bluuebunny/crossref_metadata_2025 huggingface dataset:
The middle ultraviolet spectra of proteins. II. The denatured states of RNase
Authors: A'zary Edward P. , Bigelow Charles C.
Published: December 2009

The major aim of the study was to see whether and how usefully protein denaturation can be followed by difference spectroscopy in the middle ultraviolet (220–250 nm). For this study, the enzyme RNase (bovine pancreatic ribonuclease), already extensively studied by other methods, was used. The results show that denaturation can be followed just as well in the middle ultraviolet as in the near ultraviolet, and that the interpretation of the observations is just as simple as it was in the near ultraviolet (about 280 nm). This is in part fortuitous because, while histidine absorbs at 237 nm, in RNase all four of the histidyl residues appear to be exposed to the environment even in the native state, and hence do not undergo any significant change in their absorption properties when the protein is denatured.RNase had previously been shown to reach different denatured states depending on the denaturant used, by the techniques of difference spectroscopy in the near ultraviolet, optical rotation, and intrinsic viscosity. Our results show these changes are also accompanied by spectral changes (denaturation blue shifts) which give extrema in the difference spectra at 237 nm. At this wavelength only tyrosyl and histidyl residues should contribute significantly to the observed difference. However, data of other investigators show that all the histidyl residues and three of the six tyrosyl residues are exposed to the environment even in the native molecule. Our results therefore can be interpreted in terms of the three buried tyrosyl residues, just as the near ultraviolet data could be.Our results in the middle ultraviolet show : (a) that denaturation of proteins can be followed quantitatively; (b) that, at least in this protein, values of Δε can be assigned to individual tyrosyl residues as they are exposed in denaturation; and (c) that, in agreement with data measured by other methods, RNase is capable of forming three different denatured states, depending on the denaturant used.
Detection of carbon–phosphorus bonds by proton nuclear magnetic resonance spectroscopy
Authors: Benezra Claude , Pavanaram Sripada K. , Baer E.
Published: December 2009

Nuclear magnetic resonance spectroscopy was found, to provide a direct and convenient way to identify P—C bonds; P—CH2 protons appear at higher field than P—O—CH2 protons.
Oxidative degradation of dihydrofolate and tetrahydrofolate
Authors: Chippel Diana , Scrimgeour K. G.
Published: December 2009

7,8-Dihydrofolate has been degraded oxidatively using ferricyanide under anaerobic conditions. Three reaction pathways have been found for this oxidation, and these pathways can be controlled by variation of the pH and by the nature of the buffer anion. At pH 9, treatment of dihydrofolate with two equivalents of ferricyanide leads to quantitative formation of folate. At pH 5.6 in morpholinoethanesulfonic acid buffer, the same reactants produce p-aminobenzoylglutamate, dihydroxanthopterin, and formaldehyde. Each of these products has been identified, and isolated in high yield. 6-Formyldihydropterin can be isolated by oxidation of dihydrofolate in either phosphate or carboxylic acid buffers. This compound does not appear to be an intermediate in the formation of dihydroxanthopterin under the conditions of our experiments. Tetrahydrofolate was also degraded oxidatively using ferricyanide. Although it is more susceptible to cleavage than is dihydrofolate, tetrahydrofolate gives similar products. In addition, moderate quantities of pterin were isolated from oxidation of tetrahydrofolate at pH 5.6.
Effect of organic reducing agents and ferrous ion on thioglucosidase activity of Crambe abyssinica seed
Authors: Tookey H. L. , Wolff I. A.
Published: December 2009

The addition of L-ascorbate or 2-mercaptoethanol to aged crambe seed meal tends to restore the fresh meal pattern of epi-progoitrin hydrolysis to nitriles instead of (R)-goitrin. Neither of these reducing agents has an effect on the breakdown of epi-progoitrin to goitrin by an insoluble particulate thioglucosidase from crambe meal. The addition of ferrous ion to the insoluble particles results in the conversion of epi-progoitrin to (2S)-1-cyano-2-hydroxy-3-butene instead of (R)-goitrin over a range from pH 3.9 to 6.7.
L-Cysteinylglycine: its occurrence and identification
Authors: Tkachuk Russell
Published: December 2009

L-Cysteinylglycine is present in wheat germ. The peptide was isolated as the S-carboxymethyl-2-14C derivative. The identity of S-carboxymethyl-L-cysteinylglycine was determined by amino acid analysis, N-terminal analysis, and mass spectroscopy. L-Cysteinylglycine appears to be widespread in nature as it is found also in E. coli, murine tissue, and human serum. It does not seem to be present in yeast.
Regulation of liver tyrosine aminotransferase activity in inbred strains and mutant mice. II. Studies on the starvation-induced enzyme adaptation
Authors: Blake R. L.
Published: December 2009

The activity of liver tyrosine aminotransferase (TA) (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) in C57BL/6J mice was elevated at 0900 h two- to three-fold above the control level after 48 h of fasting. In DBA/2J mice, TA activity remained low during 48 h of fasting. However, enzyme activity could be increased in fasting DBA/2J mice by the administration of either hydrocortisone or nicotinamide. The administration of glucose to fasting C57BL/6J mice caused a rapid reduction of enzyme activity to the basal level. When hydrocortisone was administered simultaneously with the glucose, the enzyme activity was maintained (approximately 73% of the untreated 1330-h control values) and was elevated about twofold above the enzyme activity in the glucose-treated mice. Multiple injections of cycloheximide (1 μg/g body weight dose) caused a slight reduction (40%) of the starvation-induced enzyme adaptation. In adrenalectomized C57BL/6J mice, TA activity remained low during a 60 h fasting period. Significant strain differences were also demonstrated in the regulation of the apoenzyme and holoenzyme levels in C57BL/6J and DBA/2J mice after 48 h of fasting.
Quantitative estimation of bile salts in serum
Authors: Ali S. S. , Javitt Norman B.
Published: December 2009

Two major modifications of existing methods for the quantitative estimation of serum bile salts by gas–liquid chromatography have been made. A new protein precipitation step using 2,2-dimethoxypropane gives almost complete recovery of added bile salts. A purification step using thin-layer chromatography gives good recovery of mono-, di-, and tri-hydroxy bile acids. Overall recovery of bile salts is greater than 70%. The new procedure reduces the time required for serum bile salt analysis.
Effect of excision and benzimidazole treatment on folate content of wheat leaves and wheat leaf chloroplasts
Authors: Kim W. K.
Published: December 2009

Crude extracts were prepared from freshly harvested wheat leaves, and from leaves floated on water or on solutions of benzimidazole. Extracts were also prepared from chloroplasts isolated with non-aqueous solvents from similar leaves. All extracts were fractionated on DEAE-cellulose columns. Folate content was determined with Lactobacillus casei, Streptococcus faecalis, and Pediococcus cerevisiae as assay organisms.Total folates increased in leaves after excision whether benzimidazole was present or not. The increase was mainly due to an increase of conjugates of 5-methyltetrahydrofolate. Similar folate drifts were observed in chloroplasts isolated from these samples. Peak fraction analyses indicated that methylated folate conjugates were converted to 5-methyltetrahydrofolate, and formylated folates disappeared in leaves floated on water. Benzimidazole treatment arrested the degradation of conjugated folates, except that of a formylated folate found only in chloroplasts.
RNA synthesis in polyoma virus-infected cells. I. Pattern of formation of polyribosome-associated messenger RNA during productive infection
Authors: Cheevers William P. , Sheinin Rose
Published: December 2009

Experimental conditions have been established for selective measurement of the synthesis of mRNA in mouse embryo cells. Using such conditions, it was found that productive infection of these cells by polyoma virus resulted in stimulation of mRNA synthesis. The pattern of induction of mRNA synthesis was biphasic, characterized by distinct "early" and "late" periods, as denoted by the time of initiation of progeny viral DNA replication. The formation of "early" mRNA was first detected at 9–11 h postinfection, 6 h prior to the time of onset of virus-induced synthesis of cell DNA and 9 h prior to initiation of polyoma DNA replication. The initiation of synthesis of "late" mRNA was approximately coincident with the onset of formation of viral DNA. Most of the newly synthesized "early" and "late" mRNA was of relatively small size (8–12 S) and was associated with polyribosomes which sedimented at less than 180 S. The proportion of the total "late" mRNA which was virus-specific was three times higher than that of the total "early" mRNA; however, the mRNA synthesized both "early" and "late" was predominantly cell-specific.
An examination of the relation between endogenous lipid and the activity of the phosphatidylgrycerol-forming enzyme system in beef heart mitochondria and microsomes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

The effect of endogenous lipid on the biosynthesis of phosphatidylglycerol in beef heart mitochondria and microsomes has been studied by assaying the CDP-D-diglyceride-dependent conversion of L-glycero-3-phosphate-2-3H to phosphatidylglycerol. It was found that lipid-depleted (by wafer–acetone–ammonia treatment) subcellular particles retained in mitochondria 30% and in microsomes 71% of the original activity found in lipid-containing subcellular particles. Mitochondria extracted with ether for a prolonged time (20 min) lost all activity, while mitochondria treated with water–acetone (but without ammonia) still retained 61% of the phosphatidylglycerol-forming activity. The loss of activity was found to be irreparable either by the addition of extracted endogenous lipid of subcellular particles, or by the addition of chemically well-defined lipids. Some implications of the experimental findings concerning the possible role of endogenous lipid in the activity of the enzyme system catalyzing the formation of phosphatidylglycerol in subcellular particles are discussed.
Mode of action of choline. V. Sequential changes in hepatic and serum lipids of choline-deficient rats
Authors: Chalvardjian A.
Published: December 2009

A sequential study of the alterations in phospholipid composition of liver and serum was made in rats in early choline deficiency. After 2 days on a choline-deficient (c.d.) diet, the phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) was decreased in the rough and smooth microsomal membranes relative to the choiine-supplemented (c.s.) controls. The decrease in PC/PE was also noted in total hepatic phospholipids after 1 day on the c.d. diet and persisted for at least 12 days. The serum total phospholipids and light-density lipoprotein phospholipids were significantly lower after 2 days in the c.d. rats relative to the c.s. controls. The high-density lipoprotein phospholipids showed a significant decrease after 3 days and levelled off. The earliest detectable change occurring 9 h after ingestion of a c.d. diet was an increase in hepatic triglyceride with no significant change in the PC/PE ratio either in total hepatic or microsomal phospholipids. These results indicate that an alteration in the phospholipid composition of the microsomal fraction (105 000 g postmitochondrial sediment) is not primarily responsible for triglyceride accumulation. However, they do not rule out the possibility of an alteration in some other subcellular fraction being responsible for the fatty livers.
Metabolie control mechanisms in mammalian systems. XI Pyruvate kinase modulation in the rat prostate and seminal vesicles
Authors: Vijayvargiya R. , Schwark W. S. , Singhal Radhey L.
Published: December 2009

The modulation of pyruvate kinase activity by several metabolites has been investigated in the prostate gland and seminal vesicles of normal mature rats. Whereas direct addition of fructose 1,6-diphosphate to the reaction mixture exerted no effect on pyruvate kinase activity, slight activation of the prostatic and seminal vesicular enzyme was produced by L-alanine. Pyruvate kinase activity in both accessory sexual tissues was rapidly inactivated by incubation of the supernatant fluids at 37 °C; this inactivation was prevented by L-alanine. Whereas L-phenylalanine was found to be a competitive inhibitor of prostatic and vesicular pyruvate kinase, L-alanine was capable of preventing and reversing the inhibition produced by L-phenylalanine. Pyruvate kinase activity in both tissues was also inhibited by Cu2+ in concentrations as low as 0.005 mM; 50% inhibition of the enzyme was produced by 0.2 mM Cu2+. Addition of L-alanine prevented as well as reversed the inhibition produced by low concentrations of this cation.The sulfhydryl inhibitor, p-CMB, produced a dose-dependent inhibition of prostatic and vesicular pyruvate kinase whereas addition of penicillamine resulted in a slight activation of the enzyme. In addition, penicillamine effectively reversed the inhibition produced by p-CMB as well as by Cu2+. The thermal inactivation of pyruvate kinase activity in accessory sexual tissues was also prevented effectively when the supernatant fluids were preincubated in the presence of penicillamine.
Evidence for the coupling of the regulations of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase
Authors: Lane E. A. , Mavrides C.
Published: December 2009

An experimental design which simulated a light–darkness schedule reproduced the daily rhythm of tyrosine aminotransferase in rat liver. A similar rhythm was identified for phosphoenolpyruvate carboxykinase. Both enzyme rhythms were profoundly and similarly modified by the amount of dietary protein fed to the animals. The liver glycogen content fluctuated in a manner that produced plots which were virtually the mirror images of the enzyme rhythm plots. The oscillations of phosphoenolpyruvate carboxykinase were less pronounced than those of tyrosine aminotransferase and the same quantitative relationship was found in response to infused nonpharmacological amounts of Cortisol.A relationship in the regulations of the two enzymes suggested by the present and other experiments is envisaged and discussed.
Inorganic pyrophosphatase from Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans)
Authors: Howard Adele , Lundgren D. G.
Published: December 2009

An inorganic pyrophosphatase was isolated from Ferrobacillus ferrooxidans and purified 21-fold. The cation Mg2+ was required for maximum activity; Mn2+, Zn2+, and Co2+ supported less than 10% of the activity with Mg2+. The pH optimum of the enzyme was between 7.5 and 8.5, using a magnesium to pyrophosphate ratio of one. The purified enzyme was unable to hydrolyze adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, or various other monophosphates; p-nitrophenyl-phosphate, the substrate for alkaline pyrophosphatase, was not hydrolyzed. Sulfhydryl binding agents did not inhibit enzyme activity, and the enzyme, in the presence of Mg2+, was heat-stable.
Cholinesterase bonded to paper
Authors: Stasiw R. O. , Brown H. D. , Hasselberger F. X.
Published: December 2009

Cholinesterase has been bonded to Procion brilliant orange – DEAE-cellulose. The matrix-supported enzyme has a lower activity than the free enzyme in solution; thermal stability, however, is much greater. A marked difference in the Km (apparent) value of the derivatized protein was observed (substrate used was acetylcholine chloride, free Cholinesterase Km = 9.6 × 10−4 M, bound Cholinesterase Km = 1.0 × 10−2 M).
Increased Subcellular Hyaluronidase in the Rat after Hydrocortisone Injection
Authors: Bowness J. M. , Carpenter N. , Barry D. N.
Published: December 2009

The activity of hyaluronidase per milligram protein was found to be significantly greater in subcellular fractions from kidneys of rats injected 12 or 18 h previously with 10 mg hydrocortisone than it was in control rats. The difference was greater at 12 h than at 6 or 18 h, and at 12 h those fractions containing lysosomes showed the greatest increase. A higher specific activity of hyaluronidase in some subcellular fractions from liver and spleen was also found after hydrocortisone. Cycloheximide, given 5 h after hydrocortisone, abolished the increase in hyaluronidase measured at 12 h. However, actinomycin D given [Formula: see text] before hydrocortisone caused only a slight decrease in the effect on hyaluronidase in kidney homogenates.
Effect of Maleylation and Amidination of Hemoglobin on its Haptoglobin and Oxygen Reactions
Authors: Lockhart W. L. , Smith David B.
Published: December 2009

Maleic anhydride and ethyl acetimidate reacted readily with all ε-amino groups of human hemoglobin. Maleyl-hemoglobin showed evidence of structural alterations and did not bind to haptoglobin. Amidino-hemoglobin was similar to hemoglobin in electrophoresis below pH 9, sedimentation rate, and gel filtration. Its oxygen equilibrium showed no cooperative effects or Bohr effect. Since it retained capacity to bind haptoglobin, it is concluded that hemoglobin amino groups are not involved in the reaction with haptoglobin.
Biosynthesis of Gibberellins in Gibberella fujikuroi. The Sequence after Gibberellin A4
Authors: Pitel D. W. , Vining L. C. , Arsenault G. P.
Published: December 2009

Gibberellins A1, A3, A4, and A7 biosynthetically labeled with 14C were added to gibberellin-producing cultures of Gibberella fujikuroi. In cultures transferred to 10% glucose solution most of the gibberellin A4 was first dehydrogenated to gibberellin A7 before being 7-hydroxylated to gibberellin A3. A small amount of gibberellin A4 was 7-hydroxylated directly to form gibberellin A1, but none of the gibberellin A1 was subsequently metabolized to gibberellin A3. In cultures growing in a medium which supported higher yields of gibberellin A1, more of the 14C-labeled gibberellin A4 supplement was directly 7-hydroxylated to this product. Gibberellin A3 was again formed mainly via gibberellin A7, but a small amount was derived by dehydrogenation of gibberellin A1. In these cultures gibberellin A4 was also metabolized to an unidentified gibberellin-like substance, probably an isomer of gibberellin A1.
Substituent Effects on the Chymotrypsin-Catalyzed Hydrolysis of Specific Ester Substrates
Authors: Williams R. E. , Bender M. L.
Published: December 2009

The substituent effect on the chymotrypsin-catalyzed hydrolysis of several phenyl esters of specific substrates has been studied. The second-order acylation rate constants (kcat/Km(app)) obey a linear free energy relationship with ρ = +0.63 for phenyl hippurates and ρ = +0.46 for phenyl N-benzyloxycarbonyl-L-tryptophanates when substituents are introduced into the phenyl group of the ester function. These results further support the previously proposed general acid – general base mechanism for the acylation reaction and the formation of a tetrahedral intermediate in the course of the reaction.
Purification and Properties of γ-Glutamyl Cyclotransferase from Pig Liver
Authors: Adamson E. D. , Szewczuk A. , Connell G. E.
Published: December 2009

The widely occurring enzyme γ-glutamyl cyclotransferase acts on γ-glutamyl peptides to effect the release of the terminal glutamyl residue as the cyclic derivative pyrrolidone carboxylic acid. The enzyme has been purified from pig liver by (1) ammonium sulfate precipitation from the supernatants of homogenates, (2) CM-cellulose treatment, (3) DEAE-Sephadex chromatography, and (4) preparative polyacrylamide gel electrophoresis. The homogeneity of the highly purified enzyme was demonstrated by ultracentrifugation and electrophoretic analyses. By means of (5) isoelectric focusing, two forms of the enzyme with isoelectric points 4.87 and 4.95 were separated. These forms proved to have very similar sedimentation velocities, molecular weights, and amino acid compositions, and to have the same amino acid, glycine, as the N-terminal residue.
Studies on Mammalian and Molluscan Steroid Sulfatase. Solubilization and Properties
Authors: Bleau G. , Chapdelaine A. , Roberts K. D.
Published: December 2009

Steroid sulfatase from the microsomal fraction of rat liver exhibits properties which are suggestive of an allosteric type of enzyme. The kinetics of dehydroisoandrosterone sulfate cleavage are normal while the cleavage of cholesterol sulfate presents a cooperative effect beyond 0.8 × 10−6 M at the protein concentration used. This abnormal kinetic behavior is normalized by the addition of an analogue of this substrate, sodium lauryl sulfate. This analogue elicits a biphasic effect on the cleavage of cholesterol sulfate while the cleavage of dehydroisoandrosterone sulfate and pregnenolone sulfate is inhibited at all of the concentrations tested.Using polyacrylamide gel electrophoresis in the presence of sodium lauryl sulfate, the molecular weight of what is believed to be the monomeric form of the enzyme was estimated to be approximately 23 000.Steriod sulfatase from Helix pomatia cleaves the sulfate of dehydroisoandrosterone while cholesterol sulfate remains intact. This enzyme was found to have a molecular weight near or below 50 000 and was isolated as a single band of sulfatase activity by polyacrylamide gel electrophoresis.
Synthesis of octadeca-trans-2,cis-9,cis-12-trienoic acid and its evaluation as a honey bee attractant
Authors: Starratt A. N. , Boch R.
Published: December 2009

Octadeca-trans-2,cis-9,cis-12-trienoic acid has been synthesized from linoleic acid and shown to have activity similar to that of the natural honey bee attractant isolated from pollen. Some related compounds were inactive.
Nucleoside Transport. I. A Mediated Process in Human Erythrocytes
Authors: Oliver J. M. , Paterson A. R. P.
Published: December 2009

The transport of nucleosides by human erythrocytes has been studied by measuring disappearance of 14C-labelled uridine and thymidine from incubation media; saturation kinetics and competitive inhibition of uridine uptake by formycin B were observed. Uridine and thymidine transport were nonconcentrative under incubation conditions in which cellular ATP concentrations were maintained. Efflux of uridine or thymidine from preloaded cells was induced by an inward flow of various nucleosides, but not by purine or pyrimidine bases. It is concluded that the transport of nucleosides across the plasma membrane of human erythrocytes is accomplished by a nonconcentrative, 'facilitated diffusion' mechanism of broad specificity.
Nucleoside Transport. II. Inhibition by p-Nitrobenzylthioguanosine and Related Compounds
Authors: Paterson A. R. P. , Oliver J. M.
Published: December 2009

p-Nitrobenzylthioguanosine (NBTGR) was found to be a potent inhibitor of nucleoside transport by human erythrocytes; initial rates of uridine uptake were reduced to zero upon exposure of cells to 10−6 M NBTGR. The inhibitor was firmly bound because repeated washing did not restore uridine transport capability to NBTGR-treated cells. NBTGR inhibited the influx of uridine, inosine, and cytidine, without inhibiting the uptake of the corresponding bases, or that of D-glucose or L-leucine. Uridine antagonized the NBTGR inhibition of uridine transport in a concentration-dependent manner. NBTGR and related compounds appear to interact with the mechanism for the facilitated transport of nucleosides.
Synthesis of Diphytanyl Ether Analogues of Phosphatidic Acid and Cytidine Diphosphate Diglyceride
Authors: Kates M. , Park C. E. , Palameta B. , Joo C. N.
Published: December 2009

The chemical synthesis of 2,3-di-O-phytanyl-sn-1-glycerophosphate was carried out in two ways: (a) by phosphorylation of 2,3-diphytanyl-sn-glycerol with diphenylphosphoryl chloride in pyridine, followed by catalytic hydrogenolysis of the phenyl groups; and (b) by condensation of 1-iodo-2,3-di-O-phytanyl-sn-glycerol with silver di-p-nitrobenzyl phosphate in anhydrous benzene, followed by catalytic hydrogenolysis of the p-nitrobenzyl groups. The free diether phosphatidic acid obtained was converted to the dipotassium and disodium salts. The diether phosphatidic acid was also converted to the diether analogue of cytidine diphosphate diglyceride by condensation with cytidine monophosphate morpholidate in pyridine. The physical properties of these diether analogues are described, as well as their stabilities towards acid hydrolysis.
Analysis of the Organic Acids of Bryophyllum calycinum
Authors: Marriage Paul B. , Wilson David G.
Published: December 2009

Major and minor acids present in dried leaf tissue of Bryophyllum calycinum were identified or characterized using the combined methods of ion exchange chromatography, paper and thin-layer chromatography, gas chromatography, and infrared spectroscopy. After esterification of the acids in methanol containing H+ form cation exchange resin, their amounts were determined by gas chromatography. The acids identified and their approximate percentage amounts of the total acid content were: malic 32.5%, citric 10.1%, isocitric 46.5%, succinic 1.0%, fumaric 0.9%, pyruvic 1.0%, oxalacetic 0.4%, α-ketoglutaric 0.5%, glyoxylic 0.1%, lactic 0.2%, oxalic 0.2%, and cis-aconitic 1.6%. Tentative characteristics were proposed for previously undetermined acids present mainly in amounts from 0.05% to 0.6%. These were apparently mono-, di-, and tricarboxylic acids containing from three to seven carbons in the carbon chain. Most of these evidently had additional functional groups, including methyl, keto, and hydroxyl groups. The boiling points of the methyl esters of these acids ranged from 140° to 300°.
Occurrence of UDP-N-acetylglucosamine: Glycoprotein N-Acetylglucosaminyltransferase Activity in Human and Rat Sera
Authors: Mookerjea Sailen , Chow A. , Hudgin R. L.
Published: December 2009

The enzyme for transferring N-acetylglucosamine into an exogenous acceptor glycoprotein pre-treated with appropriate glycosidase has been found to be present in rat and human sera. The enzyme is present in soluble form and does not require Triton X-100 for activation. The pH optima, divalent cation requirements, and linearity with time and enzyme concentrations have been established. Normal human serum has three times higher activity than normal rat serum.
Stimulation of Sphingomyelin Synthetase by Sulfhydryl Reagents
Authors: Sribney Michael
Published: December 2009

Chicken liver mitochondrial sphingomyelin synthetase (CDPcholine:ceramide cholinephosphotrans-ferase, EC 2.7.8.3) was shown to be able to utilize erythro- as well as threo-ceramides as substrates for sphingomyelin synthesis; the former was incorporated only if sulfhydryl reagents were present in the incubation mixture. It was found that the most potent activator was menadione, while N-ethylmaleimide, oxidized glutathione, oxidized CoA, and CoQ0 were active, but to a much lesser extent. In contrast to the chicken liver synthetase, which showed broad specificity for sulfhydryl reagents, the synthetase from rat liver was only activated by oxidized CoA. The mechanism of activation is considered to occur as a result of a conformational change in the enzyme, subsequent to sulfhydryl alkylation, such that it allows the erythro- as well as threo-ceramide to penetrate to the active site.
Intrastrand Bifunctional Alkylation of DNA in Mammalian Cells Treated with Mustard Gas
Authors: Walker I. G.
Published: December 2009

When mustard gas reacts with DNA one of the products consists of two guanines linked by a mustard molecule (GMG). This compound can arise from a pair of guanines lying in opposite strands of the DNA molecule and in this form is the basis of the cross-linking effect of mustard gas on the DNA molecule. The present study shows that GMG arises also in significant amounts from the reaction of two guanines that lie in the same strand. Using DNA isolated from mustard gas treated mammalian cells or alkylated in vitro, the number of GMG's per unit length of DNA was determined. The extent of renaturability which measures the degree of cross-linking was also determined. The number of GMG's exceeded the number of cross-links. When alkylated DNA was separated into a renaturable (cross-linked) fraction and one that contained single strands, analysis showed that both fractions contained GMG.
The Carboxy-Terminal Region of Sturgeon Muscle Aldolase
Authors: Anderson P. J.
Published: December 2009

The modification of the carboxy-terminal of sturgeon muscle aldolase with carboxypeptidase produces similar kinetic effects to those observed on modification of the rabbit muscle enzyme. The nature and molar ratios of amino acids released indicate that differences between the two enzymes occur in the carboxy-terminal region.
Sodium–Potassium Stimulated Adenosinetriphosphatase of Vascular Smooth Muscle
Authors: Wolowyk Michael W. , Kidwai A. M. , Daniel Edwin E.
Published: December 2009

A Na+ and K+ stimulated, Mg2+-requiring ATPase has been demonstrated in the homogenate and microsomal fractions of aortic smooth muscle. Tris–HCl buffer interfered with the Na+ plus K+ stimulatory effect revealing only a less specific nucleotide phosphatase activity. Also, in order to demonstrate stimulation with Na+ and K+, a soluble activating cofactor isolated from the 100 000 × g supernatant fraction was required. The highest activity was obtained from a density gradient prepared microsomal fraction with soluble activator added in the presence of 50 mM Na+, 2 mM K+, and 50 mM histidine buffer at pH 7.6, with 4 mM Mg-ATP as substrate. The greater part of the monovalent cation stimulated activity was due to a ouabain-insensitive Na+-stimulated ATPase activity. However, the remainder of the total stimulation was ouabain-sensitive and attributed to a Na+–K+-ATPase.
Influence of Gonads on the Sulfurylation of 11-Deoxycorticosterone and Corticosterone by Rat Liver Cytosol
Authors: Torday J. S. , Klein G. P. , Giroud C. J. P.
Published: December 2009

When incubated in the presence of ATP, the cytosol fraction of rat liver, kidney, and gut homogenate is capable of sulfurylating 11-deoxycorticosterone, the extent of sulfurylation by liver cytosol being three to four times more efficient in the female than in the male. This sex-linked difference in liver sulfokinase activity is further demonstrated by the finding that sulfurylation of corticosterone is decreased by ovariectomy and brought back to the level observed in normal females by injection of 17β-estradiol (at the daily dose of 6 μg/100 g body weight) to castrated females; this effect is increased by orchidectomy and further enhanced by the administration of the same dose of 17β-estradiol to orchidectomized rats.
Comparative Uptake of Selenium by Low Cystine and High Cystine Proteins
Authors: Jenkins K. J. , Hidiroglou M.
Published: December 2009

Purified preparations of skin collagen, tendon gelatin, and thymus histones, isolated from various animals administered 75Se-labeled selenite, contained only a trace of cystine (plus [Formula: see text]-cystine) and incorporated very little 75Se activity. Keratins and milk proteins, which contained relatively high levels of cystine, also had high radioselenium levels. The data indicate that the cystine content of a protein may be a factor which affects selenium incorporation, possibly by the formation of selenotrisulfides (P–S–Se–S–P).
Properties of Inosine-5′-phosphate Dehydrogenase from Bacillus subtilis
Authors: Wu Tai-Wing , Scrimgeour K. G.
Published: December 2009

Inosine-5′-phosphate (IMP) dehydrogenase has been purified to apparent homogeneity from extracts of Bacillus subtilis. The enzyme is inhibited by GMP in a complex manner which suggests cooperative interaction between protein subunits of IMP dehydrogenase. Several techniques have been used to desensitize the enzyme to inhibition by GMP. IMP dehydrogenase has a high molecular weight, and is composed of subunits each with a molecular weight of approximately 100 000. The properties of IMP dehydrogenase from B. subtilis are compatible with its being a regulatory enzyme in the biosynthesis of GMP from IMP.
Protein Synthesis by Ribosomes from Rat Kidney Cortex: Effect of Aldosterone and Bilateral Adrenalectomy
Authors: Majumdar Adhip P. Nandi , Trachewsky Daniel
Published: December 2009

Experiments with renal cortical ribosomes from adrenalectomized rats have shown that the incorporation of phenylalanine into polypeptide by ribosome preparations from aldosterone-treated animals is greater than incorporation by control ribosomes by a range of 20–24%. When incorporation was directed by poly U the hormone-stimulated ribosomes produced a range of 15–32% greater incorporation than control ribosomes. This latter difference might be due to a change in the functional capacity of the ribosomes induced by the hormone treatment since the poly U directed binding of phenylalanyl-tRNA to the ribosomes from aldosterone-treated rats was greater than that to the control ones by a range of 16–35%. Furthermore, ribosomes from the aldosterone-stimulated animals had an average of 26% more ribonuclease activity than the control ribosomes.
Ox Brain Aminopeptidases, Further Purification of Three Bovine Enzymes
Authors: Sobel R. E. , Brecher A. S.
Published: December 2009

Four peptidases capable of acting upon Leu∙Gly∙Gly have been separated from bovine brain extracts, and three have been highly purified by ammonium sulfate fractionation and by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. A Mn2+-stimulated aminopeptidase (E2) was purified 180-fold over an initial 16 300 × g supernatant fraction and has a M.W. of approximately 117 250 as estimated by sucrose density gradient centrifugation. Two peptidases which are inhibited by heavy metals (E1P1 and E1P2) were purified approximately 2683-fold and 2461-fold over the initial extracts and have approximate M.W.'s of 61 500 and 85 700, respectively. E1P1 and E1P2 have Km's of 0.36 and 0.38 mM, respectively. The pH optima for E1P1, E1P2, and E2 are 6.6–7.8, 7.2–8.1, and 7.8–9.6. E2 is more heat-labile than E1P1 and E1P2 since it is completely inactivated by heating at 55° for 30 min at pH 6.5 under which conditions the latter two enzymes retain full activity. The ΔEa for the hydrolysis of Leu∙Gly∙Gly was calculated to be 10 800, 13 420, and 15 480 for E1P1, E1P2, and E2. E1P1 and E1P2 hydrolyze tripeptides but do not cleave dipeptides. E2 cleaves tripeptides, and only cleaves Leu∙Gly, of a series of dipeptides examined, having a specificity differing notably from leucine aminopeptidase. All the enzymes were inactive towards leucine-p-nitroanilide and leucine-β-naphthylamide, suggesting that they should not be classed among the arylamidases. On the basis of differential heat stability, it is estimated that a peptidase sedimented by ammonium sulfate at 20% saturation contains 40% of the original tripeptidase activity; E2 represents 30% of the activity; E1P1 and E1P2 contribute 12 and 18%, respectively, to the tripeptidase activity in the original extracts.
Studies on Spermatogenesis in Rats. I. Application of the Sedimentation Velocity Technique to an Investigation of Spermatogenesis
Authors: Go V. L. W. , Vernon R. G. , Fritz I. B.
Published: December 2009

A sedimentation velocity chamber at unit gravity (Staput fractionation) was employed to separate cells obtained from suspensions of testes from adult rats, and the differential cell count was determined to estimate the sedimentation profiles of cell populations in various fractions. The incorporation of thymidine-3H into acid-insoluble portions of cell fractions was measured at varying times following the intratesticular administration of thymidine. The kinetic profiles of thymidine-3H incorporation into spermatogonia, primary spermatocytes, spermatids, and spermatozoa were compared with radioautographs prepared from histological sections of testes obtained from the same animals. Good correlations were observed between radioactivity peaks in Staput fractions and grain localization in cells of the spermatogenesis cycle at varying time periods after thymidine-3H injection. Data obtained with the Staput technique can be rapidly quantitated. The limits of the Staput fractionation technique for the separation of cells from the rat testis were evaluated with respect to the possible isolation of homogeneous populations for biochemical studies, and to the currently more amenable separation of heterogeneous classes of cells for kinetic studies. The usefulness of the method was validated for application to subsequent investigations.
Studies on Spermatogenesis in Rats. III. Effects of Hormonal Treatment on Differentiation Kinetics of the Spermatogenic Cycle in Regressed Hypophysectomized Rats
Authors: Go V. L. W. , Vernon R. G. , Fritz I. B.
Published: December 2009

The general hormonal requirements for the restoration of spermatogenesis in regressed hypophysectomized rats were investigated. With the aid of the Staput fractionation technique, it was established that thymidine-3H was readily incorporated into spermatogonia and resting spermatocytes. Labeled cells did not progress to form appreciable numbers of primary spermatocytes or spermatids in the absence of hormonal replacement. The inhibition of formation of pachytene primary spermatocytes in hypophysectomized rats was overcome by administration of follicle-stimulating hormone (FSH), luteinizing hormone (LH), or testosterone, but a combination of either FSH plus LH, or FSH plus testosterone, was required for the progression of pachytene primary spermatocytes to spermatids and spermatozoa. Carnitine acetyltransferase (CAT) measurements in testes from various groups of animals provided ancillary evidence consistent with the conclusion that either FSH, LH, or testosterone was required for the normal restoration of pachytene-diplotene spermatocyte formation. However, one or more additional blocks in spermatogenesis existed in hypophysectomized animals, since elevation of depressed testicular CAT levels in hypophysectomized rats to normal levels required FSH plus LH, or FSH plus testosterone. Cortisone and thyroxin treatment had no measurable effects on testicular function in hypophysectomized rats.
Changes in the Amount and Properties of Adenyl Cyclase in Brown Adipose Tissue during Acclimation of Rats to Cold
Authors: Muirhead Michelle , Himms-Hagen Jean
Published: December 2009

Adenyl cyclase of interscapular brown adipose tissue of rats undergoing acclimation to cold was measured in order to find out whether the amount of the nor adrenaline-stimulated enzyme might be increased in cold-acclimated rats, in which a characteristic large increase in the overall calorigenic response to noradrenaline (nonshivering thermogenesis) is induced by the prolonged exposure to cold (4–8 weeks). It was found that the growth of the brown adipose tissue in the acclimating rats is accompanied by an increase in the total amount of noradrenaline-stimulated adenyl cyclase activity. Since this increase does not keep pace with the growth of the tissue a reduction in specific activity of the noradrenaline-stimulated adenyl cyclase was observed. Thus no evidence was obtained for an increased concentration of noradrenaline-responsive structures in the cell membrane of the brown adipose tissue of cold-acclimated rats.In contrast, fluoride-stimulated adenyl cyclase activity increased almost twofold after only 2 days of exposure to cold and before the growth of the tissue had started. Thereafter the activity of this enzyme increased in parallel to the growth of the tissue. The extra activity that developed during the first 2 days persisted throughout the period of acclimation to cold (8 weeks); it disappeared within 1 day when the cold-acclimated rats were returned to room temperature.These changes in adenyl cyclase activity are more rapid than any hitherto reported. They presumably reflected a change in the properties of the cell membrane in response to an environmental stress. It is not known whether the increase in measurable fluoride-stimulated adenyl cyclase activity reflects an increase in the synthesis of a specific protein or an unmasking of the activity of an enzyme already present.No change in the specific activity of cyclic AMP phosphodiesterase was observed; during acclimation to cold the total amount of this enzyme increased in parallel with the growth of the tissue.
A Gastric Inhibitory Polypeptide II: The Complete Amino Acid Sequence
Authors: Brown John C. , Dryburgh Jill R.
Published: December 2009

Porcine gastric inhibitory polypeptide is a 43 amino acid residue polypeptide with the amino acid sequence Tyr–Ala–Glu–Gly–Thr–Phe–Ile–Ser–Asp–Tyr–Ser–Ile–Ala–Met–Asp–Lys–Ile–Arg–Gln–Gln–Asp–Phe–Val–Asn–Trp–Leu–Leu–Ala–Gln–Gln–Lys–Gly–Lys–Lys–Ser–Asp–Trp–Lys–His–Asn–Ile–Thr–Gln. Fifteen of the first 26 amino acids occur in the same position as they do in porcine glucagon, and nine of the first 26 in the same position as in porcine secretin. The calculated molecular weight of the polypeptide is 5105.
Interaction of [4-14C]Estradiol and its Metabolites with Polynucleotides in the Presence of Peroxidase
Authors: Jellinck P. H. , Fletcher Rosemarie
Published: December 2009

Incubation of [4-14C]estradiol with either horseradish peroxidase or uterine preparations resulted in the formation of water-soluble products if certain polynucleotides were also present. It was shown that both systems required hydrogen peroxide and that binding of the steroids occurred more readily with DNA or polyadenylic acid than with RNA or polyuridylic acid. The 14C radioactivity associated with the polynucleotides was present in unidentified metabolites and a small amount of unchanged estradiol. These products were bound weakly and could be removed by mild procedures in contrast to the conjugates formed with albumin or thiols by this system. The significance of the action of peroxidase in the binding of estrogens in rat uterus is discussed.
An Immunoglobulin Light Chain of Subgroup III1 of λ Type with a Free Sulfhydryl Group
Authors: Buchwald Barbara M.
Published: December 2009

A myeloma protein, Du, (γ1)2 (λ)2, is shown to have an extra cysteine residue in the light chain. This light chain is from the same λ variable region subgroup as are two Bence-Jones proteins, X and Bau, which also have an extra cysteine residue. The position of this residue is the same in all three chains.
Preparation of Methyl Derivatives of Some Organic Acids for Analysis by Gas–Liquid Chromatography
Authors: Atkins Craig A. , Canvin David T.
Published: December 2009

One microgram to 1 mg of organic acid was completely converted to its methyl derivative when it was dissolved in methanol and treated with an excess of ethereal diazomethane. Single products were obtained from α-ketoglutaric acid, oxaloacetic acid, pyruvic acid, glyoxylic acid, or hydroxypyruvic acid on reaction with thionyl chloride – methanol. The separation of 24 organic acids by gas–liquid chromatography on a Reoplex 400 column is presented.
Biosynthesis of Furanochromones
Authors: Harrison P. G. , Bailey B. K. , Steck Warren
Published: December 2009

Radioactive khellin, visnagin, and visamminol were recovered from Ammi visnaga plants fed [1-14C]-acetate. A trapping experiment with 5,7-dihydroxy-2-methylchromone indicated that this compound was formed from acetate in the plant. It was then prepared labelled with 14C and 3H and administered to shoots, demonstrating its conversion to visamminol and furanochromones. A biosynthetic pathway is proposed: acetate → 5,7-dihydroxy-2-methylchromone → peucenin →→ visamminol →→ visnagin and khellin.The gas chromatography of some A. visnaga constituents is described. The furanocoumarins xanthotoxin and isopimpinellin were shown to be present in the plant.Administration of labelled 5,7-dihydroxy-2-methylchromone to shoots of Angelica archangelica resulted in the elaboration of labelled furanochromones, suggesting that the furan moiety of furanochromones and furanocoumarins may be formed via similar if not identical enzyme systems.
Biosynthesis of Thermopsis Alkaloids from Carbon-14 Dioxide. Evidence for the Formation of the Pyridone Bases from Lupanine Via 5,6-Dehydrolupanine
Authors: Cho Y. D. , Martin R. O.
Published: December 2009

The observed relative rates and chronology of labelling of the alkaloids of Thermopsis rhombifolia and T. caroliniana following pulse feeding of 14CO2 were consistent with the formation of the pyridone bases from lupanine, via 5,6-dehydrolupanine. Rhombifoline appears to be the first of the tricyclic bases to be formed from the tetracyclic ones. No evidence was obtained for the formation of sparteine, casting doubt on its role in the formation of these alkaloids.
Acetyl-CoA Holocarboxylase Synthetase of Rat Adipose Tissue
Authors: Desjardins P. R. , Dakshinamurti K.
Published: December 2009

A preliminary separation of acetyl-CoA holocarboxylase synthetase from acetyl-CoA apo- and holocarboxylases is reported.
The Mass Spectrometric Identification of the Isomers of Tyramine: Identification of para-Tyramine in Rat Brain
Authors: Boulton Alan A. , Majer John R.
Published: December 2009

The integrated ion current produced at an m/e value of 137 has been used to distinguish between the three isomers of tyramine. Using the integrated ion current technique it has been shown that only para-tyramine exists in rat brain.
Interrelationships in the Metabolism of Liver Arachidonoyllecithins and Plasma Cholesteryl Arachidonate in the Rat
Authors: Holub B. J. , Kuksis A.
Published: December 2009

Male rats were injected intravenously with albumin-bound 1-14C-arachidonic acid or 1-14C-palmitoyllysolecithin and the molecular species of the labeled liver and plasma lipids were identified after various periods of time. There was an extensive esterification of lysolecithin with arachidonic acid in the rat liver, which was in agreement with the earlier demonstrated specific incorporation of labeled arachidonic acid into the liver lysolecithins. The subsequent gradual release of the labeled liver arachidonoyllecithins into the plasma was accompanied by a steady rise in the radioactivity of plasma cholesteryl arachidonate. In other experiments, it was shown that radioactive arachidonic acid esters of cholesterol are readily formed in vitro from 1-14C-arachidonoyllecithin and free cholesterol, and that such esters upon subsequent injection can contribute arachidonic acid for the biogenesis of lecithin from lysolecithin in the liver as a result of hydrolysis. These results are compatible with the operation of a plasma–liver cycle which results in a net transfer of cholesterol from plasma to the liver and a return of lysolecithin and arachidonic acid to the plasma in the form of lecithin.
On the Structure of the Renin Inhibitor from Hog Kidney
Authors: Rakhit S.
Published: December 2009

The structure of the renin inhibitor isolated from hog kidney has been assigned as phosphatidylethanolamine containing a large percentage of polyunsaturated fatty acids. This is contrary to previous findings (1,2).