bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine
Authors: Pegg Anthony E. , Hui Georgiani
Published: August 2015

1. The amounts of 7-methylguanine and O6-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O6-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O6-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O6-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O6-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O6-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [3H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O6-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O6-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O6-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O6-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.
Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver
Authors: Burchell B
Published: August 2015

1. The purification to homogeneity of stable highly active preparations of UDP-glucuronyltransferase from liver of phenobarbital-treated rats is briefly described. 2. A single polypeptide was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, of mol.wt.57000. 3. Antiserum raised against the pure enzyme produces a single sharp precipitin line after Ouchterlony double-diffusion analysis. 4. The pure UDP-glucuronyltransferase isolated from livers of untreated and phenobarbital-pretreated rats appears to be the same enzyme. 5. The Km (UDP-glucuronic acid) of the pure enzyme is 5.4 mM. 6. The activity of the pure enzyme towards 2-aminophenol can still be activated 2-3-fold by diethylnitrosamine. 7. UDP-glucose and UDP-galacturonic acid are not substrates for the purified enzyme. 8. The final preparation catalysed the glucuronidation of 4-nitrophenol, 1-naphthol, 2-aminophenol, morphine and 2-aminobenzoate. 9. Activities towards 4-nitrophenol, 1-naphthol and 2-aminophenol were all copurified. The proposed heterogeneity of UDP-glucuronyltransferase is discussed.
Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement
Authors: Reid K B , Thompson E O
Published: August 2015

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.
Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver
Authors: Page M A , Tubbs P K
Published: August 2015

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified from ox liver, and obtained essentially free from 3-oxoacyl-CoA thiolases. The kinetic behaviour, like that of the synthases from chicken liver and yeast, is compatible with a reaction pathway involving condensation of an acetyl-enzyme with acetoacetyl-CoA. The Km for acetoacetyl-CoA, less than 1 micronM at pH 7.8, may possibly be low enough to permit rapid ketogenesis under physiological conditions without the need for a binary complex between the synthase and oxoacyl-CoA thiolase.
Molecular weight and subunit size of rabbit mammary-gland fatty acid synthetase
Authors: Grunnet I , Knudsen J
Published: August 2015

1. The molecular weights of fatty acid synthetases isolated from lactating rabbit, rat, cow and goat mammary glands were estimated by sucrose gradient centrifugation and compared by chromatography on Sepharose 6B. 2. The values obtained for all four enzymes were in the same range (0.40 × 10(6)-0.55 × 10(6)) as that found for other mammalian and avian fatty acid synthetases. The molecular weight found for the rabbit mammary enzyme therefore differs from published values of approx. 0.9 × 10(6). 3. The molecular weights of the subunits of these four synthetases were 225000-242000. Again, the value for the rabbit mammary enzyme differs from published values.
Structural studies on the glycoproteins from bovine cervical mucus
Authors: Roberts G P
Published: August 2015

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely ‘naked’ peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.
The effects of triton WR1339 and asialo-fetuin on the hepatic uptake of circulating native and asialo-carcinoembryonic antigen
Authors: Thomas P , Jones M
Published: August 2015

Uptake of carcinoembryonic antigen from the circulation of the mouse is inhibited by treatment of the animal with Triton WR1339, but not by asialo-fetuin. Uptake of asialo-carcinoembryonic antigen is inhibited by asialo-fetuin, but not by Triton WR1339. These results reflect the different mechanisms of uptake of the glycoproteins. The presence or absence of sialic acid does not seem to be important in governing Kupffer-cell uptake, but when terminal galactoses are exposed on a glycoprotein, uptake by hepatocytes is preferred. Kupffer-cell uptake of carcinoembryonic antigen is not due to the formation of high-molecular-weight complexes in the blood stream. The effect of asialo-fetuin and Triton WR1339 on the biliary excretion of carcinoembryonic antigen and asialo-carcinoembryonic antigen is discussed.
The lipid composition of a barley mutant lacking chlorophyll b
Authors: Bolton P , Wharfe J , Harwood J L
Published: August 2015

The acyl-lipid composition of a barley mutant that contained no detectable chlorophyll b was studied. This mutant contained chloroplasts that were much less organized than chloroplasts of normal barley. The mutant contained all the normal acyl lipids, with small increases in the relative concentration of phosphatidylglycerol and diacylsulphoquinoglycerides was unchanged, but most other lipids of the mutant barley contained lower amounts of alpha-linolenic acid compared with normal. There was no difference in the transhexadec-3-enoic acid content of phosphatidylglycerol, which was evidence against this lipid being involved in grana stacking.
A method for the analysis of protein turnover characteristics. Indirect estimation of rates of protein degradation
Authors: Paskin N , Mayer R J
Published: August 2015

A method for the calculation of rate constants of degradation of a specific protein during a period of change in protein amount is described. The calculation uses measurements of rates of synthesis and amount of the protein, together with one estimate of the protein half-life. The method is demonstrated by using data from measurements made on the accumulation of fatty acid synthetase in hormonally treated explants of mammary gland from mid-pregnant rabbits, and data from other authors. These calculations demonstrate a regulated programme of co-ordinated changes in rates of synthesis and degradation during a period of change in protein mass. The role of such changes in protein degradation during protein accumulation is discussed.
Metabolism of surfactants sodium undecyltriethoxy sulphate and sodium dodecyltriethoxy sulphate in the rat
Authors: Taylor Andrew J. , Powell Gillian M. , Howes Douglas , Black J. Gordon , Olavesen Anthony H.
Published: August 2015

The metabolic fates of the synthetic surfactants, sodium [1-14C]undecyltriethoxy sulphate and sodium [1-14C]dodecyltriethoxy sulphate were studied in the rat. Both compounds were extensively metabolized regardless of the route of administration, oral, intraperitoneal or intravenous. Short-chain radioactive products were eliminated in the urine: the major metabolite of the dodecyl homologue in the urine was identified as −O2C14CH2- (OC2H4)3OSO3− by n.m.r. and g.l.c.–mass spectrometry, whereas the major metabolite of the undecyl homologue in the urine was tentatively identified as −O2CCH214CH2- (OC2H4)3OSO3−. In contrast with experiments with the dodecyl derivative, when [1-14C]undecyltriethoxy sulphate was administered to rats, appreciable amounts of radioactivity were recovered as 14CO2 in expired air. Whole-body radioautography implicated the liver as the major site of metabolism of both surfactants. The nature of the metabolic products establishes that both compounds are degraded by ω,β-oxidation. Cleavage of the ether linkage proximal to the sulphate moiety may account for the small amounts of 14CO2 recovered in expired air after the administration of [1-14C]dodecyltriethoxy sulphate. It is suggested the substantial amounts of 14CO2 recovered after [1-14C]-undecyltriethoxy sulphate administration originate from −O214C(OC2H4)3 OSO3−, an unstable product of ω,β-oxidation. An n.m.r. spectrum of the metabolite identified as 2-(triethoxy sulphate)acetic acid and a mass spectrum of the trimethylsilyl derivative of the parent alcohol of that metabolite have been deposited as Supplementary Publication SUP50086 (5 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
The brush border of rabbit kidney, a cellular compartment free of glycolytic enzymes
Authors: Busse Dietrich , Wahle Hans Ulrich , Bartel Harald , Pohl Barbara
Published: August 2015

Activities of four enzymes of the glycolytic pathway, hexokinase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, were determined in a vesicular brush-border preparation from rabbit kidneys. The specific activities of the enzymes were decreased several-hundredfold in the brush-border preparation compared with a kidney homogenate, but the enzymes were not totally absent. Density-gradient centrifugation of the brush-border preparation yielded brush border of even higher purity and also a characteristic pattern of distribution for each of the contaminating intracellular membranes. The presence of hexokinase in the brush-border preparation could be traced to contaminating mitochondria, and that of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase to contaminating vesicles derived from the endoplasmic reticulum. The brush-border vesicles contained some ATP. An intravesicular concentration of 0.1mm was estimated, indicating that the vesicles had retained at least a part of their original content. Experiments in which fluorescein isothiocyanate-dextran (mol.wt. 20000) was present during cell lysis revealed that much, but not all, of the brush-border contents had been exchanged with the medium. The complete absence of glycolytic enzymes from brush-border vesicles, which had retained part of their original content, indicates that the brush border does not contain glycolytic enzymes in vivo and can be thought of as a compartment of its own, somehow separated from the cytoplasm.
The metabolic fate of (2-14C)folic acid and a mixture of (2-14C)- and (3′,5′,9-3h)-folic acid in the rat
Authors: Barford P A , Staff F J , Blair J A
Published: August 2015

The metabolism of [2-14C]folic acid over 13 days and a mixture of [2-14C]- and [3′,5′,9-3h]-folic acid in rats over a 6-day period is described. Both 14C and 3H are excreted in urine over the 6-day period, but 3H and 14C are only detectable in faeces for 2 days. A breakdown product of folic acid labelled with 3H only was found in some urine samples, but no metabolite corresponding to the part of the molecule containing 14C was detected. These experiments show that in the whole animal a substantial portion of orally administered folic acid undergoes scission shortly after administration [Blair Biochem. J. (1957) 68, 385-387] and that the retained folates are a shortage form for folate monoglutamates.
The specificity of the interaction of bovine pancreatic ribonuclease A with natural and halogenated purine nucleotides
Authors: Pares X , Arus C , Llorens R , Cuchillo C M
Published: August 2015

The interaction between bovine pancreatic ribonuclease A (EC 3.1.4.22) and the purine nucleotides AMP, GMP, 6-chloropurine 5′-ribonucleotide and 8-bromoadenosine 5′-monophosphate was studied by u.v. difference spectroscopy. The stoicheiometry of the binding of the halogenated nucleotides to the enzyme shows a 1:1 ratio, as for the natural ones. The binding constants, Ka, for all four nucleotides at pH 5.5 were determined. They are within the same order of magnitude, though the nucleotides with a 6-amino group show a stronger interaction. The magnitude of the binding shows a reciprocal dependence on the ionic strength, which indicates an electrostatic interaction between ligand and enzyme. Finally, solvent-perturbation experiments show that all four nucleotides bind to the enzyme in a partially hydrophobic region. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule. The special synthesis and identification of 6-chloropurine 5′-ribonucleotide are discussed extensively. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule and thus the halogenated analogues are potential reagents for the affinity labelling of the purine-binding site.
Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics
Authors: Wright C L , Warsy A S , Holroyde M J , Trayer I P
Published: August 2015

The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.
Intrasubunit nucleotide binding in ribonucleic acid polymerase
Authors: Malcolm A D B , Moffatt J R
Published: August 2015

1. Periodate oxidation of the ribose ring was used to synthesize derivatives of nucleoside triphosphates. 2. These oxidized nucleoside triphosphates. 2. These oxidized nucleoside triphosphates are competitive inhibitors of RNA polymerase. 3. On incubation, together with NaBH4, these oxidized labelled nucleotides are covalently bound to Escherichia coli RNA polymerase. 4. Nucleoside triphosphate substrates decrease the extent of labelling. 5. A lysine residue in an alpha-subunit is labelled. 6. The significance of these results in relation to the location of the nucleotide-binding site is discussed.
Modification of yeast ribosomal proteins. Methylation
Authors: Kruiswijk T , Kunst A , Planta R J , Mager W H
Published: August 2015

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.
Purification and properties of nitrogenase from Rhodospirillum rubrum, and evidence for phosphate, ribose and an adenine-like unit covalently bound to the iron protein
Authors: Ludden P W , Burris R H
Published: August 2015

1. The molybdenum-iron (Mo-Fe) protein, iron (Fe) protein and the activating factor of nitrogenase from Rhodospirillum rubrum were purified. 2. The Mo-Fe protein has properties similar to those of the Mo-Fe proteins of other nitrogen-fixing organisms. 3. The Fe protein is similar to other Fe proteins with respect to its molecular weight, metal composition and e.p.r. signal. 4. The Fe protein is different from other Fe proteins in that it apparently has two types of subunits rather than one, its u.v. spectrum has an extra peak, and phosphate, ribose and an adenine-like unit are covalently bound to the protein. The presence of these non-protein groups on the protein may explain the requirement for activation of R. rubrum Fe protein.
Intermolecular specificity of pancreatic lipase in the lipolysis of triacylglycerols containing phytanic acid
Authors: Needs E C , Welch V A
Published: August 2015

Treatment of mixtures of glycerol tripalmitate and rac-glycerol 1,2-dipalmitate 3-phytanate with pancreatic lipase demonstrated that phytanic acid retarded the release of palmitic acid esterified in the same triacylglycerol.
A comparative approach to protein-and ligand-dependence of the Root effect for fish haemoglobins
Authors: Giardina Bruno , Giacometti Giorgio M. , Coletta Massimo , Brunori Maurizio , Giacometti Giovanni , Rigatti Giorgio
Published: August 2015

Ligand-binding equilibria, kinetics and 13C n.m.r. spectra of bound 13CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the 13CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O2 strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the α and β chains in the tetramer were obtained for both haemoglobins from 13CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.
The metabolic modification of low-density lipoproteins in normal and hypercholesterolaemic guinea pigs
Authors: Weech P K , Mills G L
Published: August 2015

1. Low-density lipoproteins were isolated by ultracentrifugation from the serum of guinea pigs that were fed either on a normal diet, or on a diet supplemented with corn oil and cholesterol. 2. After labelling with tracer amounts of radioactive iodine, these lipoproteins were injected into the bloodstream of guinea pigs that were fed either on the normal or on the supplemented diet. 3. In all cases, the density of the labelled lipoproteins was increased by exposure for 24-48 h to the metabolic processes of the guinea pig. 4. The final density reached by lipoproteins isolated from fat-fed guinea pigs was less than that reached by lipoproteins from normal animals. 5. Fat-fed guinea pigs were unable to increase the density of either normal lipoproteins, or those from fat-fed guinea pits, to the same extent as animals fed on the normal diet. 6. It is concluded that the lipid-rich diet brings about a modification of lipoprotein metabolism in the guinea, pig, which plays an important part in determining the nature of the nature of the low-density lipoprotein that is present in the plasma.
Hepatic storage of oligosaccharides and glycolipids in a cat affected with GM1 gangliosidosis
Authors: Holmes E W , O'Brien J S
Published: August 2015

1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated.
Evaluation of rate constants for enzyme-catalysed reactions by the jackknife technique. Application to liver alcohol dehydrogenase
Authors: Cornish-Bowden A , Wong J T
Published: August 2015

Steady-state measurements of enzyme-catalysed reactions are capable of providing more information about the rate constants of the individual steps than is commonly obtained. We have applied a combination of the jackknife and non-linear regression techniques to measurements of the rate of oxidation of ethanol by NAD+, catalysed by alcohol dehydrogenase from horse liver. This has permitted values and confidence intervals to be assigned to the eight rate constants that characterize the binding of ethanol and NAD+ in random order to the enzyme, and to the net rate constant kcat. for the breakdown of the ternary complex.
Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues
Authors: Mäkinen K K , Mäkinen P L
Published: August 2015

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.
α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro
Authors: Williams J , Villarroya H , Petek F
Published: August 2015

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).
The biosynthetic pathway of new polyamines in Caldariella acidophila
Authors: De Rosa Mario , De Rosa Salvatore , Gambacorta Agata , Cartenì-Farina Maria , Zappia Vincenzo
Published: August 2015

1. Spermidine and sym-nor-spermine (1,11-diamino-4,8-diazaundecane) were identified as the major components of the polyamine pool in Caldariella acidophila, an extreme thermoacidophilic bacterium. A minor component, a new polyamine, sym-nor-spermidine (1,7-diamino-4-azaheptane) was isolated and characterized. 2. To elucidate the biosynthetic pathway, labelled methionine, putrescine, spermidine and spermine were fed to Caldariella acidophila. Incubation of the bacterium in the presence of putrescine or spermidine labelled in the tetramethylene moiety gave unlabelled sym-nor-spermidine and sym-nor-spermine, whereas the radioactivity of propylamine-labelled methionine or spermidine was incorporated into these molecules. No radioactivity was recovered in the polyamines pool when spermine was fed to Caldariella acidophila. 3. S-Adenosylmethionine and S-(5′-adenosyl)-3-methylthiopropylamine were identified as intermediates of the biosynthetic pathway; the cellular contents of the two sulphonium compounds, measured with a new isotope-dilution technique, are 60 and 15nmol/g wet wt. of cells respectively. 4. The above results are indicative of a new pathway characterized by three propylamine-transfer reactions, decarboxylated S-adenosylmethionine being the common donor of the propylamine moiety. The reactions yielding sym-nor-spermidine and sym-nor-spermine are reported for the first time. 5. The probable intermediates related to the catabolism of the tetramethylene moiety of spermidine, γ-aminobutyraldehyde, γ-aminobutyric acid or Δ1-pyrroline were not detectable. Experiments with [3-aminopropyl-3(n)-3H]spermidine trihydrochloride plus [tetramethylene-1,4-14C]spermidine trihydrochloride gave rise to an amount of labelled CO2 equivalent to the spermidine catabolized.
Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion
Authors: Chandler C S , Ballard F J
Published: August 2015

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.
Calcium accumulation during sporulation of Bacillus megaterium KM
Authors: Hogarth C , Ellar D J
Published: August 2015

Accumulation of Ca2+ in Bacilli occurs during stages IV to VI of sporulation. Ca2+ uptake into the sporangium was investigated in Bacillus megaterium KM in protoplasts prepared in stage III of sporulation and cultured to continue sporulation. These protoplasts and whole cells exhibit essentially identical Ca2+ uptake, which is compared with that of forespores isolated in stage V of sporulation. Ca2+, uptake into both sporangial protoplasts and isolated forespores occurs by Ca2+-specific carrier-mediated processes. However, protoplasts exhibit a Km value of 31 micrometer, and forespores have a Km value of 2.1 mM. Sporangial protoplasts accumulate Ca2+ against a concentration gradient. In contrast, Ca2+ uptake into isolated forespores is consistent with downhill transfer in which both rate and extent of uptake are affected by the external Ca2+ concontration. Dipicolinic acid has no effect on Ca2+ uptake by isolated forespores, apart from decreasing the external Ca2+ concentration by chelation. A model for sporulation-specific Ca2+ accumulation is proposed, in which Ca2+ is transported into the sporangium, resulting in a concentration of 3–9 mM in the mother-cell cytoplasm. This high concentration of Ca2+ enables carrier-mediated transfer down a concentration gradient into the forespore compartment, where a low free Ca2+ concentration is maintained by complexing with dipicolinic acid.
The mechanism of haem degradation in vitro. Kinetic evidence for the formation of a haem-oxygen complex
Authors: Brown S B , Thomas S E
Published: August 2015

The rate of haem degradation in aqueous pyridine shows a saturation dependence on O2 concentration. CO competitively inhibits the reaction. This is evidence for formation of an iron-O2 complex and is consistent with an intramolecular pathway for haem degradation.
Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements
Authors: Huvos P , Fey S , Hardwicke P M D
Published: August 2015

1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo.
The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain
Authors: Irvine R F , Hemington N , Dawson R M C
Published: August 2015

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.
Initiation of transcription in normal and phosphorylated rat liver nucleosomes
Authors: Ord M G , Stocken L A
Published: August 2015

Initiation sites were enumerated in rat liver nucleosomes with Echerichia coli RNA polymerase. One site was present in approx. 3.5 mononucleosomes and in a 1200-base-pair length of polynucleosomes. S-phase nuclei or normal nuclei phosphorylated in vitro showed increased numbers of sites; the elongation rates were unchanged.
The development of ketogenesis at birth in the rat
Authors: Ferré P , Pégorier J P , Williamson D H , Girard J R
Published: August 2015

In the suckling newborn rat, blood ketone bodies begin to increase slowly 4h after birth and then rise sharply between 12 and 16h, whereas the major increase in plasma non-esterified fatty acids and liver carnitine occurs during the first 2h of life, parallel with the onset of suckling. In the starved newborn rat, which shows no increase in liver carnitine unless it is fed with a carnitine solution, the developmental pattern of the ketogenic capacity (tested by feeding a triacylglycerol emulsion, which increases plasma non-esterified fatty acids by 3-fold) is the same as in the suckling animal. This suggests that the increases in plasma non-esterified fatty acids and liver carnitine seen 2h after birth in the suckling animal are not the predominant factors inducing the switch-on of ketogenesis. Injection of butyrate to starved newborn pups resulted in a pattern of blood ketone bodies which was similar to that found after administration of triacylglycerols, but, at all time points studied, the hyperketonaemia was more pronounced with butyrate. It is suggested that, even if the entry of long-chain fatty acids into the mitochondria is a rate-limiting step, it is not the only factor controlling ketogenesis after birth in the rat. As in the adult rat, there is a reciprocal correlation between the liver glycogen content and the concentration of ketone bodies in the blood.
A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein
Authors: Gomez-Fernandez Juan C. , Harris David A.
Published: August 2015

1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (α–ε) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the Kd is of the order of 10−8m. 3. The enthalpy of the ATPase–inhibitor complex-formation is positive, the value of Kd decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase–inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K2SO4, the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase–inhibitor complex concentration. The temperature coefficients of the ‘on’ and ‘off’ processes were also measured. 6. A simple kinetic model for the ATPase–inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg2+, reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.
Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland
Authors: Speake B K , White D A
Published: August 2015

1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction.
Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus
Authors: Clarke C M , Hartley B S
Published: August 2015

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).
The binding and catalytic activities of forms of ligandin after modification of its thiol groups
Authors: Carne T , Tipping E , Ketterer B
Published: August 2015

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.
The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide
Authors: Price N C
Published: August 2015

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.