bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Transport of Glucose, Fructose, and Mannitol by Pseudomonas aeruginosa
Authors: Eagon R. G. , Phibbs Jr. P. V.
Published: December 2009

The transport of glucose, fructose, and mannitol by specifically induced cells of Pseudomonas aeruginosa was energy and temperature dependent, obeyed saturation kinetics, and was inducible. These properties were commensurate with active transport mediated by an enzyme-like carrier.Cells of P. aeruginosa were induced to active transport of glucose by growth on either glucose, fructose, or mannitol, but not on succinate. Active transport of fructose was induced by growth on either fructose or mannitol, but not on glucose; and active transport of mannitol was induced by growth on mannitol, but not on fructose or glucose. Facilitated diffusion was not detected via counterflow experiments by induced cells that had been poisoned by sodium azide plus iodoacetate. It was hypothesized that transport of sugars by P. aeruginosa occurred by active transport and by passive diffusion only.When induced cells were incubated with these sugars for 10 s and then extracted with hot water, either free sugar alone or, in the case of fructose or mannitol, free sugar plus fructose 6-phosphate were detected. These data were interpreted to indicate that carbohydrates were transported as free sugars by P. aeruginosa and were trapped intracellularly by phosphorylation.
Adenine as Substrate for Purine Nucleoside Phosphorylase
Authors: Zimmerman T. P. , Gersten N. B. , Ross A. F. , Miech R. P.
Published: December 2009

Purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from four sources (rat liver and brain, human erythrocytes, and calf spleen) has been shown to exhibit a low intrinsic activity towards adenine in the presence of ribose 1-phosphate. The Km for adenine determined with purine nucleoside phosphorylase from rat liver and human erythrocytes was 3.4 × 10−4 M and 4.1 × 10−4 M, respectively. Depending upon the source of the enzyme, the Vmax for ribosylation of hypoxanthine was 274–1630 times greater than the Vmax for ribosylation of adenine.Adenine is a competitive inhibitor of purine nucleoside phosphorylase from Sarcoma 180 ascites cells. An inhibition constant, Ki, of 5.5 × 10−4 M was determined for adenine with this enzyme and with selenoguanine as the variable substrate.
Studies on Plasminogen. IX. Purification of Human Plasminogen from Cohn Fraction III by Affinity Chromatography
Authors: Liu T. H. , Mertz Edwin T.
Published: December 2009

Human plasminogen was prepared from Cohn fraction III by affinity chromatography with 35-fold purification and 70% recovery. The solubility of this plasminogen at pH 7.4 depends on the pH of the solutions in which it was kept during the purification procedure. Thus, a native and an altered form can be obtained by using 0.1 M lysine at pH 7.4 and 0.1 M acetic acid at pH 3.0, respectively, as eluting agents. The altered plasminogen has high activity when suspended in acidic solution. It shows little activity when suspended in pH 7.4 buffer; its activity is partially restored with addition of 0.1 M lysine. The specific activity of these plasminogens is in the same range as that of plasminogen prepared directly from human plasma by affinity chromatography.
Phosphorylation of Histones in Normal Goose Erythrocytes
Authors: Seligy V. L. , Neelin J. M.
Published: December 2009

Phosphate is taken up by erythrocytes in normal goose blood and incorporated into purified, characterized histones. The firmly bound radiophosphorus was not in the form of inorganic phosphate, free nucleotide, or phospholipid. That it was not due to contaminating nucleic acid or nonhistone protein was shown by chromatographic and electrophoretic localization.Most of the histone-bound radioactivity was recovered in slightly lysine-rich histone IIb1 (or f2a2) and in erythrocyte-specific histone V (or f2c), with the highest specific activity in the former. In these proteins the incorporated radiophosphorus was found only in phosphoserine.The differential incorporation did not correspond to alkali-labile phosphate contents, or to synthesis of the different proteins, and may reflect at least two different roles of phosphorylation in cell maturation and control of gene expression.
Evaluation of Methods for Preparing Proteins from Plasma High Density Lipoproteins
Authors: Sodhi H. S. , Bhatnagar R. K. , MacKenzie S. L.
Published: December 2009

The proteins from plasma high density lipoproteins have been prepared by three methods and the products are compared by electrophoresis on or in the media of paper, agarose, cellulose acetate, and polyacrylamide gel and by analytical ultracentrifugation. The capacities of the proteins to bind phospholipids, free fatty acids, and cholesterol are compared. The method based on that of Shore and Shore is the easiest to perform. The peptides are less likely to be denatured since no precipitation step is involved; this is supported by their generally greater affinity for lipids.
Effect of L-Histidine on Human and Rat Jejunal Pyruvate Kinase Activity
Authors: Stifel Fred B. , Herman Robert H.
Published: December 2009

L-Histidine was found to be an in vitro activator of both human and rat jejunal pyruvate kinase activity and rat hepatic pyruvate kinase activity. Jejunal and hepatic pyruvate kinase activities were higher in rats fed a histidine-complete diet than a histidine-deficient diet. Feeding groups of rats successively larger doses of oral histidine produced a progressive increase in rat jejunal pyruvate kinase activity. An inverse relationship was seen between the pyruvate kinase activity without histidine added and the percent stimulation with histidine added in vitro.The histidine effect appears to be a ubiquitous one in the rat, since stimulation was observed in each of the seven tissues tested. The broad range of the histidine effect suggests that it may play a fundamental role in the regulation of glycolysis in mammalian systems.
The Sulfite Oxidase of Thiobacillus ferrooxidans (Ferrobacillus ferrooxidans)
Authors: Vestal J. Robie , Lundgren D. G.
Published: December 2009

The sulfite oxidase (sulfite: cytochrome c oxidoreductase) from sulfur-grown Thiobacillus ferrooxidans was isolated and partially purified, and its properties were studied. The enzyme was purified 7.3-fold and was 75–85% of the protein present. Sulfite oxidase required SO32− for activity, and could use horse heart cytochrome c and ferricyanide as electron acceptors. The molecular weight was 41 500. The enzyme had a Km for sulfite of 0.58 mM with either ferricyanide or cytochrome c as the electron acceptor. The Km for ferricyanide was 0.25 mM. 5′-AMP did not stimulate enzyme activity. Other properties of the enzyme were similar to the enzyme from Thiobacillus thioparus and Thiobacillus novellus. A metabolic scheme of sulfur utilization for energy production in Thiobacillus ferrooxidans is presented.
The Specificity of Penicillopepsin
Authors: Mains G. , Takahashi Miho , Šodek J. , Hofmann T.
Published: December 2009

The specificity of penicillopepsin was investigated with a number of different substrates. In agreement with earlier work (1), no action was observed on di- and tripeptide and ester substrates. However, the pepsin substrate carbobenzoxyglycylglycylphenylalanylphenylalanine 3-(4-pyridyl)propyl-1 ester was hydrolyzed between the two phenylalanine residues. No cleavage of homopolymers of glycine, alanine, and glutamic acid and the random copolymer of glutamic acid and tyrosine was observed. Polymers of lysine, lysine and tyrosine (2:1), and lysine, glutamic acid, and tyrosine (13:20:1) were, however, hydrolyzed giving mainly tripeptides to pentapeptides. Polylysine hydrolysis showed a pH dependence centering about a group of pK between 3 and 4. In glucagon and the S-sulfo B-chain of insulin, penicillopepsin hydrolyzed the same peptide bonds as the other acidic proteases, including pepsin and rennin, with very few exceptions. There are, however, distinct differences between the action of penicillopepsin and that of other proteases of low specificity. Peptide bonds which have a hydrophobic amino acid in the P1′ position (as defined by Berger and Schechter, Ref. 2) are preferentially cleaved by penicillopepsin. A kinetic study of the hydrolysis of bovine serum albumin showed very high rate constants for the initial cleavages. The present study shows the requirement for an extended binding site in penicillopepsin.
The Purification and Some Properties of Soluble Trehalase and Sucrase from Drosophila melanogaster
Authors: Huber R. E. , Lefebvre Y. A.
Published: December 2009

Soluble trehalase (EC 3.2.1.28) from Drosophila melanogaster was purified to a specific activity of about 112 units/mg and only one band was detected by analytical disc-gel electrophoresis. Soluble sucrase (EC 3.2.1.26) having a specific activity of about 10 units/mg was obtained. The two enzymes had similar molecular weights, pH optima, and responses to mercaptoethanol, and neither enzyme was activated by ions or ethanol. Para-chloromercuribenzoate inhibited sucrase to a greater extent than trehalase while iodoacetate did not affect either enzyme. Sucrase was less stable to a variety of denaturing conditions, the Km's of the two enzymes differed greatly, and Tris and Tricine were much stronger competitive inhibitors of sucrase than of trehalase. Both enzymes underwent a large reduction in molecular weight during the purification procedure.
Rabbit Immunoglobulin Allotypy: The Physiochemical Nature of a-Group Determinants
Authors: Stemke G. W.
Published: December 2009

Allotypic specificity Aal (al) of the a-group of rabbit immunoglobulin is at least partially conserved in the Fd fragment of IgG as shown by quantitative complement fixation. However, al reactivity is not on tryptic digest peptides. Allotype al reactivity is also conserved when the IgG was amidinated. The addition of poly-DL-alanine branches to IgG has a variable quantitative effect on al reactivity; this activity is not present on poly-DL-alanine H-chain which was prepared by extensive denaturation, reduction, and S-alkylation of the poly-DL-alanine IgG. It is concluded that al reactivity is due to protein tertiary structure rather than prosthetic group addition.
Pressure Jump Studies of Proteins. I. Isomerization of Bovine Serum Albumin at Neutral pH
Authors: Goldsack D. E. , Waern P. M.
Published: December 2009

Pressure jump kinetic studies of the conformational change occurring in bovine serum albumin in neutral solutions have been carried out over the pH range 6.5–9.5. Two distinct relaxation effects are observed at each pH. The faster relaxation is attributed to binding of the dye to the protein, and the slower relaxation is related to the conformational change occurring in the protein. This slower relaxation effect is pH dependent with a maximum value near pH 8. Detailed analysis of these data leads to a mechanism for the conformational change which indicates that the one form of the protein has an ionizable group with a pK of 8.7 which changes to a pK of 6.7 when the protein undergoes the conformational change. A simple iterative procedure is given for analyzing the pH dependence of a relaxation time constant for a cyclic mechanism involving only one ionizing group controlling the conformational change.
A Factor Capable of Dissociating Rat Liver Ribosomes
Authors: Lawford G. Ross , Kaiser Jutta , Hey W. C.
Published: December 2009

A factor capable of dissociating rat liver monomeric ribosomes into 60 S and 40 S subunits has been partially purified and characterized.The factor was prepared by extracting a fraction of rat liver enriched in its content of native subunits with 0.05 M triethanolamine–HCl, 1.0 M KCl, 0.01 M MgSO4, and 2 mM dithiothreitol. The activity of the preparation was assayed by testing its ability to dissociate monomeric ribosomes into subunits which were detected by sucrose density gradient analysis. The ribosomes used as substrate were prepared by dissociating polysomes in the presence of puromycin, 0.5 M KCl, and 3 mM MgSO4 and subsequently reassociating the subunits into monomers by lowering the ionic strength. The factor acts only on ribosomes freed of both messenger RNA and nascent protein by associating with the small subunit. The activity was time and temperature dependent, reaching a plateau after 30 min at 30 °C.The factor has been partially purified by ammonium sulfate fractionation between 35% and 65% saturation and by treatment at 40 °C for 15 min to precipitate ribosome-aggregating substances.
Some Properties of Ribosomes from Myxobacter 495
Authors: Sendecki Witold , Mikulik Karel , Matheson A. T.
Published: December 2009

The ribosomes of Myxobacter 495 are similar to other bacterial ribosomes in that the 70 S monomer is made up of a 50 S subunit (containing 23 S and 5 S RNA) and a 30 S subunit (containing 16 S RNA). The base composition of the ribosomal RNA is very similar to that of E. coli. The Myxobacter 495 ribosomes contain nucleases that are not removed by washing with 1 M (NH4)2SO4 and which are relatively insensitive to inhibition by Macaloid or sodium dodecyl sulfate. Only in the presence of diethyl pyrocarbonate was it possible to isolate intact 16 S and 23 S RNA. The (NH4)2SO4-washed ribosomes were much less stable than the unwashed ribosomes due apparently to activation of a nuclease which was located mainly in the 30 S subunit.
Structural and Metabolic Interrelationships Among Glycerophosphatides of Rat Liver In Vivo
Authors: Holub B. J. , Kuksis A.
Published: December 2009

The specific activities of individual molecular species of rat liver diacylglycerylphosphorocholine (PC), diacylglycerylphosphoroethanolamine (PE), and diacylglycerophosphorylinositol (MPI) were determined and compared following intravenous injection of glycerol-14C. PC, PE, and MPI contained 41, 51, and 83%, respectively, tetraenoic species, and 40,17, and 9% combined mono-, di-, and trienoic species. The rest of the phosphatide mass of PC, PE, and MPI was contributed by 18, 32, and 8% penta- and hexaenoic species, respectively. The proportions of chemical classes of the glycerophosphatides differed by 1.1- to 18-fold while the fatty acid associations within the unsaturation classes common to these phosphatides varied 2.2- to 17-fold. After 5 min exposure to radioactive glycerol, the mono-, di-, and trienoic species of the PC, PE, and MPI possessed 13–18, 15–50, and 6–42 times, respectively, the specific activity of the tetraenes of the corresponding phosphatide classes. While the pentaenoic and hexaenoic species of PC and MPI had specific activities three to five times those of the respective tetraenes, the higher polyenes of PE were considerably more radioactive and approached the specific activity of the dienoic species of this phosphatide. With progressing time up to 60 min, the tetraenoic species of PC, PE, and MPI showed increases in relative specific activity of 50, 64, and 109%, respectively, in the three phosphatides. These results are consistent with an effective de novo synthesis of the oligoenoic species and a transacylation of the tetraenoic species of all liver glycerophosphatides tested. The proportional contribution of de novo synthesis in comparison to acyl transfer is apparently greater to the formation of PC and PE than to that of MPI.
Studies of Dlalkyl Ether Phospholipids. II. Requirement for a Liquid-Crystalline Substrate for Hydrolysis by Cabbage Leaf Phospholipase D
Authors: Chen Jung-shou , Barton Peter G.
Published: December 2009

The hydrolysis of 1,2-ditetradecyl-, 1,2-dihexadecyl-, and 1,2-dioctadecyk-sn-glycero-3-phosphoryl-cholines by the soluble phospholipase D (EC 3.14.4) from cabbage leaf has been compared with that of egg lecithin. When a diethyl ether – chloroform mixture was used as activator low hydrolysis rates were observed for the dialkyl ether phospholipids because of their poor solubility in diethyl ether. Under these conditions the compounds did not inhibit the hydrolysis of egg lecithin. The hydrolytic reaction appeared to take place at the solvent–water interface resulting in a gradual denaturation of the enzyme. When sodium dodecyl sulfate was used as activator faster hydrolysis was evident due in part to a reduction in mesomorphic transition temperature (Tm) revealed by differential thermal analysis. Rates of hydrolysis of the homologous dialkyl ether phospholipids were inversely proportional to their Tm values in excess water. Under conditions required for effective hydrolysis of these compounds they inhibited the hydrolysis of egg lecithin. From studies of hydrolysis rates obtained with various combinations of phosphatidylcholine and phosphatidic acid analogues it was concluded that binding of enzyme to phospholipids and their subsequent hydrolysis required both an appropriate surface charge and a gel to liquid-crystalline phase transition in the substrate.
Serum Cholinesterase Activity in Hyperlipidemia and the In Vitro Effect of Isoniazid on Serum Cholinesterase
Authors: Kutty K. M. , Jacob J. C.
Published: December 2009

Increased serum Cholinesterase activity was observed in hyperlipidemic patients. When hyperlipidemia was induced in rabbits by injecting the lipopolysaccharide of Escherichia coli, a significant rise in serum low density lipoproteins and Cholinesterase activity occurred.In vitro experiments demonstrated that isoniazid produced proportionate decreases in serum low density lipoprotein concentration and in serum Cholinesterase activity.
Amino Acid Sequence Studies of Horseradish Peroxidase. I. Tryptic Peptides
Authors: Welinder K. G. , Smillie L. B. , Schonbaum G. R.
Published: December 2009

Commercially available horseradish peroxidase (RZ 3.1) was characterized with respect to its homogeneity by (1) chromatography on CM-cellulose, (2) disc gel electrophoresis in alkaline and acidic buffers, (3) micro-scale sucrose gradient isoelectrofocusing in pH 3–10 and pH 8–10 gradients, (4) gel isoelectrofocusing in pH 3–10 and pH 8–10 gradients, and (5) amino acid and hexosamine analyses. The preparation was found to be highly homogeneous except by pH 8–10 gel isoelectrofocusing which resolved it into several very close bands. This heterogeneity has been assumed to reflect differences in carbohydrate composition rather than in the amino acid sequence or composition. Amino acid analyses after performic acid oxidation yielded eight cysteic acid residues per mole of enzyme. Since no S-carboxymethylcysteine was recovered after treatment of the protein in 8 M urea with iodoacetic acid, it was concluded that the enzyme has four disulfide bridges. Peptides resulting from a tryptic digest of the heme-free enzyme were purified by high-voltage paper electrophoresis and subjected to sequence analysis. Several half-cystine sequences were elucidated after isolation of the radioactive peptides from a tryptic digest of the reduced and 14C-S-carboxymethylated protein. The complete sequences of 21 and partial sequences of three tryptic peptides were determined. These account for 203 of the approximately 300 amino acid residues of this protein. Several sites of carbohydrate attachment were observed.
Movement of [U-14C]glucose Carbon into and Subsequent Release from lipids and High-Molecular-Weight Constituents of Rat Brain, Liver, and Heart In Vivo
Authors: Vrba R. , Winter Anna
Published: December 2009

After subcutaneous injection of [U-14C]glucose into rats the amount of 14C incorporated in vivo into proteins was always higher than into lipids in brain, liver, and heart. The specific radioactivity of brain proteins was higher than those of liver and heart. Blood-brain comparisons show that protein carbon is derived continuously from glucose in the brain in situ and not as a result of deposition of amino acids or proteins from the circulation. Seventy-two percent of 14C in purified brain protein fractions was found in the amino acids of the hydrolysates of these fractions, mainly in alanine, glutamic, and aspartic acids. Maximum labelling was reached about 4 h after injection of [U-14C]glucose. Elimination of 14C from three classes of brain proteins (high-speed supernatant, particulate deoxycholate extractable, and residual) followed a biphasic time-course. The extent of labelling of, and the rate of elimination of 14C from, the three classes of rat brain proteins were very similar. The fate of 14C in the other investigated tissue fractions of brain, liver, and heart was compared with the fate of 14C in brain proteins.The results lend further support to the previously published suggestion that: (a) brain does not contain appreciable amounts of metabolically inert proteins or of proteins with turnover rates significantly higher than the mean for the bulk of brain proteins; (b) glucose carbon participates at a different rate and to a different extent in the metabolism of high-molecular-weight constituents of brain as compared to liver, heart, and plasma proteins; (c) the continuous conversion of glucose carbon into protein is an important part of the maintenance of the homeostasis of tissue proteins in vivo.
Procedure for Simultaneous Determination of Secretion Rates of Deoxycorticosterone and Corticosterone by Double-Isotope Derivative Assay
Authors: Seth K. , Nowaczynski W. , Kuchel O. , Mancheno-Rico E. , Genest J.
Published: December 2009

A procedure for the simultaneous determination of secretion rates (S.R.) of deoxycorticosterone (DOC) and corticosterone (B) by double-isotope derivative assay is described. In a detailed assessment of the method the importance of careful purification and isolation of the metabolites of both steroids is emphasized. The S.R. of DOC was 174.6 ± 14.6 S.E. μg/24 h in 19 determinations in 15 normal controls and 7712 ± 2072 S.E. in three determinations after metapyron administration. Corticosterone S.R. was 2778 ≠ 232 S.E. μg/24 h in 15 determinations in 12 normal controls. All the experiments were done on normal sodium intake.
Occurrence and Biosynthesis of Ceramide Phosphorylethanolamine in Chicken and Rat Liver
Authors: Muehlenberg Bernd A. , Sribney Michael , Duffe Marilyn Kemp
Published: December 2009

Ceramide phosphorylethanolamine has been found to occur in chicken and rat liver. An enzyme (CDP-ethanolamine:ceramide ethanolaminephosphotransferase) has been found in a number of tissues which catalyzes the biosynthesis of this lipid. The enzyme catalyzes the transfer of the phosphorylethanolamine moiety of CDP-ethanolamine to the free primary hydroxyl group of a ceramide (N-acylsphingosine). The chicken liver enzyme requires 0.010 M manganese ions for optimal activity and has a pH optimum of 7.7. The Km for the substrate N-octanoyl-threo-sphingosine was found to be 2.5 × 10−4 M. A study of the effect of increasing CDP-ethanolamine concentration on the reaction rate indicates from sigmoid kinetics that the coenzyme modulates and possibly regulates PE-ceramide transferase activity. The enzyme differs from sphingomyelin synthetase (CDP-choline:ceramide cholinephosphotransferase) in that it will only utilize the unnatural threo isomer of N-acylsphingosines (threo-ceramides) as acceptors for the phosphorylethanolamine moiety of CDP-ethanolamine. Sphingomyelin synthetase has been shown to utilize erythro-ceramides the presence of sulfhydryl reagents (Sribney, M.: Can. J. Biochem. 49, 306 (1971)); the enzyme catalyzing the biosynthesis of ceramide phosphorylethanolamine, however, does not do so, even in the presence of a variety of sulfhydryl reagents tested.
A Fluorescence Assay for DNA with Covalently Linked Complementary Sequences
Authors: Morgan A. Richard , Paetkau Verner
Published: December 2009

An assay which measures polydeoxyribonucleates containing covalently linked complementary sequences is described. It is based on the fluorescence enhancement of ethidium bromide bound to bihelical polydeoxyribonucleates. This dependence of fluorescence enhancement upon bihelicity has been further documented. Synthetic, defined polymers, such as d(A)n∙d(T)n, d(AT)n∙d(AT)n, d(TC)n∙d(GA)n, and d(TTG)n∙d(CAA)n, have been studied, as well as naturally occurring DNAs. At moderate ionic strengths, the synthetic polymers will re-anneal following heat denaturation. Their facility to renature is due to the self-complementarity between strands throughout their lengths. This facility is lost at very low ionic strengths (Γ/2 < 0.003 M), where ionic repulsion prevents the formation of "nucleation" sites for annealing. However, a covalent linkage between strands provides a nucleation site for renaturation. The fluorescence seen with ethidium bromide after heating and cooling such polymers is then a measure of DNA which has covalently linked complementary sequences. By this assay, natural DNAs appear to contain short, relatively unstable bihelical regions following heat denaturation and cooling, in agreement with models based on other physical measurements.
Effects of Adrenalectomy, Adrenalectomy–Ovariectomy, and Cortisol and Estrogen Therapies Upon Enzyme Activities in Lactating Rat Mammary Glands
Authors: Korsrud G. O. , Baldwin R. L.
Published: December 2009

The effects of adrenalectomy and adrenalectomy–ovariectomy on the 5th day of lactation followed by cortisol and estrogen therapies on enzyme activities in rat mammary glands were investigated. This stage of lactation was selected because mammary secretory cell proliferation is essentially complete at this time thereby enabling study of the effects of cortisol and estrogen on enzyme levels in a nonproliferating secretory cell population. Eighteen enzymes were selected for study on the bases of their respective roles in milk biosynthesis and carbohydrate and energy metabolism and/or on the basis of previous studies indicating that their activities increase during midlactation or are regulated, in part, by steroid hormones. After adrenalectomy on the 5th day of lactation, cortisol therapy was required for normal increases in the activities of succinic dehydrogenase, citrate cleavage enzyme, malic enzyme, UDPglucose pyrophosphorylase, UDPglucose 4-epimerase, and glucose-6-phosphate dehydrogenase. The activities of UDPglucose pyrophosphorylase and glucose-6-phosphate dehydrogenase were higher than normal in cortisol-treated animals. Cortisol therapy during the last 2 days of the experiment increased the activity of UDPglucose pyrophosphorylase and possibly citrate cleavage enzyme. The activities of α-glycerolphosphate dehydrogenase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, pentose phosphate metabolizing ability, hexokinase, phosphofructokinase, fructose-1,6-diphosphate aldolase, pyruvate kinase, lactic dehydrogenase, aspartate aminotransferase, isocitrate dehydrogenase, and extramitochondrial malate dehydrogenase were not notably affected by adrenalectomy or cortisol therapy. The activities of 6-phosphogluconate dehydrogenase, pentose phosphate metabolizing ability, phosphofructokinase, and pyruvate kinase may have increased after the 5th day of lactation in adrenalectomized as well as in normal animals. Combining ovariectomy with adrenalectomy reduced pup weight gains more than adrenalectomy alone, but did not further decrease significantly the activities of any of the enzymes measured. Ovariectomy had no effect when cortisol was administered. Cortisol therapy completely reversed adverse effects of estrogen given to adrenalectomized–ovariectomized animals. On the bases of these and previous data, it was concluded that cortisol regulates the rates of synthesis of several mammary gland enzymes during midlactation.
Biochemical Changes in Progressive Muscular Dystrophy. IX. Synthesis of Native Myosin, Actin, and Tropomyosin in the Skeletal Muscle of Mouse as a Function of Muscular Dystrophy
Authors: Srivastava Uma
Published: December 2009

The synthesis of native myosin, actin, and tropomyosin in the skeletal muscle of normal and hereditary dystrophic mice was studied with the help of direct counting as well as acrylamide-gel electrophoresis and protein purification procedures.Labelling of the nascent protein indicated that heavier polysomes from the normal muscle were able to incorporate more radioactivity into the protein than the heavier polysomes from the dystrophic muscles. Contrary to this, lighter polysomes in the dystrophic muscle demonstrated higher incorporation as compared to the normal.Results of in vivo and in vitro incorporation as well as those of acrylamide-gel electrophoresis and protein purification procedures indicated that synthesis of myosin decreased in the dystrophic muscle. The synthesis of actin did not show a significant change either in normal or dystrophic muscle, whereas that of tropomyosin increased sharply in the dystrophic mouse muscle.
Pyridine–Adenine Dinucleotide Transhydrogenase Activity in Cells Cultured from Rat Hepatoma
Authors: Luca C. De , Gioeli R. P.
Published: December 2009

Preparations from cells cultured from a minimal-deviation hepatoma in the rat exhibit pyridine nucleotide transhydrogenase (NAD(P)H: NAD(P) oxidoreductase, EC 1.6.1.1) activity. The pH optimum, its release by digitonin, and its apparent lack of dependence on steroids for activity tentatively classify it as a transhydrogenase of the type first described for animal tissue.Enzyme preparations from digitonin-treated homogenates were very unstable. The time necessary for the loss of one-half the activity was 16–18 h when the enzyme was stored at 5 °C; this was reduced to 4 h when storage was in polycarbonate tubes.The enzyme apparently transferred hydrogen directly and with equal ease from NADH to both the 3-acetyl-pyridine and thionicotinamide analogues of NAD. Half-saturation values for NAD and its acetylpyridine analogue were 0.99 × 10−5 M and 3.55 × 10−4 M, respectively. The enzyme exhibited its maximum activity in phosphate buffer at pH 5.8. It was inhibited by 50–60% over the pH range 7.0–8.5 in Tris buffer. This could be reversed by dithiothreitol; reversal was complete between pH 8.0 and 8.5.
A Study of the Structural Subunits of Urease Obtained During Controlled Dissociation
Authors: Contaxis C. C. , Reithel F. J.
Published: December 2009

Native urease, an enzyme with a preparative molecular weight of 480 000, has been dissociated stepwise to 60 000 and 30 000 molecular weight subunits. The 60 000 dalton species was obtained by exposure to 9 M urea. It is enzymatically active, with a specific activity approximately 45% of the native enzyme, and possesses a substantially less ordered structure. A presumptive dissociation to 90 000 dalton species was obtained by exposure to 8 M urea. This dissociation proceeds with conservation of approximately 90% of the enzymic activity of native urease and conservation of the ordered structure. Both 60 000 and 90 000 dalton species were also obtained by exposure of urease to 3 M or 6 M guanidine hydrochloride. These preparations were heterogeneous, enzymatically inactive, and suffered severe structural loss. Dissociation to a 30 000 dalton structural subunit was observed after treatment of urease with acetate–acetic acid at pH 2.2. The dissociation appeared to be a symmetrical process leading to physically identical subunits and conserving the ordered structure of the enzyme. Under the normal assay conditions the 30 000 dalton species polymerizes and this prevents conclusive proof of enzymic activity at this time.
Succinyl Coenzyme A Synthetase of Pig Heart: Studies of the Subunit Structure and of Phosphoenzyme Formation
Authors: Brownie E. R. , Bridger W. A.
Published: December 2009

Succinyl coenzyme A synthetase from pig heart (molecular weight approximately 75 000) gives rise to two species of subunits when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights of the constituent subunits are estimated to be about 42 500 and 34 500, suggesting that the enzyme is a dimer. The presence of saturating concentrations of guanosine 5′-triphosphate (GTP) results in the formation of one phosphohistidine residue per molecule of enzyme; the site of phosphorylation is in the smaller subunit. The standard free energy change for phosphorylation of the enzyme by GTP is approximately −400 cal/mol. The results point to both interesting similarities and differences in succinyl-CoA synthetases isolated from mammalian and bacterial sources.
New Reaction Vessel for Solid-Phase Peptide Synthesis
Authors: Park W. K. , Regoli D.
Published: December 2009

The vessel for the synthesis of peptides by the solid-phase method consists of a round flask with one side-arm and three stopcocks. The side-arm is used to attach the vessel to a wrist-shaker and to insert a gas dispersion tube for the cleavage of the synthesized peptide. Solvents and reagents are introduced from the stopcock on the top and removed from the one at the bottom, by applying negative pressure and by opening the lateral stopcock at the same time.
Studies on the Dissociation of Porcine Haptoglobin
Authors: Lockhart W. L. , Chung W. P. , Smith David B.
Published: December 2009

Porcine haptoglobin was tested for reversible dissociation in dilute solution by gel filtration. Elution volume in Bio-Gel P-150 was independent of concentration and shapes of elution profiles failed to show dissociation. Molecular weight in dilute solution was estimated as 96 500. Interchain disulfide bridges were assayed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Partially reduced samples showed a series of intermediates but fully reduced samples showed only heavy and light chains. Intermediates were tentatively identified as to chain composition from their electrophoretic mobilities.
Prolylhydroxyproline Absorption in Hamsters
Authors: Hueckel H. J. , Rogers Q. R.
Published: December 2009

The intestinal transport of the dipeptide prolylhydroxyproline was investigated using hamsters and segments of hamster small intestines. The feeding of prolylhydroxyproline to hamsters resulted in the urinary excretion of 2% of the ingested dipeptide after 3 h or 7% after 24 h. In vitro perfusions of prolylhydroxyproline through the hamster small intestine resulted in the movement of the dipeptide across the intestinal wall. The rate of absorption was proportional to the concentration of prolylhydroxyproline on the mucosal side (0.17 μmol Pro∙Hyp/mM in lumen/g tissue/h). Anoxia, 2,4-dinitrophenol, cyanide, or an excess of proline or hydroxyproline did not change this absorption rate. These results suggest that prolylhydroxyproline is absorbed by "simple passive diffusion", that it reaches the site of intestinal absorption because of its relative resistance to enzymatic hydrolysis, and that it is excreted by glomerular filtration without subsequent active reabsorption.
L-Fucose Metabolism in Mammals. I. Pork Liver L-Fuconate Hydro-lyase
Authors: Yuen Raymond , Schachter Harry
Published: December 2009

Pork liver has been shown to contain a soluble enzyme, L-fuconate hydro-lyase (L-fuconate dehydratase), capable of dehydrating L-fuconate to 2-keto-3-deoxy-L-fuconate. The enzyme has been partially purified. The Km for L-fuconate is 1.0 mM; D-arabonate is also an excellent substrate (Km 1.3 mM) but the enzyme is inactive with D-fuconate, L-arabonate, D-gluconate, D-gulonate, D-galactonate, L-mannonate, and D-glucuronate. The enzyme has a sharp pH optimum at pH 7.0, requires Mg2+ for activity, and is inhibited by p-hydroxymercuribenzoate. The substrate specificity of L-fuconate hydro-lyase resembles that of pork liver L-fucose: NAD oxidoreductase and it is suggested that these enzymes serve in the metabolism of L-fucose by the following pathway: L-fucose → L-fucono-1,5-lactone → L-fuconate → 2-keto-3-deoxy-L-fuconate. The metabolic fate of the latter compound is not known. D-Arabinose can be metabolized by pork liver by an analogous pathway.
Use of 4-Nitrobenzylthioinosine in the Measurement of Rates of Nucleoside Transport in Human Erythrocytes
Authors: Pickard M. A. , Paterson A. R. P.
Published: December 2009

The equilibrium exchange diffusion of uridine in human erythrocytes is abruptly terminated in the presence of 4-nitrobenzylthioinosine, an inhibitor of nucleoside transport. This compound would appear to have value as a stopping agent in studies of nucleoside transport which require measurements over short time intervals.
Hemoglobins A2-Sphakiá and Al-NYU in Canada
Authors: Vella F. , Wong S. C. , Wilson J. B. , Huisman T. H. J.
Published: December 2009

A hemoglobin A2 variant which is detectable on electrophoresis occurs frequently in the heterozygous form in Canadian Indian subjects. Structural analyses have been made on this variant obtained from two such persons living in Alberta and Manitoba, respectively, and have led to its characterization as Hb-A2-Sphakiá [Formula: see text]. Similar studies on a second delta chain variant found in a Canadian subject of Ukrainian origin from Saskatchewan have shown this variant to be Hb-A2-NYU [Formula: see text].
Enantiomeric Diglycerides as Stereospecific Probes in Triglyceride Synthesis In Vitro
Authors: O'Doherty P. J. A. , Kuksis A. , Buchnea D.
Published: December 2009

The stereospecificity of diglyceride acyltransferase (EC 2.3.1.20) was studied in the microsomes of rat intestine and liver using enantiomeric diglycerides as acceptors of labeled fatty acids. The diglyceride mixtures were prepared by combining equal amounts of synthetic 1,2- and 2,3-diglycerides of different degrees of unsaturation. The labeled triglycerides formed were resolved by argentation thin-layer chromatography, whereupon the products from each enantiomeric diglyceride moved to a different spot on the thin-layer plate.It was shown that both 1,2- and 2,3-diglycerides were utilized for direct acylation to triglycerides by both tissues. The diglyceride acyltransferase, if a single enzyme, showed a definite preference for the acylation of the 1,2-diglycerides. The intestine esterified more of the 2,3-isomer than did the liver. The yields of triglyceride depended upon the nature of the fatty acids involved, and their positional distribution on the glycerol molecule. The new method of stereospecific assessment of triglyceride biosynthesis in vitro is applicable to preparations of other subcellular fractions and to other suitable combinations of fatty acid and diglyceride precursors.
Metabolism of Intravenously Administered 17α-[6,7-3H]Estradiol-17-Glucoside in Normal Women
Authors: Williamson D. G. , Layne D. S. , Nilsen Mona , Hobkirk R.
Published: December 2009

17α-[6,7-3H]estradiol-17-glucoside was injected intravenously into two normal young women. Within 72 h, 60 and 46% of the 3H dose was excreted in the urine in the two experiments. In the first 6 h, 4 and 1.4% of the dose was excreted as the unchanged glucoside, together with 14 and 5% in the form of a double conjugate, identified as 17α-estradiol-3-glucuronide-17-glucoside. Over the periods 6–24 h and 24–48 h none of the injected glucoside and only minor amounts of the double conjugate were present in the urine. The main excretory product after 6 h was 17 α-estradiol-17-glucuronide. This latter result contrasts with that obtained in previous experiments in which 17β-estradiol-6,7-3H-17-glucoside was injected, and in which the main monoconjugates excreted were the 3-glucuronides of 17β-estradiol and estrone.
Properties of Adenyl Cyclase from Ehrlich Ascites Cells
Authors: Bär Hans-Peter , Henderson J. Frank
Published: December 2009

Properties of adenyl cyclase from Ehrlich ascites tumor cells were studied. The enzyme was stimulated by catecholamines, prostaglandin E1 (but not F1α or F1β), and NaF; it had a pH optimum around 8. Calcium and thiol reagents reduced both basal and stimulated activities. Sonication destroyed activity; storage at 0–4° for 5 h resulted in small losses of NaF and adrenaline-stimulated but not basal activity. Half maximal stimulation was seen at 0.03 μM for L-adrenaline, 0.05 μM for isopropylnoradrenaline, and 5 μM for L-phenylephrine. Propranolol blocked adrenaline and phenylephrine action, and phentolamine had no effect. Stimulatory effects by adrenaline and prostaglandin E1 were not additive. The Km values for ATP were in the range of 0.02–0.15 mM and were not changed by NaF or adrenaline; likewise, Km values for Mg2+ (0.25–0.36 mM) were not affected.
Effect of Cordycepin on Ribosome Formation and Enzyme Induction in Rat Liver
Authors: Rizzo Arthur J. , Kelly Camille , Webb Thomas E.
Published: December 2009

Cordycepin (3′-deoxyadenosine), at a dose of 10 mg/kg, inhibits the accumulation of ribosomes in rat liver cytoplasm within 2.5 h of administration. This effect is not due to an inhibition of RNA synthesis per se but rather to the premature termination of the transcription of the 45 S ribosomal precursor in the presence of the analogue. The nucleoside analogue also inhibits the corticosteroid-mediated induction of tyrosine transaminase and general protein synthesis. The latter results are consistent with the current theory that cordycepin inhibits the formation of polyadenylic acid which is involved in the nuclear processing and/or nucleocytoplasmic transport of messenger RNA.
Permeability of Phospholipid Membranes to Inorganic Phosphate
Authors: Petkau A. , Chelack W. S.
Published: December 2009

(1) Diffusion of inorganic phosphate through a model phospholipid membrane exhibits an initial time lag but is linear with time thereafter.(2) In 0.1 M NaCl the permeability coefficient k = 3.4 (± 0.14) × 10−8 cm/s and is invariant with Δc, the concentration difference of phosphate across the membrane. The diffusion lag time is 51 ± 20 min.(3) In 0.5 mM MgCl2 the permeability coefficient decreases exponentially as Δc is increased and is less than 10−8 cm/s. The diffusion lag time is 94 ± 38 min.(4) In 0.5 mM MgCl2 the activation energy for phosphate diffusion through the membrane is 20 kcal/mol.(5) The flux of inorganic phosphate through the model membrane is in good agreement with experimental values obtained on erythrocytes.
Studies on the Cytotoxicity of Myleran and Dimethyl Myleran
Authors: Mitchell M. P. , Walker I. G.
Published: December 2009

Myleran and dimethyl Myleran (DMM) are two potentially bifunctional alkylating agents which might be expected to form cross-links between guanine residues on the same or opposite strands of native DNA. When L cells were treated with these agents DMM was considerably more toxic than Myleran. When DNA, RNA, and protein were subsequently isolated and analyzed it was found that both agents reacted with DNA to the same extent, the degree of labelling being linear with the concentration of the agents. RNA and protein were labelled to a rather greater extent. When DNA was analyzed chromatographically, no evidence was found for the formation of alkylated guanine residues after treatment with DMM. With Myleran, a product corresponding to the N-7 alkylated monoguaninyl derivative was formed, but no diguaninyl product was detected. Similar results were obtained when DNA was treated in vitro. No evidence of cross-linking by either agent was found when DNA treated in vitro was subjected to alkaline denaturation and subsequent renaturation, and then analyzed by cesium chloride density gradient centrifugation. It is concluded that neither Myleran nor DMM causes cross-linking of DNA strands, nor do they form links between adjacent guanine residues in one strand of DNA.
Glycoprotein Biosynthesis: Stimulation of N-Acetylglucosaminyltransferase Activity by Cytidiee 5′-Diphosphocholine
Authors: Mookerjea Sailen
Published: December 2009

The stimulatory effect of CDP-choline on N-acetylglucosaminyltransferase activity is marked in rough microsomes but is almost absent in Golgi-rich membranes or in serum. The marked CDP-choline effect on the enzyme is evident even when the nucleotide–sugar substrate concentration is raised to near saturation. Diglyceride has an inhibitory action on the enzyme which is effectively reversed by further addition of CDP-choline. Of the other different lipid factors tested only CDP-ethanolamine has a stimulatory effect similar to CDP-choline. CDP-choline alone activates the enzyme better than Triton. CDP-choline and Triton, in different combinations of doses, show a marked synergistic effect. Cationic detergents do not activate the enzyme and inorganic pyrophosphate almost completely inhibits the enzyme activity. Phospholipase A has an inhibitory effect in the presence of CDP-choline. Phospholipase C, by itself, stimulates the enzyme activity. In the presence of CDP-choline, a higher concentration of phospholipase C partially abolishes the CDP-choline effect on the enzyme. Phosphorylcholine from labeled CDP-choline is rapidly incorporated into lecithin in the assay system used for measuring N-acetylglucosaminyltransferase activity. Capacity for lecithin synthesis is poor in Golgi membranes. However, lecithin synthesis is stimulated by adding exogenous diglyceride, but CDP-choline plus diglyceride failed to activate N-acetylglucosaminyltransferase in Golgi membranes. Finally, various possibilities have been discussed to explain the mechanism of action of CDP-choline on the enzyme.
A Role of Cytidine 5′-Diphosphocholine in the Transfer of N-Acetylglncosamine from UDP-N-acetylglucosamine into Endogenous Acceptor Lipids and Proteins in Rat and Hen Liver Microsomes
Authors: Mookerjea Sailen , Cole D. E. C. , Chow A. , Letts Pamela
Published: December 2009

Evidence is presented for the transfer of N-acetylglucosamine into lipids by cell-free preparations of rat and hen livers. CDP-choline stimulates the formation of this carbohydrate–lipid product. Triton is required for this reaction and for promoting the CDP-choline effect. Requirements for divalent cations, Triton, pH, and temperature optima have been established. The total microsomes, rough microsomes, and Golgi-depleted fractions of rat liver, but not the Golgi-rich membrane fractions, are active in the formation of carbohydrate–lipid product and are responsive to CDP-choline.The labeled lipid–carbohydrate product is stable to mild alkali but labile to mild acid hydrolysis. Following acid hydrolysis radioactive N-acetylglucosamine was recovered by thin-layer electrophoresis. The radioactive lipid has been separated by thin-layer chromatography. Autoradiography of thin-layer plates showed two or three (only in presence of CDP-choline) radioactive bands. Over 90% of the labeled carbohydrate–lipid product has been isolated by DEAE-cellulose acetate column chromatography by elution with ammoniacal chloroform–methanol mixture.A study of simultaneous incorporation of N-acetylglucosamine-1-14C into lipids and proteins in the microsomes showed that the peak incorporation into lipids and into proteins was 5 and 30 min, respectively. This suggests that N-acetylglucosamine incorporation into protein may be mediated through a lipid–carbohydrate intermediate.
Formaldehyde as an Active Site Label of α-Chymotrypsin
Authors: Martin Charles J. , Oza Narendra B. , Marini Mario A.
Published: December 2009

The effect of formaldehyde concentration on the steady state parameters of the chymotrypsin-catalyzed hydrolysis of specific ester substrates has been investigated. At the limit values which are attained in about 3 M formaldehyde, the catalytic rate constant is decreased eightfold and [Formula: see text] increased ca. 2.5-foid. The reduction in activity does not conform to competitive, noncompetitive, uncompetitive, or partially competitive inhibition. The effect of increasing formaldehyde concentration is, however, in accord with an equimolar equilibrium reaction between chymotrypsin and formaldehyde with Kassoc of 5 M−1. Spectro-photometric analysis of the same reaction (J. Biol. Chem. 242, 5736 (1967)) and binding studies with 14C-elabeled formaldehyde (Biochim. Biophys. Acta, 242, 320 (1971)) have shown that both histidine residues react with formaldehyde.From a study of the imidazole group catalyzed hydrolysis of p-nitrophenyl acetate, the effect of formaldehyde on such processes is qualitatively similar to that observed for reactions catalyzed by chymotrypsin. The Kassoc values, however, are higher: 36 M−1 for imidazole and 25 M−1 for acetylhistidine.On the basis of these and other results, it is concluded that formaldehyde reacts with the active site of chymotrypsin to form a modified enzyme with decreased catalytic efficiency. The causative event is most probably due to the formation of an N-hydroxymethyl derivative of the His-57 residue.