bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of diethylnitrosamine on ribonucleic acid and protein synthesis in the liver and lung of the Syrian golden hamster
Authors: Witschi Hanspeter
Published: August 2015

1. Syrian golden hamsters were treated with a single subcutaneous dose of 200mg of diethylnitrosamine/kg. In the liver the treatment produced a significant and early inhibition of the incorporation of orotic acid into RNA and of leucine into protein. Diethylnitrosamine also lowered basal and 20-methylcholanthrene-stimulated activities of hepatic aryl hydrocarbon hydroxylase. 2. RNA synthesis, protein synthesis and aryl hydrocarbon hydroxylase activity were also evaluated in the lungs of the same animals. In this organ only protein synthesis was affected by diethylnitrosamine, but not RNA synthesis or aryl hydrocarbon hydroxylase activity. 3. The incorporation of thymidine into DNA was inhibited in both organs early after diethylnitrosamine treatment and increased 2–3 days later. 4. Although diethylnitrosamine, injected subcutaneously, accumulates in liver and lung in toxicologically active amounts, the acute biochemical responses of the two organs are not identical.
Glucocerebrosidase: reconstitution from macromolecular components depends on acidic phospholipids (Short Communication)
Authors: Ho Mae Wan , Light Nicholas D.
Published: August 2015

The reconstitution of glucocerebrosidase activity from its inactive macromolecular components depended on acidic membrane phospholipids.
Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue
Authors: Detchon P. , Possingham J. V.
Published: August 2015

Chloroplast rRNA synthesis was studied in spinach leaf tissue cultured under sterile conditions which eliminate bacterial rRNA synthesis. The synthesis was inhibited by darkness, but concomitant cytoplasmic rRNA synthesis was unaffected. A complex pattern of labelled rRNA precursors was found in extracts from cultured leaf tissue by using polyacrylamide-gel electrophoresis. However, differences between the precursor profiles of leaf tissue cultured in the light and in the dark could not be correlated with chloroplast rRNA synthesis since large amounts of high-molecular-weight precursors of cytoplasmic rRNA dominated the pattern in both cases. A double-isotope-labelling technique was used, which enabled light-stimulated rRNA synthesis to be studied in whole leaf tissue. Two rapidly labelled RNA species of molecular weights 1.15×106and 0.65×106were detected, which were thought to have possible precursor significance in the synthesis of mature chloroplast rRNA of molecular weights 1.04×106and 0.56×106respectively. Cycloheximide treatment resulted in the accumulation of RNA of molecular weight 1.8×106, whose function is unknown.
The effects of chemical porphyrogens and drugs on the activity of rat liver tryptophan pyrrolase
Authors: Badawy Abdulla A.-B. , Evans Myrddin
Published: August 2015

1. Drugs such as phenobarbitone and phenylbutazone, which increase the concentration of microsomal haem and cytochrome P-450, also increase the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator, as does the haem precursor 5-aminolaevulinate. 2. At 4h after the administration of the porphyrogens 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and griseofulvin, the total pyrrolase activity is increased whereas the haem saturation of the apoenzyme is decreased. This decreased saturation is prevented by pretreatment of the animals with the inhibitor of drug-metabolizing enzymes, SKF 525-A. 3. Pretreatment of rats with the above porphyrogens inhibits the rise in holo-(tryptophan pyrrolase) activity produced by subsequent administration of cortisol, tryptophan and 5-aminolaevulinate with two single exceptions, the possible reasons for which are discussed. 4. At 24h after the administration, in starved rats, of a single daily injection of the above porphyrogens for 1 or 2 days, the holoenzyme activity is significantly increased. 5. It is suggested that the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator can be modified by treatment known to cause destruction, inhibition of synthesis, increased utilization and enhanced synthesis of liver haem. The possible involvement of the latter phenomenon in the aetiology of mental disorders in some patients with porphyria is discussed.
The occurrence of a stepwise-decreasing respiration rate during oxidative assimilation of different substrates by resting Klebsiella aerogenes in a system open to oxygen
Authors: Degn Hans , Lilleør Mette , Iversen J. J. Lønsmann
Published: August 2015

The addition of an oxidizable substrate to a continuous culture of Klebsiella aerogenes is known to cause an increased respiration rate that decreases in discrete steps as the added substrate is being exhausted. We have used a simple new technique to show that this phenomenon is also produced by washed, resting cells harvested from batch or continuous growth culture. The stepwise-decreasing respiration rate is caused by the exhaustion of different pools of intermediates. Each plateau of respiration rate is a measure of the activity of one or more enzymes that are rate limiting in the exhaustion of pools of intermediates. If the identities of the enzymes that are rate limiting at the different plateaux are known, the method may allow the determination, in one experiment, of the activities of up to six different enzymes in the intact bacteria. Integration of the respiration-rate measurements yields the total amount of O2 taken up. After the addition of glucose to the washed resting bacteria 37% of the amount of O2 required for the complete oxidation of the glucose was taken up. Acetate, pyruvate and succinate were all oxidized to the extent of 51%.
The effect of ethylene on [1-14C]glycerol incorporation into phospholipids of etiolated pea stems
Authors: Irvine Robin F. , Osborne Daphne J.
Published: August 2015

The effect of ethylene (10 p.p.m.) on the rate of incorporation of [1-14C]glycerol into phospholipids of etiolated pea stems was studied. After 2–3h treatment with ethylene, incorporation was decreased by 50%. It remained at this value for as long as ethylene was supplied (8h). Handling the plants also caused a temporary decrease in incorporation, which we attribute to the production of endogenous ‘wound’ ethylene. The percentage decrease in incorporation was the same in four major phospholipid fractions, i.e. phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol.
Guanine deaminase inhibitor from rat liver. Isolation and characterization
Authors: Ali Shahid , Sitaramayya A. , Kumar K. Sree , Krishnan Padmanabhan S.
Published: August 2015

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ‘heavy mitochondrial’ fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH4)2SO4 and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH4)2SO4 in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor–phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.
Effect of pH, ionic strength and univalent inorganic ions on the reconstitution of aspartate aminotransferase
Authors: Gianfreda Liliana , Marino Gennaro , Palescandolo Rosaria , Scardi Vincenzo
Published: August 2015

1. The effect of pH change on the reconstitution of aspartate aminotransferase (EC 2.6.1.1), i.e. the reactivation of the apoenzyme with coenzyme (pyridoxal phosphate and pyridoxamine phosphate), was studied in the pH range 4.2–8.9 by using three buffer systems at concentrations ranging from 0.025 to 0.1m. 2. Although the profile of the reconstitution rate–pH curve in the range pH5.2–6.8 (covered by sodium cacodylate–HCl buffer) reflects the influence of the H+ concentration on the reconstitution process, the profile of the curve in the pH ranges 4.2–5.6 and 7.2–8.25 (covered respectively by sodium acetate–acetic acid and Tris–HCl buffers) appears to be influenced by the ionic strength of the buffer. 3. The reconstitution is also influenced by univalent inorganic ions such as halide ions and, to a lesser extent, alkali metal ions, which are known to alter the water structure.
A specific collagenase from rabbit fibroblasts in monolayer culture
Authors: Werb Zena , Burleigh Mary C.
Published: August 2015

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24°C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH2Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.
Dihydrofolate reductase: low-resolution mass-spectrometric analysis of an elastase digest as a sequencing tool
Authors: Morris Howard R. , Batley Karen E. , Harding Nigel G. L. , Bjur Richard A. , Dann John G. , King Rodney W.
Published: August 2015

An elastase digest of a protein of unknown structure, dihydrofolate reductase, was studied by mass spectrometry. This soluble digest contained a large number of small peptides in different yields, within the ideal molecular-weight range (200–1200) for mixture-analysis mass spectrometry. Sequences of the major component peptides in the digest are reported.
Fluorescence studies of protein–sterol relationships in human plasma lipoproteins
Authors: Smith Rory J. M. , Green Colin
Published: August 2015

Cholesta-5,7,9(11)-trien-3β-ol and its oleate ester were incorporated into human low-density lipoprotein and reconstituted high-density lipoprotein. The unesterified sterol was more efficient than its ester in quenching tryptophan fluorescence, especially in low-density lipoprotein. The results, which indicate that in such lipoproteins unesterified sterols are more closely associated with peptide than are esterified sterols, are used to assess possible structures for the lipoproteins.
Enhanced phosphatidylinositol labelling in rat parotid fragments exposed to α-adrenergic stimulation
Authors: Michell Robert H. , Jones Lynne M.
Published: August 2015

1. Adrenergic agonists provoke a marked increase in labelling of phosphatidylinositol in fragments of rat parotid gland. 2. Adrenaline and phenylephrine (an adrenergic α-agonist) are effective stimulants, but isoprenaline (an adrenergic β-agonist) is relatively ineffective. 3. The response evoked by phenylephrine or adrenaline is prevented by prior incubation of the tissue with phenoxybenzamine (an α-receptor blocking agent), but not by prior incubation with pindolol (a β-receptor blocking agent). 4. Adrenergic stimulation of phosphatidylinositol metabolism in parotid gland is therefore mediated through α-receptors, in common with the adrenaline-induced K+ efflux. It is not linked to enzyme secretion, which is triggered by stimulation of β-receptors. 5. It is suggested that the stimulation of phospholipid metabolism that occurs in several other tissues in the presence of adrenaline or noradrenaline may also involve α-receptors.
Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation
Authors: Hradec J. , Dušek Z. , Mach O.
Published: August 2015

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform–methanol mixture (2:1, v/v) extracted 1.87±0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92±0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.
A protein kinase inhibitor in brown adipose tissue of developing rats
Authors: Skala Josef P. , Drummond George I. , Hahn Peter
Published: August 2015

A heat-stable protein was extracted from brown adipose tissue of infant rats that inhibited both purified bovine heart protein kinase and a crude preparation of protein kinase from brown fat. It did not act as an adenosine triphosphatase nor did it exert its effect by proteolytic action. Inhibition of protein kinase was affected in both the presence and the absence of cyclic AMP. Most of the inhibitory activity was present in the 100000g supernatant fraction of tissue homogenates. Inhibitory activity was highest perinatally and it declined 10 days after birth. It is suggested that the protein kinase inhibitor may play a role in regulating cyclic AMP actions.
Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes
Authors: Halliwell Barry , Butt Vernon S.
Published: August 2015

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of 14CO2 from [1-14C]glycollate and [1-14C]glyoxylate were investigated. 2. At pH8, 14CO2 production from [1-14C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H2O2, which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. 14CO2 is shown to be generated from [1-14C]glycollate at pH8 by a similar reaction, but the H2O2 is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.
Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis
Authors: Parrilla R. , Goodman M. N.
Published: August 2015

Nitrogen balances were measured in isolated perfused rat livers in the presence and absence of nitrogen donors. In all instances the balance apparently was incomplete. The expression [alanine][α-oxoglutarate]/[pyruvate][glutamate] remained fairly constant under the metabolic conditions studied, indicating that it may be at near-equilibrium. The source of the extra nitrogen seems to be derived from increased hepatic proteolysis. The addition of a nitrogen donor to the perfusate arrested proteolysis, as did the addition of pyruvate. The free mitochondrial [NAD+]/[NADH] ratio, calculated from the glutamate dehydrogenase and β-hydroxybutyrate dehydrogenase reactants, showed similar values and exhibited parallel changes under most metabolic situations studied. These results suggest that, under the reported experimental conditions, both dehydrogenases share a common mitochondrial NAD pool. Glutamate dehydrogenase plays an important role in hepatic nitrogen metabolism in vivo.
Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation
Authors: Mellenberger R. W. , Bauman D. E.
Published: August 2015

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO2 production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77–86% as C8:0+C10:0 acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO2 production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP+–isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP+–malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).
Regulation of hexose phosphate metabolism in Acetobacter xylinum
Authors: Weinhouse Haim , Benziman Moshe
Published: August 2015

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO2 were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO2 was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically 14C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly 14C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO2. The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO2 was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.
The tryptic peptides of rabbit muscle triose phosphate isomerase
Authors: Corran P. H. , Waley S. G.
Published: August 2015

1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.
Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants
Authors: Shaw W. H. , Anderson J. W.
Published: August 2015

1. ATP sulphurylases were partially purified (20–40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PPi–ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PPi–ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and Km(selenate)/Km(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PPi–ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PPi is the first product released. 4. Synthesis of adenosine 5′-[35S]sulphatophosphate from [35S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg2+-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [75Se]selenate as substrate could not be demonstrated.
Purification and characterization of the seven cyanogen bromide fragments of human serum transferrin
Authors: Sutton Michael R. , Brew K.
Published: August 2015

1. Procedures are described for the isolation of seven distinct cyanogen bromide fragments in high yield from human serum transferrin. 2. Cyanogen bromide-cleaved transferrin is separated into three fragments (CN-A, CN-B and CN-C) by gel filtration with Sephadex G-100. 3. Four peptides are obtained from CN-A (the largest fragment) after reduction and carboxamidomethylation, by gel filtration in acidic solvents. Two peptides are similarly obtained from fragment CN-B, whereas fragment CN-C is a single cystine-free peptide. 4. The molecular weights of the seven peptides, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, by sedimentation-equilibrium ultracentrifugation and by sequence studies, range from 3100 to 27000. Together they account for a molecular weight of 76200 for transferrin. 5. The two largest fragments contain the carbohydrate attachment sites of the protein, and the smallest fragment is derived from the N-terminus. 6. The amino acid compositions and N-terminal groups of the fragments are reported and the results compared with those of previous investigations.
The selective isolation of an active-site histidine peptide from chymotrypsin-α by diagonal peptide ‘mapping’. An Nτ-carboxymethyl-histidine diagonal peptide ‘map’
Authors: Stevenson Kenneth J.
Published: August 2015

1. The selective isolation of an `active' histidine peptide from reduced and cyanoethylated chymotrypsin-α inhibited with Tos-Phe-CH2Cl (l-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained with a His(τCm) (Nτ-carboxymethylhistidine) diagonal peptide-`mapping' technique. Performic acid vapours, used between the first and second dimensions of the diagonal peptide `map', resulted in a peracid rearrangement of the alkylated (Tos-Phe-CH2)-histidine-57 residue into an Nτ-carboxymethylhistidine residue. The consequent change in electrophoretic mobility allowed isolation of peptides that contained the `active' histidine. 2. Peptides containing methionine or S-cyanoethylcysteine were oxidized to their sulphones during the treatment. Peptides in which these residues were N-terminal were selectively isolated on the basis of the change in electrophoretic mobility at pH6.5 which was due to the depression of the pK of the terminal amino group by the inductive effect of the sulphonyl group. 3. An attempt to apply the method to subtilisin BPN′ inhibited with l-1-benzyloxycarbonylamido-2-phenylethyl bromomethyl ketone failed to yield a peptide containing Nτ-carboxymethylhistidine, although peptides containing N-terminal methionine were isolated by the procedure.
Subunit interactions in hybrids of native, carboxypeptidase-treated and citraconylated rabbit muscle aldolase
Authors: Gibbons Ian
Published: August 2015

1. The kinetic properties of hybrids of native (or carboxypeptidase-treated) and citraconylated rabbit muscle aldolase are compared with those of equivalent mixtures of the parental enzymes. 2. In the hybrids, the native subunits function slightly less well than in the homotetramer, but the citraconylated subunits have enhanced activity. 3. Subunits of carboxypeptidase-treated aldolase behave essentially as expected in a hybrid environment, but the citraconylated subunits do not show the same enhancement of activity found in the hybrids of native and citraconylated enzyme. The apparent affinity for fructose 1,6-diphosphate of the citraconylated subunits in hybrids of carboxypeptidase-treated and citraconylated aldolase is increased. 4. These results are interpreted in terms of a substrate-induced conformational difference between native and carboxypeptidase-treated aldolase. 5. This conformational change can take place within a single native subunit in the hybrids and does not require a similar conformational change to occur simultaneously in the other three subunits.
A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli
Authors: Wilkinson Susan , Knowles Jeremy R.
Published: August 2015

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNAVal from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNAVal to valyl-tRNA synthetase from B. stearothermophilus were determined between 5° and 60°C.
The reaction of an α-aza-amino acid derivative with chymotrypsin and its use as a ligand for covalent affinity purification
Authors: Barker S. A. , Gray C. J. , Ireson J. C. , Parker R. C. , McLaren J. V.
Published: August 2015

1. Chymotrypsin is inactivated by N-acetyl-α-azaphenylalanine phenyl ester (phenyl N2-acetyl-N1-benzylcarbazate) in a stoicheiometric reaction. 2. The inactivation is reversible spontaneously (first-order rate constant is 1.2×10-4s-1) and accelerated by the presence of hydroxylamine. 3. Polymers based on polyacrylamide and carrying ligands containing the α-azaphenylalanine phenyl ester group were prepared. 4. Chymotrypsin reacts with these polymers and is removed by them from solution. Trypsin reacts less rapidly. 5. Chymotrypsin is slowly released from the polymer spontaneously and more rapidly on treatment with hydroxylamine. 6. The reaction of trypsin can be inhibited by competitive inhibitors. 7. Chymotrypsin was separated from trypsin by the selective bonding of chymotrypsin on to and its subsequent liberation from one of the polymers described.
Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall
Authors: Ward J. Barrie , Perkins Harold R.
Published: August 2015

A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by 14C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.
X-ray evidence for metal–N-7 bonding in a hydrated manganese derivative of guanosine 5′-monophosphate
Authors: de Meester Patrice , Goodgame David M. L. , Jones T. Jeffrey , Skapski Andrzej C.
Published: August 2015

Single-crystal X-ray studies of a manganese(II) derivative of guanosine 5′-monophosphate, [Mn(5′-GMP)(H2O)5],3H2O, have shown that it is isostructural with its nickel analogue. The manganese atom therefore is bonded to five water molecules with the remaining octahedral co-ordination site being occupied by N-7 of the nucleotide base. No direct metal–phosphate bonding is involved, but there are structure-stabilizing intramolecular hydrogen bonds between two phosphate oxygen atoms and co-ordinated water molecules.
Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain
Authors: Clark John B. , Land John M.
Published: August 2015

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a Ki of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (Ki~7.2mm) and 2-oxo-3-methylbutanoate (Ki~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (Ki~930μm) and 2-oxo-3-methylpentanoate (Ki~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.
Pathways of glyceride glycerol synthesis
Authors: Rognstad Robert , Clark Dallas G. , Katz Joseph
Published: August 2015

Isolated rat liver parenchymal cells were incubated for various periods with [U-14C,2-3H]glycerol and the radioisotopic yields in the major products were determined, as well as the 3H/14C ratios in glyceride glycerol and intracellular glycerol phosphate. Under the conditions used (0.1mm-glycerol+10mm-l-lactate or 10mm-glycerol as substrates), only small differences were found between these 3H/14C ratios. The results suggest a minor role for a pathway of glyceride glycerol synthesis involving reduction of acylated dihydroxyacetone phosphate, under these experimental conditions.
The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum
Authors: Green Anita A. , Newell Peter C.
Published: August 2015

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.
Studies on the lipid composition of the rat liver endoplasmic reticulum after induction with phenobarbitone and 20-methylcholanthrene
Authors: Davison Susan C. , Wills Eric D.
Published: August 2015

1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120–125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125–140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the β position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.
Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions
Authors: Spivey H. Olin , Flory Wayne , Peczon Benigno D. , Chandler John P. , Koeppe Roger E.
Published: August 2015

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, kapp.=0.24s-1 with 0.02mm-fructose 1,6-diphosphate (t0.9 ≃ 10s where t0.9 is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20μm). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t0.9=0.5–1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.
Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster
Authors: Rolton Hilary A. , Keir Hamish M.
Published: August 2015

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).
Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid
Authors: Hiwada Kunio , Wachsmuth Ernst D.
Published: August 2015

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg2+ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of Km and Vmax. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.
A new assay procedure for monoglyceride acyltransferase
Authors: Short Verena J. , Brindley David N. , Dils Raymond
Published: August 2015

1. A new assay system is described for monoglyceride acyltransferase (acylglycerol palmitoyltransferase, EC 2.3.1.22) in which palmitoyl-CoA is generated from palmitoyl-(-)-carnitine. 2. With the microsomal fraction from homogenates of guinea-pig intestinal mucosa, the Vmax. of this enzyme decreased with different acyl acceptors in the order 2-monopalmitoylglycerol>2-hexadecylglycerol>rac-1-monopalmitoylglycerol. 3. There were highly significant correlations between the monoglyceride acyltransferase activity as measured with these three substrates. This demonstrates that each of these substrates can be used to measure the same enzyme activity. 4. The advantages of using generated palmitoyl-CoA with 2-hexadecylglycerol and rac-1-monopalmitoylglycerol as model substrates for this enzyme are discussed.
The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias
Authors: Lowry Philip J. , Bennett Hugh P. J. , McMartin Colin , Scott Alexander P.
Published: August 2015

An adrenocorticotrophic hormone (ACTH) was isolated from extracts of the pars distalis of the pituitary of the dogfish Squalus acanthias by gel filtration and ion-exchange chromatography. It had 15% of the potency of human ACTH in promoting cortico-steroidogenesis in isolated rat adrenal cells. Sequence analysis revealed it to be a nonatria-contapeptide with the following primary structure: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met-Gly-Arg-Lys-Arg-Arg-Pro-Ile-Lys-Val-Tyr-Pro-Asn-Ser-Phe-Glu-Asp-Glu-Ser-Val-Glu-Asn-Met-Gly-Pro-Glu-Leu. The N-terminal tridecapeptide sequence was identical with the proposed structure of dogfish α-melanocyte-stimulating hormone (α-MSH). On comparison with human ACTH eleven amino acid differences were seen, nine of which are in the 20–39 region of the molecule which is not essential for the steroidogenic activity of ACTH. A peptide identical with the 18–39 portion of this new ACTH was similarly isolated from the neurointermediate lobe of the pituitary where considerable amounts of dogfish α-MSH were found. This supported our view that ACTH as well as having a distinct biological role of its own is also the precursor of α-MSH.
The reduction of carboxymethyl-cytochrome c by chromous ions
Authors: Brittain Thomas , Wilson Michael T. , Greenwood Colin
Published: August 2015

The reduction of ferricytochrome c and ferricytochrome c carboxymethylated at the haem-linked methionine (residue 80) by Cr2+ ions was studied by stopped-flow techniques. At pH6.2 the kinetics of reduction of ferricytochrome c are simple and correspond to a second-order rate constant of 1.21×103m-1·s-1. Under identical conditions the kinetics of reduction of the carboxymethyl derivative, carboxymethyl-cytochrome c, are complex; two Cr2+-concentration-dependent processes (1.5×104m-1·s-1 and 1.3×103m-1·s-1) lead to the formation of an intermediate which decays in monomolecular fashion (0.15s-1) to form the normal fully reduced material. The kinetic difference spectrum for the overall process corresponds to that found statically, whereas the kinetic difference spectrum of the intermediate minus the oxidized form resembles that of the low-spin ferrous form of carboxymethyl-cytochrome c minus oxidized carboxymethyl-cytochrome c. A model is proposed in which the reduction of low-spin ferric carboxymethyl-cytochrome c to high-spin ferrous carboxymethyl-cytochrome c involves a low-spin ferrous intermediate. The monomolecular step involving the decay of this low-spin ferrous intermediate is associated with an activation energy of approx. 126kJ·mol-1 and is thought to involve both a change of spin state and a protein-conformational event. Although carboxymethyl-cytochrome c represents a mixture of species separable on a charge basis, the above observations were independent of which species was chosen for study.
A more detailed study of bile salt evolution, including techniques for small-scale identification and their application to amphibian biles
Authors: Anderson Ian G. , Haslewood Geoffrey A. D. , Oldham Robert S. , Amos Bernard , Tökés Laszlo
Published: August 2015

1. Methods have been developed for the isolation and identification of small amounts of bile salts and of bile acids and alcohols obtained by solvolysis. These methods involve preparative and analytical t.l.c., purification on columns of protonated Al2O3 and Sephadex LH-20 and also g.l.c.–mass spectroscopy of solvolysis products. 2. Application to 29 species of frogs and toads has confirmed the constancy of bile salt patterns in a single species, including colour phases in two instances, and has revealed great variations between different species in some genera (e.g. Rana, Ptychadena) and little difference between widely distributed species in others (e.g. Bufo). 3. Taxonomic deductions should be made with caution and with regard to the physiological significance of the biochemical character considered. The molecular differences found might be interpreted as indicating variations in the rate of evolution.
Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine α- and β-trypsin
Authors: Roberts D. V. , Elmore D. T.
Published: August 2015

Several esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid and α-N-toluene-p-sulphonyl-l-homoarginine by α- and β-trypsin were compared. On the basis of values of the specificity constants (kcat./Km), the two enzymes display similar catalytic efficiency towards some substrates. In other cases α-trypsin is less efficient than β-trypsin. It is possible that α-trypsin possesses greater molecular flexibility than β-trypsin.
Salt-soluble elastin from lathyritic chicks
Authors: Sykes Bryan C. , Partridge S. Miles
Published: August 2015

The isolation of a salt-soluble homogeneous elastin from the aortas of lathyritic chicks by chromatography on DEAE-cellulose and salt precipitation is described. These new techniques, as well as some previously published by other workers, were evaluated with the help of antiserum raised in sheep against insoluble chick elastin. The purified elastin was very basic and behaved in a predictable manner in coacervation studies. The protein migrated in sodium dodecyl sulphate–polyacrylamide gels as a single band moving slightly faster than pyruvate kinase (mol.wt. 57000).