adenylosuccinate synthetase plus lyase (9%).
Reduction and Reactivation of the Bowman-Birk Soybean Inhibitor
Authors: Hogle James M. , Liener Irvin E.
Published: December 2009

Experimental conditions were established whereby it was possible to control the extent of reduction of the disulfide bonds of the Bowman-Birk soybean inhibitor (BBI) with sodium borohydride. Losses in the antitryptic and antichymotryptic activities of BBI were directly dependent on the average number of disulfide bonds which had been reduced. Both activities were completely lost upon the reduction of an average of four out of the seven disulfide bonds of BBI. Disc gel electrophoresis of partially reduced, carboxymethylated preparations of BBI revealed a complex mixture of the native and fully reduced protein as well as partially reduced intermediates present in varying proportions depending on the state of reduction. It was possible to regenerate the activity of a solution of fully reduced BBI by dilution with a disulfide–thiol redox buffer similar to that which had been employed by Saxena and Wetlaufer (Biochemistry, 9, 5015, 1970) for the reactivation of reduced lysozyme. It was concluded that BBI probably has four reducible disulfide bonds which are essential for inhibitory activity against trypsin as well as chymotrypsin.
Role of 2,4-Dichlorophenol in the Oxidation of Estradiol by Uterine and Horseradish Peroxidase
Authors: Lyttle C. R. , McNabb T. , Jellinck P. H.
Published: December 2009

The role of 2,4-dichlorophenol in enhancing the conversion of [4-14C]estradiol to water-soluble products by a uterine preparation in the presence of hydrogen peroxide has been investigated. The addition of this phenol to a solution of uterine or horseradish peroxidase in 8 M urea restored the activity of the enzyme and also that of horseradish peroxidase inactivated by heating. It also protected the enzyme from inactivation during incubation. It is proposed that 2,4-dichlorophenol exerts its effect by activating peroxidase and protecting the enzyme from inactivation by the products of the reaction.
The Cleavage of Adenylosuccinate and 5-Amino-4-imidazole-N-succino-carboxamide Ribonucleotide by an Adenylosuccinate Lyase from Ehrlich Ascites Tumor Cells
Authors: Brox L. W.
Published: December 2009

Adenylosuccinate lyase (EC 4.3.2.2), which catalyzes the conversion of either adenylosuccinate (AMPS) or 5-amino-4-imidazole-N-succino-carboxamide ribonucleotide (SAICAR) to fumarate and either AMP or 5-amino-4-imidazole carboxamide ribonucleotide (AICAR), was purified in excess of 80-fold by a one-step procedure involving column chromatography on phosphocellulose. The AMPS and SAICAR were prepared enzymatically from either AMP or AICAR. The Michaelis constants were 8.4 μM and 10.5 μM for AMPS and SAICAR, respectively. Product inhibition experiments were consistent with an ordered Uni Bi kinetic model with fumarate being the first product released.
Selective Simultaneous Proton Magnetic Resonance Labelling of Hydrophilic and Hydrophobic Regions of Phospholipid Vesicles. Effect of Polar and Apolar Reagents
Authors: Nogrady Thomas
Published: December 2009

Methyl-12-dimethylsilastearate was synthesized and incorporated by sonication into lecithin vesicles. In the resulting mixed vesicle the nuclear magnetic resonance line width of the Si-methyl resonance line is proportional to the lecithin–silastearate ratio, indicating interaction between the hydrocarbon chains of the phospholipid and the labelled fatty acid ester reporter molecule. The Si-methyl line reports selectively perturbations of the structure of hydrophobic regions as shown by addition of hydrocarbons. The N-methyl resonance line of lecithin is not affected by these changes, but indicates selectively effects on the hydrophilic exterior of the micelle produced by salts. The mixed vesicle may therefore be used as a membrane model allowing simultaneous observation of effects on the exterior and interior of the lipid bilayer.The silastearate label was successfully incorporated into Acholeplasma laidlawii membrane, which however failed to give a sufficiently resolved nuclear magnetic resonance spectrum.
Goblet Cell Mucin of Rat Small Intestine. Chemical and Physical Characterization
Authors: Forstner Janet F. , Jabbal Inderjit , Forstner Gordon G.
Published: December 2009

Goblet cell mucin (GCM) of rat small intestine has been isolated previously, and its location established by immunofluorescence techniques. In the present study, GCM was characterized more fully by chemical and physical methods. It was found to be a flexible negatively charged macromolecule with a molecular weight of about 2.0 × 106, an intrinsic viscosity of 15.3 dl/g, and an axial ratio of about 225:1. Its composition was protein 12%, total hexose 23%, hexosamine 22.4%, sialic acid 10%, fucose 6.6%, and sulfate < 1% of the dry weight. It contained approximately 34 disulfide bonds per molecule. Like most mucin glycoproteins it was rich in serine, threonine, and proline (45.5 mol %) and poor in hydrophobic and sulfur containing amino acids. One major and two minor components were identified by acrylamide disc gel electrophoresis and analytic ultracentrifugation. The components appeared to represent different molecular weight species of GCM. No evidence of subunit structure could be obtained using a variety of techniques, including the disruption of ionic, hydrophobic, and disulfide bonds by detergents, denaturants, or reducing agents.
Changes in Acidic Chromatin Proteins During Mammary Cell Differentiation
Authors: Kadohama Nobuyuki , Turkington Roger W.
Published: December 2009

Chromatin was prepared from mouse mammary epithelial cell nuclei and histones were removed by extraction with 2.0 M NaCl, pH 6.0. Electrophoresis of the residual acidic chromatin proteins in polyacrylamide gels containing sodium dodecyl sulfate revealed a heterogeneous banding pattern which was tissue specific. Six or more bands among the proteins present in differentiated, lactational cells were not detectable in undifferentiated, virginal cells.In organ culture studies insulin stimulated the incorporation of 3H-tryptophan or 3H-aspartic acid and 3H-leucine into all electrophoretic components of the mammary acidic chromatin protein. Two peak rates of synthesis, preceding and following the peak rate of DNA synthesis, were observed. Synthesis was unaffected by hydrocortisone or prolactin. Synthesis of the six bands characteristic of the lactational cells was initially undetectable in undifferentiated, virginal cells, but was induced after incubation with insulin for 42 h. These results, taken in correlation with previous studies on this system, are consistent with a proposed model in which new species of acidic chromatin proteins may participate in chromatin reconstitution during mammary cell differentiation.
Synthesis of Glycerol-1,3-diphosphonic Acid
Authors: Robinson Ranga , Baer Erich
Published: December 2009

The synthesis of glycerol-1,3-diphosphonic acid, the phosphonic acid analogue of the naturally occurring glycerol-1,3-diphosphoric acid, is described. The phosphonic acid analogue was obtained in an overall yield of 29.7%, by heating a mixture of 1,3-dibromo-2-hexadecanoyloxypropane and triethyl phosphite to 140° for 48 h, distilling off in vacuo the excess of triethyl phosphite, hydrolyzing the condensation product, 1,3-bis(diethylphosphono)-2-hexadecanoyloxypropane, with 2 N hydrobromic acid at 100° for 5 h, and isolating the glycerol-1,3-diphosphonic acid in form of the dibarium salt. The glycerol-1,3-diphosphonate differs from glycerol-1,3-diphosphate and other phosphate esters of glycerol and glyceric acid by its greater stability towards acid hydrolysis. The glycerol-1,3-diphosphonic acid is readily distinguished from glycerol-1,3-diphosphoric acid and glycerol-1,2-diphosphoric acid by paper chromatography.
Structural Studies on Antigen E of Ragweed Pollen
Authors: Griffiths B. W.
Published: December 2009

The analysis of antigen E of ragweed pollen by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) resulted in the demonstration of two subunits of molecular weights of about 20 000 and 14 500. These results confirm earlier studies on antigen E by gel chromatography that demonstrated a two-polypeptide subunit composition. Treatment of antigen E with 1% SDS (in the absence of reducing agent) resulted in the dissociation of the two polypeptide chains, which indicates that the bonds bridging the subunits are non-covalent in nature.
Some Properties of a DNA -Dependent RNA Polymerase from Halobacterium cutirubrum
Authors: Chazan L. L. , Bayley S. T.
Published: December 2009

A DNA–protein–membrane complex, which incorporated ribonucleoside triphosphates (rNTP's) into a cold trichloracetic acid insoluble product, was isolated from an homogenate of the extremely halophilic Halobacterium cutirubrum by high speed centrifugation. After digestion with DNase and chromatography on a Sephadex column, the complex yielded an enzyme preparation which incorporated rNTP's in the presence of added DNA. Incorporation by both the complex and the enzyme preparation depended on all four rNTP's and was sensitive to DNase and RNase. Nearest-neighbor analyses showed that the product from the partially purified enzyme contained all four rNTP's in roughly equal proportion. The enzyme was inhibited by actinomycin D and acriflavin but not by rifampicin or streptovaricin. It showed no specificity for DNA templates. From Sephadex chromatography and centrifugation on glycerol gradients, its molecular weight was estimated to be roughly 300 000 – 400 000 daltons.For incorporation, the optimum Mg2+ concentration was 40 mM. The complex was active at all concentrations of KCl or NH4Cl up to saturation, but the partially purified enzyme was active only below 0.4 M. The lack of activity at higher concentrations was not due to irreversible denaturation of the enzyme but concentrated salt may have inhibited the reaction by altering the configuration of the DNA template.
The Enzymic Phosphorylation of N6-(Δ2-Isopentenyl)adenosine in Chick Liver Homogenates
Authors: McLennan B. D. , Pater A.
Published: December 2009

Enzyme preparations from chick kidney and liver rapidly phosphorylate the modified nucleoside, N6-(Δ2-isopentenyl) adenosine. The resulting metabolite behaves electrophoretically as a mononucleotide and is quantitatively hydrolyzed to the nucleoside by 5′-nucleotidase. The isopentenyl side chain is not altered in the phosphorylated derivative. It is concluded that the metabolite is N6-(Δ2-isopentenyl) adenosine-5′-monophosphate. The properties of the enzyme activity responsible for the phosphorylation resemble the properties of adenosine kinase isolated from sarcoma-180 cells, and from rabbit liver, with respect to pH optimum, Km value, and sensitivity to magnesium ion and ATP. Thus, it seems probable that the phosphorylation observed in the chick tissue preparations is due to adenosine kinase. The mononucleotide could not be further phosphorylated in vitro by adenylate kinase. This observation, coupled with the fact that isopentenyladenosine is biosynthesized at the macromolecular level, suggests that the 5′-monophosphate ester may be the form through which some of the biological effects of this modified nucleoside are manifested.A major end product of N6-(Δ2-isopentenyl) adenosine catabolism in chick liver and kidney homogenates is uric acid.
Kinetic Studies on Antibody–Hapten Reactions. II. Reactions with Antibodies and their Polypeptide Chains
Authors: Kelly K. A. , Sehon A. H. , Froese A.
Published: December 2009

Kinetic and equilibrium studies were performed on the reactions of the hapten ε-dinitrophenyl-lysine with specific intact antibodies, reduced, alkylated, and polyalanylated antibodies, and reduced, alkylated, and polyalanylated γ-chains. No reaction was detected between the hapten and light chains. The γ-chains were found to have 0.5 combining sites per chain, and thin layer gel chromatography revealed that they existed as monomers. The rate constant of association for the reaction of γ-chains with hapten was found to be almost 1000 times lower than that for the corresponding reaction with the parent antibody. Differences in the rate constants of dissociation were much less pronounced. These results suggested that the combining site in the separated γ-chain had undergone a change in conformation.
Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. I. Purification and Physical Properties
Authors: Wu Tai-Wing , Scrimgeour K. G.
Published: December 2009

IMP (inosinic acid or inosine-5′-phosphate) dehydrogenase has been purified to apparent homogeneity from Bacillus subtilis. The purification method yields an enzyme preparation that retains a constant level of inhibition by guanosine 5′-phosphate. The enzyme is membrane bound, and can be removed from membrane material after treatment either with detergents or with phospholipase A. Both the membrane-bound and solubilized forms of IMP dehydrogenase have similar kinetic properties. The soluble enzyme can occur in a number of oligomeric forms, with molecular weights that are multiples of 100 000 daltons. Although both the tetramer and the dimer appear to be catalytically active, no conclusions can yet be drawn about the quaternary structure of the enzymically active form(s).
Excision Properties of DNA Polymerase
Authors: Hoepfinger L. M. , Morgan C. D. , Lakanen C. L.
Published: December 2009

The excision of nucleotides from altered DNA or d(A-T) copolymer by Escherichia coli DNA polymerase I was measured. Altered DNA or d(A-T) copolymer was produced by treating radioactive DNA or d(A-T) copolymer with nitrous acid, formaldhehyde, or acridine orange. The resultant products were subjected to DNA polymerase I treatment. Excision of nucleotides occurred to a greater extent from the nitrous acid and formaldehyde treated materials than it did from untreated material. DNA treated with acridine orange snowed no significant differences in excision relative to untreated DNA.
The Effect of Oxygen on Metabolism in Astasia
Authors: Morosoli Rolf , Bégin-Heick Nicole
Published: December 2009

When Astasia is grown in the presence of O2–CO2 (95%: 5%), ethanol is incompletely utilized and an acidic substance accumulates in the medium. We have shown that this acidic substance can be utilized for growth if the gas phase is changed to air–CO2 (95%: 5%) and we have identified it as acetate. The results point to a defect in the utilization of acetate which is not due to the inhibition of acetate:CoA ligase (AMP) (EC 6.2.1.1), since the activity of this enzyme is increased in oxygen-treated cells. On the other hand, exposure to oxygen caused a diminution of the intracellular concentration of CoASH, which could result in the inhibition of 14CO2 production via the oxidative assimilation pathways. In addition, the oxidation of NADH by mitochondrial preparations was inhibited by exposure to oxygen. This inhibition coupled with the increased transformation of ethanol into acetate by these cells may lead to an increase in the levels of reduced pyridine nucleotides in the cell with a subsequent inhibition of metabolism.
The Reaction of N-ethylmaleimide with Different Forms of Cytochrome Oxidase
Authors: Kornblatt Jack A. , Baraff Georgia A. , Williams G. R.
Published: December 2009

The reaction of N-ethylmaleimide (NEM) with three different forms of beef heart cytochrome oxidase was studied. Whereas denatured oxidase binds 4–5 mol NEM per heme a, native oxidase and a new form of the oxidase prepared by extensive dialysis against water each binds 0.8–1.0 mol NEM per heme a. Oxidase in the form of coupled vesicles binds only half as much NEM as the native molecule. Six different polypeptides are associated with cytochrome oxidase. They have molecular weights of (A) 45 000; (B) 25 000; (C) 16 000; (D) 12 500; (E) 10 500; and (F) 8500. Four of the six different subunits appear to contain groups that bind NEM. In the native oxidase subunits A, B, D, and F react with NEM, subunit B being the most reactive, subunit F the least. Water-dialyzed oxidase binds NEM to subunits A, B, and F, the binding to D being greatly diminished. Vesicular oxidase binds NEM to only subunit B.