bluuebunny/crossref_metadata_2025 huggingface dataset:
Agents affecting lipid metabolism. XXXVII. Separation of rat serum lipoproteins with dextran sulfate
Authors: Hill P. , Dvornik D.
Published: December 2009

Dextran sulfate was used to separate rat serum lipoproteins into insoluble low-density and soluble high-density fractions. Lipid composition of the separated fractions was compared to that of lipoprotein fractions prepared by ultracentrifugation; data indicate that dextran sulfate precipitates lipoproteins with a density lower than 1.063.In rats treated with ethyl p-chlorophenoxyisobutyrate and estradiol-17β comparable, though not identical, changes in the composition of serum lipoproteins were observed with both methods of separation. The observed differences may reflect the fundamental difference in the basis of separation used by the methods. Data obtained with dextran sulfate may indicate changes in the protein moieties of lipoproteins.
A note on the status of 16-ketoestrone as a conversion product of injected estrone in hen's urine
Authors: Mulay Shree , Henneberry G. O. , Common R. H.
Published: December 2009

A conversion product of injected 14C-labelled estrone in hen's urine which was formerly identified tentatively as 16-ketoestrone was shown to contain approximately 90% nonketonic material that corresponded in chromatographic mobility with estradiol-17α, a known urinary conversion product of injected estradiol-17β or estrone. Contrary to the previous suggestion, therefore, 16-ketoestrone is not a major urinary conversion product of injected estrone.
The isolation and characterization of a new form of porcine pancreatic carboxypeptidase A (carboxypeptidase Ae)
Authors: Narayanan A. Sampath , Anwar R. A.
Published: December 2009

A new porcine carboxypeptidase A, designated as Ae, has been isolated from twice crystallized elastase. The enzyme preparation appeared homogeneous in the ultracentrifuge and gel electrophoresis, and had an s20,w of 3.34 S. A molecular weight of 34 700 was obtained from sedimentation equilibrium and the zinc content was 0.93 mole/mole enzyme. The new enzyme differs from the known porcine carboxy-peptidases (Aj, A2, A3) in solubility properties and amino acid composition. The amino acid compositions of Ai, A2, and Ae, which mainly differ in the content of isoleucine, are suggestive of their being genetic variants due to several amino acid replacements in the molecule.
Lipid metabolism in rabbit lungs
Authors: Wolfe B. M. J. , Anhalt B. , Beck J. C. , Rubinstein D.
Published: December 2009

The incorporation of palmitate-3H into phospholipids and neutral lipids by alveolar slices from rabbit lung rose with increasing medium palmitate concentration, but glycerol-U-14C incorporation was inadequate to account for all of the palmitate-3H esterified. The highest specific activities with respect to palmitate-3H were found in the triglycerides, diglycerides, and phosphatidylcholine fractions, although that of the phosphatidyl-N,N-dimethylethanolamine was greatest in the early stages of incubation. Incubation of slices with the labelled precursors for 5 min followed by a chase of 175 min resulted in a rapid drop in the specific activity of the phosphatidyl-N,N-dimethylethanolamine and diglycerides with concomitant increases in phosphatidylcholine and triglycerides. However, the radioactivity of the lecithin was too great and the specific activity of the phosphatidylethanolamine too low for the methylation pathway to be of importance in the synthesis of lecithin.Lysophosphatidylcholine could be converted to phosphatidylcholine by lung slices but its addition did not increase the esterification of palmitate. Methionine-methyl-14C was incorporated into phospholipids only slightly, phosphatidylserine having the highest specific activity.Following the intravenous injection of the labelled precursors, half the radioactivity in the lung was found in the phosphatidylcholine fraction of the alveolar tissue, little radioactivity being found in the bronchiolar tissue and the alveolar cell-free saline washings. The highest specific activities were found in the triglycerides and phosphatidylcholine fractions in all three tissue components.
Phosphonolipids. XXI. Synthesis of phosphonic acid analogues of diether L-α-(N,N-dimethyl)cephalins
Authors: Baer Erich , Rao K. V. Jagannadha
Published: December 2009

A synthesis of phosphonic acid analogues of diether L-α-(N,N-dimethyl)cephalins, viz. di-O-tetradecyl, di-O-hexadecyl, and di-O-octadecyl L-α-glyceryl-(N,N-dimethyl-2-aminoethyl)phosphonate, is described. They were obtained by phosphonylating the appropriate D-α,β-dialkyl glycerol ethers with 2-bromoethylphosphonic acid monochloride and triethylamine at 0°, and treating the reaction products with dimethylamine at 50–55° for 3 days. The procedure is applicable to the synthesis of saturated and unsaturated dialkyl α-(N,N-dimethyl)phosphonocephalins in both their optical active forms.
A rapid method for the assay of dextranase
Authors: Koh T. Y. , Khouw B. T.
Published: December 2009

A method, based on the liberation of a dye complex from Blue Dextran 2000, is described for the assay of dextranase. The reaction was conducted at 40 °C for 5 min at pH 4.0 in a mixture containing 0.5% Blue Dextran 2000 and the enzyme. The Km for the hydrolysis of Blue Dextran 2000 was estimated at 2.5 × 10−6 M.
Effects of various conditions on the movement of carbon atoms derived from glucose into and out of protein in rat brain
Authors: Yoshino Y. , Elliott K. A. C.
Published: December 2009

The time course of entry of radioactive carbon from intravenously administered [U-14C]-glucose into protein in five brain regions in rats was studied using an automatic amino acid analyzer coupled, through a flow cell, with a scintillation counter. Radioactivity appeared in protein-bound alanine and in glutamic and aspartic acids and, more slowly and to a much lesser extent, in serine, proline, and glycine; none was detected in any other bound amino acids. There appear to be two main groups of proteins, one of which turns over more slowly than the other. In pentobarbital narcosis, radioactivity in protein-bound alanine increased but no other changes were obvious. In convulsions induced by pentylenetetrazol or picrotoxin, and during hypoxia, the entry of radioactive carbon into protein-bound alanine, glutamic acid, and aspartic acid decreased. At 24 h after the injection of radioactive glucose, radioactivity in free amino acids had almost completely disappeared in normal animals though protein remained radioactive. Pentobarbital narcosis did not affect this situation. Radioactivity was released from protein into free amino acids during hypoxia, during convulsions induced by pentylenetetrazol, picrotoxin, or oxygen at high pressure, and after insulin treatment. Evidence was obtained that free alanine occurs in more than one pool.
Oxygen toxicity and carbon deprivation in Astasia longa
Authors: Bégin-Heick Nicole
Published: December 2009

The changes in metabolic activity of Astasia longa during oxygen treatment and carbon deprivation were examined. In many respects (growth, cell composition, and some enzymatic activities) the two treatments produce similar effects. Other parameters such as cellular respiration are affected differently. Oxygen treatment inhibits the ethanol-stimulated respiration but affects endogenous respiration very little. At the same time, the total alcohol dehydrogenase activity of the cell is increased at least 10-fold. In starved cells, the endogenous respiration is decreased parallel to the cellular content of carbohydrate reserves, but the ability of the cell to utilize ethanol stays constant. The NADH : cytochrome c oxidoreductase activity of the oxygen-treated cells is decreased by 70% whereas this activity is decreased less than 30% in starved cells. It is proposed that the inhibition of the NADH : cytochrome c oxidoreductase activity is the primary activity affected by oxygen treatment in both acetate- and ethanol-grown cells. The characteristic effects of oxygen treatment on ethanol-grown cells are probably the result of the combination of increased NAD-dependent alcohol dehydrogenase activity and decreased NADH : cytochrome c oxidoreductase activity. Such a modification of these two activities would tend to keep NAD in the reduced state and therefore limit cellular reactions which require oxidized NAD.
Preparation of gibberellin A1-3,4-3H
Authors: Pitel D. W. , Vining L. C.
Published: December 2009

An improved procedure is described for preparing gibberellin A1-3,4-3H by catalytic reduction of gibberellin A3 with tritium.
Developmental changes in the fatty acid composition of the larval lipid of the house cricket Acheta domesticus (L.)
Authors: Lipsitz E. Y. , McFarlane J. E. , Henneberry G. O.
Published: December 2009

The fatty acid composition of larvae of Acheta domesticus (L.) at two distinct ages (12 days and 30 days) was determined. No appreciable age differences were observed in the composition of fatty acids from lipid extracts; palmitic, stearic, oleic, and linoleic acids were found to be the major fatty acids, together accounting for about 90% of the total. However, distinct age differences were found in the fatty acid composition of neutral lipid components. The most marked developmental change was a reduction from 34.5% to 10.9% in the myristic acid content of the monoglyceride. High concentrations of short-chain fatty acids were found in at least three neutral lipid components, and considerable alterations in the concentration of these fatty acids occurred during development.
On the specificity of cytidine diphosphate diglycerides in monophosphoinositide biosynthesis by rat brain preparations
Authors: Bishop H. H. , Strickland K. P.
Published: December 2009

Some properties of the enzyme cytidine diphosphate diglyceride : inositol transferase which catalyzes the reaction[Formula: see text]have been studied with various preparations from rat brain. The enzyme was found to be primarily located in the microsomal fraction (either the 12 000 × g supernatant or the 90 000 × g pellet). Optimal conditions were established for pH and concentrations of each of the reactants and Mn2+ ion. A number of cytidine diphosphate diglycerides with different fatty acids in the 1- and 2-positions were synthesized chemically and used as substrates for the above reaction. The substrates included those with the following distribution of fatty acids: 1,2-dipalmitoyl; 1,2-distearoyl; 1-palmitoyl,2-oleoyl; 1-stearoyl,2-oleoyl; 1-oleoyl,2-stearoyl and 1,2-dioleoyl. It was observed that those substrates with oleic at the 2-position were more readily utilized than those containing palmitic or stearic acids at the 2-position or at both the 1- and 2-positions. This selectivity for cytidine diphosphate diglyceride with an unsaturated fatty acid at the 2-position was consistent with the pattern of fatty acid distribution found for isolated rat brain monophosphoinositide. In the latter, saturated fatty acids (stearic and palmitic acids) were predominantly located at the 1-position and unsaturated fatty acids (20:4, arachidonic acid) were predominantly located in the 2-position.
The regulation of protein synthesis in nephrotic rat kidney ribosome preparations
Authors: Nicholls D. M. , Ryan M. P. , Miall S. H. , Westall C. G. , Cappon I. D.
Published: December 2009

The incorporation of 14C-leucine and 14C-phenylalanine into protein was measured in ribosomes following the deoxycholate treatment of microsomes from control and nephrotic rat kidney homogenates. The pH 5 enzyme fraction was obtained from postmicrosomal supernatant (S105) of control and nephrotic kidney homogenates. The incorporation was markedly increased in the nephrotic preparations and depended on changes in both the ribosomes and pH 5 enzymes. The increase was present in the presence of either endogenous mRNA or synthetic mRNA (poly U). It was associated with increased binding of aminoacyl-tRNA as well as increased incorporation of aminoacyl-tRNA into peptide. Active ribonuclease activity was decreased in nephrotic kidney S105, pH 5 enzymes, and microsomes compared to comparable fractions in control kidney. Total ribonuclease activity was also decreased in nephrotic kidney in these fractions. No increase in polysome content was found in these ribosomes prepared with deoxycholate but when deoxycholate was eliminated, a crude homogenate of nephrotic kidney had twice as many polysomes.
Inactivation of bovine thrombin by nitrous acid
Authors: Magnusson S. , Hofmann T.
Published: December 2009

Bovine thrombin is rapidly inactivated by nitrous acid at pH 4.35 and 0°. The clotting activity falls off more rapidly than the activity towards N-benzoylarginine ethyl ester. The loss of esterase activity parallels the loss of the N-terminal isoleucine residue. N-Benzoylarginine ethyl ester gives almost full protection of the esterase activity and the N-terminal isoleucine, but only partial protection of the clotting activity. These results suggest strongly that this residue is essential for activity and indicate that thrombin resembles the pancreatic serine proteinases in this respect. The initial rate of inactivation of thrombin by nitrous acid is higher than that of trypsin, and considerably higher than that of elastase and α-chymotrypsin under the same conditions, suggesting that the N-terminal residue is more readily accessible to the reagent. The experiments strengthen the hypothesis that all mechanisms of prothrombin activation are proteolytic and suggest that they involve the liberation of the essential N-terminal isoleucine.
Glyoxylate aminotransferases from oat leaves
Authors: Brock B. L. W. , Wilkinson D. A. , King J.
Published: December 2009

Alanine : glyoxylate, glycine : 2-oxoglutarate (EC 2.6.1.4), and serine : glyoxylate aminotransferases were isolated and partially purified by two different procedures from oat leaves (Avena sativa L. var. Garry). Initial velocity, enzyme resolution and reconstitution, pyridoxal 5-phosphate activation, and enzyme inhibitor studies showed that the serine : glyoxylate aminotransfer reaction operates by the Ping Pong Bi Bi mechanism and involves the coenzyme pyridoxal phosphate, as do other aminotransferases, but does not appear to involve metal ions or sulfhydryl groups. Enzyme specificity determinations suggested that glyoxylate aminotransfer reactions are catalyzed by protein(s) distinct from other aminotransferases. Results are discussed in relation to earlier work with comparable enzymes from wheat leaves and to similar studies with enzymes from animal sources.
Isolation and preservation of cell nuclei for studies on RNA polymerase activity
Authors: Read R. S. D. , Mauritzen C. M.
Published: December 2009

The suitability of saponin for the isolation and of glycerol for the preservation of mammalian cell nuclei has been investigated. The nonionic detergent saponin was found to be a useful cell lytic agent in a procedure for the isolation of nuclei from several mammalian cell types. The RNA polymerase activity of rat liver nuclei was not affected by treatment with saponin or with some other nonionic detergents that were tested. Low concentrations of ionic agents also did not affect the activity of the enzyme though higher concentrations caused lysis of the nuclei. The preservation of structure and enzyme activity in the isolated nuclei was achieved by storage at low temperature in a medium containing 70% glycerol together with a suitable concentration of a divalent metal and phosphate buffer.
Mg2+-activated ATP hydrolysis and sulfhydryl groups in membranes from human erythrocytes
Authors: Smith Frances M. , Verpoorte Jacob A.
Published: December 2009

The ATP-hydrolyzing activity of membranes prepared from human erythrocytes depends on the presence of Mg2+ ions. Maximum activity is observed when the concentrations of Mg2+ and ATP are equal. The pH–rate profile of the enzymatic reaction shows a maximum at pH 7.8. The ATP-hydrolyzing activity has decreased to half the maximal activity at pH 6.3 and 9.8, respectively. The enzyme is inhibited by sulfhydryl reagents like p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide. Membranes that are titrated with NaOH or treated with p-CMB lose up to 30% of their protein content. This loss is reversed when Mg2+ or Mg2+ plus ATP is added. However, neither Mg2+ nor Mg2+ plus ATP detectably alters the rate of reaction between the sulfhydryl groups and specific reagents, nor protects the ATP-hydrolyzing activity from inhibition by p-CMB. The amount of protein which dissociates from the membrane increases with pH. The curve describing this extraction has a sigmoid shape with an inflection point at pH 10.2. Maximum solubilization is obtained at pH 11.2. Thus the results indicate that sulfhydryl groups and also the structure of the membrane are important for the ATP-hydrolyzing activity.
The reaction with nitrous acid of the "active site" N-terminal isoleucine in chymotrypsin and derivatives
Authors: Dixon Joan W. , Hofmann T.
Published: December 2009

Bovine α-chymotrypsin, δ-chymotrypsin, homoarginine-δ-chymotrypsin, and bovine chymotrypsin B were inactivated by nitrous acid at pH 3.8–4.4 and 0°. The potential activity of bovine chymotrypsinogen A was not affected under these conditions. The inactivation rates as measured with the substrates α-N-acetyl-L-tyrosine ethyl ester, carbobenzoxyglycine p-nitrophenyl ester, and p-nitrophenyl acetate, and by 3H-di-isopropyl phosphorofluoridate incorporation were identical with the deamination rates of the amino group of the N-terminal isoleucine-16, but were slower than the deamination rates of the amino groups of the N-terminals half-cystine-1 and alanine-149. It is concluded that the deamination of isoleucine-16 is directly responsible for the inactivation. Third-order deamination rate constants of the N-terminal isoleucine-16 were measured and the following values (in min−1M−2) were obtained: α-chymotrypsin, 0.4–0.6; homoarginine-δ-chymotrypsin, 0.05; di-isopropyl phosphoryl-α-chymotrypsin, [Formula: see text]; tosyl-α-chymotrypsin, 0.05; chymotrypsin B, 0.3; α-chymotrypsin in guanidinium chloride, 30–50; homoarginine-δ-chymotrypsin in guanidinium chloride, > 20. The deamination rate constants for the model dipeptides isoleucylvaline and valylvaline are 40 and 46, respectively (Kurosky, A., and Hofmann, T.: to be published). A comparison shows that the constants for the dipeptides and the two chymotrypsins in guanidinium chloride are very close and are probably those of a fully exposed amino group. The much lower constants for the other enzymes and derivatives represent the varying degrees of accessibility of the amino group and show the usefulness of the reagent as a conformational probe. The results are fully compatible with the proposed structure of α-chymotrypsin (1) and the proposed function of the N-terminal isoleucine (2).
Differential inhibition of bacterial tRNA methylases by S-adenosylethionine and other adenine derivatives
Authors: Moore B. G.
Published: December 2009

The inhibition of bacterial tRNA methylases by S-adenosylethionine resulted in apparent preferential inhibition of cytosine and adenine methylation by S-adenosylmethionine. Uracil and guanine methylases were more resistant to the inhibitor. Adenine and dimethylallyladenine specifically inhibited guanine methylases with only slight to negligible inhibition of adenine and cytosine methylation.
The dissociation in vitro of the α- and β-lipoprotein components of human and rat very low density lipoproteins
Authors: Kook Abraham I. , Eckhaus Anita S. , Rubinstein David
Published: December 2009

Heparin precipitates of human and rat serum lipoproteins, dissolved, dialyzed, and analyzed by paper electrophoresis, were found to migrate as β-lipoproteins. The pre-β-lipoproteins disappeared. Using specific anti-human β- and α-lipoprotein sera it was shown that the heparin precipitate of human very low density lipoproteins (VLDL) contained β-lipoproteins, whereas the supernatant contained α-lipoproteins. This indicates that these components are dissociated by the heparin precipitation procedure. Following partial delipidation of the heparin precipitate, a procedure which exposes the antigenic site of the α-lipoprotein of the VLDL, it can be demonstrated that the α-lipoprotein component is completely removed by the heparin precipitation procedure. Partially delipidated rat VLDL showed four immuno-reactive components when reacted against rabbit anti-rat VLDL serum, but only one, corresponding to the β-lipoprotein, when reacted with rabbit antiserum to heparin precipitate.Chemical analysis of the lipid components of the heparin precipitate and supernatant of human and rat VLDL reveals that over 90% of the triglycerides are precipitated by the heparin and that the supernatant fraction contains lipoproteins having a phospholipid to protein ratio higher than any of the other lipoproteins.Upon reincubation of the dissolved and dialyzed human serum heparin precipitate and its supernatant a pre-β band reappeared on paper electrophoresis. The recombined pre-β-lipoprotein was isolated and found to react with anti-β-, but not with anti-α-lipoprotein serum. This is a characteristic of native pre-β-lipoproteins. Evidence has also been obtained that the high density lipoproteins combine only with the β-lipoproteins originally present in the VLDL.
Some reactions of sterculic and malvalic acids. A new source of malvalic acid
Authors: Roomi M. W. , Hopkins C. Y.
Published: December 2009

Oxidation of sterculic acid by aqueous alkaline permanganate gave 9,11-dioxononadecanoic acid. Oxidation of sterculic acid by peracetic acid gave a mixture of 9-oxononadec-10-enoic and 11-oxononadec-9-enoic acids. Acetolysis of malvalic acid gave a mixture of 8-hydroxy-9-methylene-heptadecanoic and 9-hydroxy-8-methylene-heptadecanoic acids. Seed oil of Pterospermum acerifolium Willd. (family Sterculiaceae) contained malvalic acid as its major cyclopropenoid component. It comprised about 16% of the total fatty acids.
Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase
Authors: Hétu C. , Gianetto R.
Published: December 2009

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.
A potentially unique type of lipid storage in the migrating pink salmon
Authors: Robinson J. Stephen , Mead James F.
Published: December 2009

Analysis of the composition of the hump developed by the pink salmon (Oncorhynchus gorbuscha) during the spawning migration has revealed that it consists largely of lipid at the start of the migration and that lipid is largely replaced by water at the end. Quantitation of the individual lipids reveals that the high triglyceride present at the start decreases, resulting in a relative increase of cholesterol ester and free fatty acid during the migration.
Biosynthesis of linear furanocoumarins
Authors: Brown Stewart A. , El-Dakhakhny M. , Steck Warren
Published: December 2009

Labelling experiments with 14C and 3H have shown that linear furanocoumarins of Ammi majus, Angelica archangelica, Heracleum lanatum (Umbelliferae), and Ruta graveolens (Rutaceae) are formed from umbelliferone via the dihydrofuranocoumarin, marmesin. The unsubstituted furanocoumarin psoralen is formed first, and hydroxylation and O-methylation then lead to methoxy-substituted derivatives, with other alkylated psoralens assumed to be formed analogously. No evidence was obtained for oxygenation of the coumarin nucleus before attachment of a side chain, since oxygenated umbelliferones were poorly utilized for furanocoumarin biosynthesis. A detailed biosynthetic route to linear furanocoumarins, consistent with known stereochemistry, is proposed.A separation of furanocoumarins by column partition chromatography on formamide-impregnated silica, and syntheses of [3-14C]umbelliferone, [2-14C]- and [3-14C]skimmin, [3-14C]daphnetin, [2-14C]-7-hydroxy-8-methoxycoumarin, and [2-14C]5-methoxy-7-hydroxycoumarin are described.
Circular dichroism studies on Mengo virus variants and their constituent ribonucleates
Authors: Kay Cyril M. , Colter John S. , Oikawa Kimio
Published: December 2009

A comparison of the circular dichroic spectra of three variants of Mengo virus and their constituent ribonucleates shows that the curves are essentially identical to one another, with respect to crossover points and the positions and amplitudes of troughs and peaks. This would suggest that the manner in which the proteins are arranged around the RNA chain in the virus is precise and similar in all three cases, and that there are no significant differences in the density of packing among the virions. By subtracting the RNA contribution from that of the virus, the circular dichroic curve of the protein in situ was obtained. The small amplitude of the ellipticity bands at ~205 and 220 mμ (transitions characteristic of the right-handed α-helix) suggests that the protein, as it exists in the virion, possesses a low α-helical content. This observation is consistent with the fact that the viral protein contains a large content of proline and non-α-helix-forming amino acids.
Amino acid sequence around the active site aspartic acid in penicillopepsin
Authors: Šodek J. , Hofmann T.
Published: December 2009

Penicillopepsin, the acid proteinase of Penicillium janthinellum, was specifically inactivated with diazoacetylnorleucine methyl ester. The peptide containing the glycollylnorleucine methyl ester group was isolated from a peptic digest. The amino acid sequence was found to be Ile∙Ala∙β(glycollyl-Nle OMe)-Asp∙Thr∙Gly∙Thr∙Thr∙Leu and is thus almost identical with the active site peptide of porcine pepsin: Ile∙Val∙Asp∙Thr∙Gly∙Thr∙Ser. This finding provides strong evidence for an evolutionary homology between penicillopepsin and porcine pepsin.
Isolation and characterization of β-lipolytic hormone from porcine pituitary gland
Authors: Gilardeau C. , Chrétien M.
Published: December 2009

A lipolytic substance was isolated from porcine pituitary glands. It's amino acid composition, molecular weight, N-terminal amino acid, isoelectric point, and biological activities are reported. These results are compared to the corresponding values of sheep β-lipolytic hormone.
The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group
Authors: Purdie Jocelyn E. , Benoiton N. Leo
Published: December 2009

The action of α-chymotrypsin on L- and D-phenylalanine ethyl esters (PEE), L- and D-phenylalanine p-nitrobenzyl esters (PNBE), L-phenylalanine methyl and isopropyl esters, and N-methyl-L-phenylalamne methyl ester has been studied using a pH-stat. The D-esters were not hydrolyzed but acted as competitive inhibitors of the hydrolysis of the L-isomers. The N-methyl ester was very slowly hydrolyzed due to its low Kcat. For L-PEE (pK 7.23) and L-PNBE (pK 6.93), the activity of α-chymotrypsin is displaced to a more acid region relative to that for the N-acyl amino acid esters. The Km increases sharply below pH 6.5 while the kcat and kcat/Km show maxima at pH 6 and 7.6, respectively. On the acid side kcat is controlled by a basic group of pK 4.86 for L-PNBE and pK 5.1 for L-PEE, and kcat/Km by a basic group of pK 6.4 for L-PNBE and pK 6.6 for L-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the catalytically active entities of the enzyme–substrate complex and acyl enzyme, the α-amino group of the substrate is protonated, (iii) the pK of the basic group on the acyl enzyme is considerably lowered by the presence of the [Formula: see text] of the substrate, and (iv) the increase in Km and Ki below pH 6.8 is due to the development of unfavorable charge interactions.
Role of glucose cycloacetoacetate hydrolysate on tyrosine oxidation
Authors: Nath M. C. , Bhai Idrees
Published: December 2009

In the livers of the scorbutic guinea pigs the activity of p-hydroxyphenylpyruvic acid oxidase was found to remain unchanged but profoundly reduced after the feeding of extra tyrosine. Glucose cycloacetoacetate hydrolysate, a reducing compound, when ingested simultaneously with tyrosine was found to prevent the reduction in the enzyme activity. This effect was comparable to that of ascorbic acid.
Peroxidase-catalyzed conjugation of [4-14C] estradiol with albumin and thiols
Authors: Jellinck P. H. , Fletcher Rosemarie
Published: December 2009

The mechanism of conjugation of estradiol with either albumin or various thiols in the presence of horseradish peroxidase was studied and these two systems were shown to differ in a number of ways. With albumin, the reaction was slow, required Mn2+, and was inhibited by catalase but not iodoacetamide. In contrast, the reaction with thiols was rapid, did not require Mn2+, and was relatively insensitive to catalase. The yield of conjugates with different thiols and the effect of various inhibitors were also determined. The nature of the water-soluble steroid conjugate was investigated and the product obtained with glutathione shown to differ from that formed by rat liver microsomes. The possible extension of this model system to the situation existing in estrogen-stimulated uteri is discussed.
Formation of p-tyramine from DOPA and DOPamine in rat brain
Authors: Boulton Alan A. , Quan Lillian
Published: December 2009

Labelled p-tyramine was isolated and identified in rat brain 15 min after an intraventricular injection of DL-3(3,4-dihydroxyphenyl)alanine-2-14C. Chromatographic and mass spectrometric techniques indicated that the dehydroxylation occurred at the meta position.
A ribosomal-boand aminopeptidase in Escherichia coli B: substrate specificity
Authors: Matheson A. T. , Dick A. J. , Rollin F.
Published: December 2009

The substrate specificity of the ribosomal-bound aminopeptidase from Escherichia coli B has been studied using di-, tri-, and tetrapeptides. The enzyme shows strong activity to leucyl, methionyl, threonyl, and lysyl peptides. Of the other dipeptides tested considerable hydrolysis was observed only if the C-terminal amino acid was leucine or methionine. In a given series of peptides the rate of hydrolysis of the N-terminal peptide bond increased as the size of the peptide increased. Although leucyi dipeptides were hydroiyzed more rapidly than the corresponding methionyl dipeptide the reverse was true with the tripeptides tested. No carboxypeptidase activity was observed and peptides containing D-amino acids were not hydroiyzed. The substrate specificity of the aminopeptidase was compared with the known N-terminal sequences of E. coli proteins to determine whether the enzyme may be involved in the removal of N-formylmethionyl from newly synthesized polypeptides.
Secretion rate of dehydroepiandrosterone and dehydroepiandrosterone sulfate in benign essential hypertension as compared to normal subjects
Authors: Shao A. , Nowaczynski W. , Kuchel O. , Genest J.
Published: December 2009

A study of the secretion rate of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) in normal subjects and patients with benign essential hypertension by a double isotope dilution method showed a fivefold increase in the secretion rate of dehydroepiandrosterone (from 9.8 mg/day ± 3.1 mg/day S.D. to 52.0 mg/day ± 15.5 mg/day S.D.), and a sixfold increase of dehydroepiandrosterone sulfate in hypertensive patients (from 10.7 mg/day ± 2.9 mg/day S.D. to 60.7 mg/day ± 18.6 mg/day S.D.). This study was carried out following the administration of 14C-labelled DHEA and 3H-labelled DHEA-S and involved an initial column chromatographic separation of urinary DHEA-glucuronide and sulfate.
A comparison of brain and liver catechol O-methayltransferase
Authors: Rock Gail Dolan , Tong J. H. , D'Iorio A. D.
Published: December 2009

Partially purified catechol O-methyltransferase (COMT) was obtained from both rat liver and brain and the properties of the two preparations were compared. The enzymes had the same pH optimum, heat and pH stability, ionic requirements, and response to inhibitors. Both had a molecular weight of approximately 25 000. They differed only in the fact that the brain COMT had one less activity band and [Formula: see text]th the specific activity of the liver enzyme.
State of iron(III) in normal human serum: low molecular weight and protein ligands besides transferrin
Authors: Sarkar Bibudhendra
Published: December 2009

A fraction of Fe(III) in normal human serum is bound to both low molecular weight as well as protein ligands besides transferrin. Citrate was shown to be the major Fe(III)-binding substance in the low molecular weight fraction. Amino acids, sugars, and organic acids, such as ascorbate, pyruvate, and lactate, showed very little or no binding to Fe(III) in normal serum. Iron(III)-binding proteins other than transferrin were shown to be present in normal serum when the native serum with [59Fe(III)] was fractionated by (NH4)2SO4 and Sephadex G-150. The presence of these proteins was observed when trace amounts of Fe(III) were added to the normal serum and when the iron-binding capacity was saturated with Fe(III) to 50% and 100%. These proteins were eluted in the void volume of Sephadex G-150 and none of them corresponded electrophoretically to transferrin. The results of the gel filtration of a mixture of [131I]-transferrin and the proteins eluted in the void volume of Sephadex G-150 were strongly in favor of the Fe(III)-proteins as being neither transferrin aggregates nor transferrin adducts with other proteins. Immunoelectrophoresis of the Sephadex G-150 void volume proteins on agar gel against the antibody to transferrin revealed the absence of transferrin. The presence of at least six proteins in this fraction was shown by immunoelectrophoresis. Positive precipitin reactions were obtained with the antibodies to α2-macroglobulin, γG-globulin, γA-globulin, and γM-globulin. At least two more proteins in this fraction remained unidentified. When the same fraction containing [59Fe(III)] was treated with the whole antisera and the precipitates were counted for radioactivity, a typical antigen-antibody reaction curve was obtained as the antibody concentration was increased. Similar experiments with this fraction and antibodies to α2-macroglobulin, γG-globulin, γA-globulin, and γM-globulin failed to show any significant radioactivity in the precipitate. Since this fraction did not contain any transferrin, it was concluded that there are proteins besides transferrin which can act as ligands for Fe(III) in normal blood plasma.
Fungal metabolism of flavonoids. Purification, properties, and substrate specificity of an inducible laccase from Polyporus versicolor PRL 572
Authors: Pickard M. A. , Westlake D. W. S.
Published: December 2009

Laccase (o-diphenol: O2 oxidoreductase, EC 1.10.3.1) was obtained from culture filtrates of P. versicolor grown under a variety of conditions. The effect of gratuitous and nongratuitous inducers was examined and the general characteristics of the enzyme are reported. Purification of the enzyme, including the separation of two isoenzymic forms on DEAE-Sephadex A-50, and the substrate specificity of the two forms, are described.Kinetic data indicated that the enzyme had a higher affinity for flavonoid compounds than substituted phenols and phenylenediamines. To act as a substrate, flavonoids did not require a saturated C-2—C-3 bond, but did require a substituent at the C-3 position and a phenyl group at the C-2 position. Highest activity was obtained using flavonols as substrates. Flavonols glycosylated at the C-3 position were oxidized more slowly than the parent aglycon. No correlation was observed between the pattern of flavonol hydroxylation or flavonol glycosylation and ability to act as a substrate.
Fluorescent Substances in Protein Hydrolysates. II. Comparison of Some Protein and Peptide Hydrolysates
Authors: Ledvina M. , LaBella F. S.
Published: December 2009

Fluorescence was measured in the hydrolysates of several proteins and peptides of known amino acid composition. The most intense fluorescence (emission maximum at 445 nm) was found in acid, but not enzymic, hydrolysates of tryptophan-rich proteins; nine fluorescent fractions were resolved from acid hydrolysates of these proteins by gel filtration. Fluorescence of substances in tryptophan-containing proteins is largely eliminated by reduction, but little affected by oxidation or ultraviolet irradiation. The substances are thermostable and apparently are not amino acids.Tyrosine, tyrosine-containing peptides, and tryptophan-free proteins yielded fewer fluorescent products than did tryptophan-rich proteins. The number and amount of fluorescent derivatives produced from tyrosine during acid hydrolysis depends on the particular protein; also, intra- or intermolecular interactions involving tyrosine or its degradation products appear to influence the nature of fluorescence in protein hydrolysates.The present findings suggest possible quantitation of the tryptophan content of proteins by fluorometric measurement of tryptophan derivatives produced by acid hydrolysis.
Molecular Species of Glycerophosphatides and Triglycerides of Egg Yolk Lipoproteins
Authors: Gornall D. A. , Kuksis A.
Published: December 2009

Four major density classes of lipoproteins were isolated from hen's egg yolk by conventional ultracentrifugation and each class was subjected to thin-layer chromatography on hydroxylapatite. Lipid extracts were obtained from the total egg yolk, the density classes recovered at each stage of centrifugation and from the fractions resolved on hydroxylapatite. The proportions of the lipid classes in the total lipid extracts were determined by direct gas chromatography. The molecular species of the glycerides and phospholipids were identified by means of specific enzymic hydrolyses following isolation of the lipid classes by thin-layer chromatography.The results indicate that the major differences in the lipid composition of the egg yolk lipoproteins are confined to variations in the proportions of cholesterol, triglycerides, and phospholipids. There are lesser differences in the proportions of the various phospholipid classes among the different lipoproteins. All lipoprotein classes, except the water-soluble fraction, contained the same species of each phospholipid and triglyceride. The lipids of the water-soluble fraction were more saturated than those of the other lipoproteins.
Fragmentation of Haptoglobin by Plasmin
Authors: Ofosu F. , Campbell D. W. , Connell G. E.
Published: December 2009

Human haptoglobin type Hp 1-1, is hydrolyzed by plasmin to give two dissimilar products, P1 and P2, with molecular weights of 78 000 and 17 000, respectively. The larger fragment, P1 has the same N-terminal amino acids, valine and isoleucine, as intact haptoglobin. The smaller fragment, P2, is derived from the carboxyl-terminal ends of the β-chains, and has an N-terminal serine.The haptoglobins of genetic types Hp 2-2 and Hp 2-1, which are found as a series of oligomers, are also sensitive to digestion by plasmin in an analogous manner.
Glutamate Dehydrogenase from Pea Roots: Purification and Properties of the Enzyme
Authors: Pahlich E. , Joy K. W.
Published: December 2009

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) has been purified 1250-fold from pea roots. The preparation contains only a single protein, and the molecular weight was estimated to be 208 000 ± 10 000. The enzyme shows NADH (aminating) and NAD+ (deaminating) activities, but the ratio of these activities is not constant and can be changed experimentally. NADPH activity is also present and shows a relatively constant ratio to NAD+ activity. EDTA inhibits NADH activity in intermediate concentrations, but reactivates at higher concentrations. NAD+ (and NADPH) activity is only slightly changed by EDTA. The effects of dioxane and the coenzymes on the enzyme are also reported. Mechanisms which could explain the different activity ratios, in terms of two interconvertible enzyme forms, are discussed.The pH optimum for NADH and NAD+ activities is about pH 8.0. Michaelis constants were found to be: α-ketoglutarate, 3.3 × 10−3 M; ammonium (sulfate), 3.8 × 10−2 M; glutamate, 7.3 × 10−3 M; NADH, 8.6 × 10−4 M; NAD+, 6.5 × 10−4 M. The enzyme is highly specific for the substrates glutamate and α-ketoglutarate, showing no alanine or aspartate dehydrogenase activity, and no deamination with a range of amino acids.
Metabolism of Coumarin and 4-Methylcoumarin by Rat-Liver Microsomes
Authors: Gibbs P. A. , Janakidevi K. , Feuer G.
Published: December 2009

The metabolism of coumarin and 4-methylcoumarin by rat-liver microsomes has been studied. The major metabolites of coumarin in vitro, viz. 3- and 7-hydroxycoumarin, o-hydroxyphenyllactic acid, and o-hydroxyphenylacetic acid, were identified by thin-layer chromatography, and ultraviolet and fluorescence spectroscopy. Metabolites derived from 4-methylcoumarin in vitro have shown similar chromatographic and spectral characteristics as those of coumarin, suggesting that they are the methyl analogues of corresponding coumarin metabolites.
Improved Methods for Preparing Pure Gibberellins from Cultures of Gibberella fujikuroi. Isolation by Adsorption or Partition Chromatography on Silicic Acid and by Partition Chromatography on Sephadex Columns
Authors: Pitel D. W. , Vining L. C. , Arsenault G. P.
Published: December 2009

Procedures have been developed for the separation of closely related gibberellins on a preparative scale by partition chromatography on columns of Sephadex. This technique simplifies the isolation of gibberellins which have previously been difficult to obtain, or have been overlooked. It has particular value in preparing radiochemically pure gibberellins after biosynthetic labeling. Selective fermentations were used to promote the formation of required gibberellins by Gibberella fujikuroi. These gave enriched mixtures which, after an initial fractionation by silicic acid chromatography, were conveniently separated by Sephadex partition chromatography to yield pure compounds.
The Effect of Ethanol and Acetaldehyde on the Metabolism and Vascular Resistance of the Perfused Heart
Authors: Gailis L. , Verdy M.
Published: December 2009

The effect of ethanol and acetaldehyde on the perfused nonworking rat and guinea pig hearts was studied. Ethanol (100 mM) initially increased the coronary vascular resistance, but had no appreciable effect on oxygen consumption, glucose-U-14C oxidation, or anaerobic glycolysis. Acetaldehyde (1 mM) increased the heart rate and oxygen consumption, and decreased the coronary vascular resistance. The decrease in coronary resistance was not affected by propranolol but the increase in heart rate was partially blocked. The loss of amino acids or aspartate transminase activity from the heart was not affected by the presence of ethanol. Ethanol-1-14C, either at 10 or 100 mM, was not oxidized by the perfused rat heart.
Structural Investigations on a Cell-Wall Lipopolysaccharide from Neisseria sicca
Authors: Adams G. A.
Published: December 2009

Lipopolysaccharide (LPS) prepared from Neisseria sicca in 1.5% yield contained D-glucose, D-glucosamine, D-galactosamine, 3-deoxyoctulosonic acid, protein, lipid A, and phosphate. The molecule was judged to be homogeneous as tested by free boundary electrophoresis. D-Galactosamine was associated exclusively with the polysaccharide portion of the molecule and was in fourfold excess of D-glucosamine. The latter hexosamine was primarily a constituent of the lipid A moiety in which it formed the backbone structure linked glycosidically 1 → 4. To this structure, fatty acids, principally β-hydroxymyristic acid and β-hydroxylauric acid, were linked along with phosphate groups. The D-glucosamine units in the polysaccharide portion of the LPS molecule were also attached by 1 → 4 glycosidic linkages. D-Galactosamine units did not survive the methylation procedures due presumably to the lack of acyl protecting groups on its amino groups. Methylation results showed that approximately one-third of the D-glucose units were nonreducing end groups, approximately one-third were linked α1 → 2, a small proportion was linked 1 → 4, and the remainder was branched through C-3, C-4, and C-6. Periodate oxidation results were in agreement with the structure proposed on the basis of the methylation data. The LPS of N. sicca was considerably simpler than that of N. perflava and lacked heptose, rhamnose, and ethanolamine components.
The Biosynthesis of Chelidonic Acid
Authors: Malcolm M. J. , Gear J. R.
Published: December 2009

A method was devised for the isolation of chelidonic acid from species of Narcissus and from Chelidonium majus, and for the degradation of the isolated chelidonic acid so as to isolate separable carbon atoms. Radioactive phenylalanine, tyrosine, pyruvate, aspartate, and acetate were poor precursors of chelidonic acid, indicating that it does not arise from aromatic amino acids, from members of the Krebs cycle, or via a polyketide intermediate. Radioactive glucose, ribose, and carbon dioxide were good precursors of chelidonic acid in flowering Chelidonium majus, and the distribution of activity indicated that chelidonic acid arises by the condensation of a three-carbon unit with a four-carbon unit. The feeding results further suggest that these units are produced from the photosynthetic reduction cycle of the plants.
Interaction of Proteoglycans and Chondroitin Sulfates with Calcium or Phosphate Ions
Authors: MacGregor E. A. , Bowness J. M.
Published: December 2009

Equilibrium dialysis was used to study the distribution of calcium or phosphate ions at equilibrium in dialysis cells containing proteoglycan or chondroitin sulfate in one compartment. A higher concentration of calcium or lower concentration of phosphate was found in the cell compartment containing the polymer than in the compartment separated from it by a semipermeable membrane. The difference in calcium concentration across the boundary represents bound calcium. The formation constant (K) for the complex of bound calcium with chondroitin sulfate was investigated and found to be affected by two main factors: ionic strength and calcium/glucuronate ratio. K decreased rapidly with increasing ionic strength up to 0.15. At low ionic strength and high Ca2+/glucuronate ratios, evidence has been obtained that more calcium is bound by the polymers than can be accounted for by the simple equilibrium system, involving the combination of one calcium per disaccharide unit period, whose operation is consistent with the K values found at low Ca/glucuronate ratios over a wide range of ionic strengths. Infrared spectra obtained at high and low Ca2+/glucuronate ratios also indicate the existence of two calcium forms of proteoglycans. Viscosity and ultracentrifugal data show that differences exist between proteoglycans in calcium and sodium solutions. The data for disaggregated proteoglycan preparations indicate that their calcium-binding properties are very similar to those of chondroitin 4-sulfate and are determined by the same factors. One aggregated puppy rib proteoglycan, however, showed significantly greater K values than chondroitin 4-sulfate; these decreased after disaggregation.