bluuebunny/crossref_metadata_2025 huggingface dataset:
The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339
Authors: Borensztajn J , Rone M S , Kotlar T J
Published: August 2015

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.
Erythrocyte calcium metabolism. Calcium exchange in normal and sickle-cell-anaemia erythrocytes
Authors: Cameron B F , Smariga P E
Published: August 2015

Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.
Kinetics and reaction mechanism of yeast alcohol dehydrogenase with long-chain primary alcohols
Authors: Schöpp W , Aurich H
Published: August 2015

Kinetic studies of yeast alcohol dehydrogenase with NAD+ and ethanol, hexanol or decanol as substrates invariably result in non-linear Lineweaver-Burk plots if the alcohol is the variable substrate. The kinetic coefficients determined from secondary plots are consistent with an ‘equilibrium random-order‘ mechanism for extremely low alcohol concentrations and for all alcohols, the transformation of the ternary complexes being the rate-limiting step of the reaction. This mechanism also applies to long-chain substrates at high concentrations, whereas the rate of the ethanol-NAD+ reaction at high ethanol concentrations is determined by the dissociation of the enzyme-NADH complex. The dissociation constants for the enzyme-NAD+ complex and for the enzyme-alcohol complexes obtained from the kinetic quotients satisfactorily correspond to the dissociation constants obtained by use of other techniques. It is suggested that the non-linear curves may be attributed to a structural change in the enzyme itself, caused by the alcohol.
The role of tetrahydrofolate dehydrogenase in the hepatic supply of tetrahydrobiopterin in rats
Authors: Stone K J
Published: August 2015

The reduction of 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin by rat liver tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is competitively inhibited by trimethoprim lactate (apparent Ki 0.285 muM). An apparent Michaelis constant of 43 muM for dihydrobiopterin was obtained, which is 430 times higher than the reported Km for dihydrofolate with this enzyme. The reduction of dihydrobiopterin is thus more susceptible to inhibition by trimethoprim lactate than is the reduction of dihydrofolate. However, intraperitoneal administration of trimethoprim had no significant effect on the hepatic supply of tetrahydrobiopterin in rats.
The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease
Authors: Emmens M , Welling G W , Beintema J J
Published: August 2015

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.
Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin
Authors: White H B , Dennison B A , Della Fera M A , Whitney C J , McGuire J C , Meslar H W , Sammelwitz P H
Published: August 2015

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.
Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase
Authors: London J W , Shaw L M , Fetterolf D , Garfinkel D
Published: August 2015

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.
Isolation of membrane-bound renal enzymes that metabolize kinins and angiotensins
Authors: Ward P E , Erdös E G , Gedney C D , Dowben R M , Reynolds R C
Published: August 2015

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..
Rabbit muscle myogen. Interactions with phosphate as the source of non-enantiography in moving-boundary electrophoresis
Authors: Lovell S J , Winzor D J
Published: August 2015

Rabbit muscle myogen has been subjected to moving-boundary electrophoresis and velocity sedimentation in 0.0187 M-potassium phosphate buffer, pH7.7, I = 0.05. The ascending and descending and descending electrophoretic patterns are sufficiently non-enantiographic to suggest the existence of rapid, reversible interactions in the myogen solutions. However, no evidence of pronounced macromolecular association was obtained in velocity-sedimentation experiments. The source of the non-enantiography in electrophoresis has been traced to interactions of phosphate with components of myogen, which should therefore be considered as a mixutre, rather than a complex, of glycolytic enzymes.
The interaction of reduced nicotinamide–adenine dinucleotide phosphate with reduced nicotinamide–adenine dinucleotide–ubiquinone reductase from bovine heart mitochondria
Authors: Ragan C I
Published: August 2015

Reduction of the chromophores of mitochondrial NADH-ubiquinone reductase by NADPH reaches only 50% of the extent of reduction by NADH, monitored at 450 nm. This effect is due to autoxidation of an enzyme component at a higher rate than its reduction by NADPH.
Comparison of renal and hepatic glutathione S-transferases in the rat
Authors: Kaplowitz N , Clifton G , Kuhlenkamp J , Wallin J D
Published: August 2015

Renal and hepatic GSH (reduced glutathione) S-transferase were compared with respect to substrate and inhibitory kinetics and hormonal influences in vivo. An example of each of five classes of substrates (aryl, aralkyl, epoxide, alkyl and alkene) was used. In the gel filtration of renal or hepatic cytosol, an identical elution volume was found for all the transferase activities. Close correspondence in Km values was found for aryl, epoxide- and alkyl-transferase activities, with only the aralkyl activity significantly lower in kidney. Probenecid and p-aminohippurate were competitive inhibitors of renal aryl-, aralkyl-, epoxide- and alkyl-transferase activities and inhibited renal alkene activity. Close correspondence in Ki values for inhibition by probenecid of these activities in kidney and liver was found. In addition, furosemide was a potent competitive inhibitor of renal alkyl-transferase activity. Hypophysectomy resulted in significant increases in aryl-, araklyl-, and expoxide-transferase activities in liver and kidney. The hypophysectomy-induced increases in renal aryl- and aralkyl-transferase activities (approx. 100%) were more than twofold greater than increases in hepatic activities (approx. 40%). Administration of thyroxine prevented the hypophysectomy-induced increase in aryltransferase activity in both kidney and liver. The renal GSH S-transferases, in view of similarities to the hepatic activities, may play a role as cytoplasmic organic-anion receptors, as previously proposed for the hepatic enzymes.
The regulation of folate and methionine metabolism
Authors: Krebs H A , Hems R , Tyler B
Published: August 2015

1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the ‘methylfolate-trap hypothesis’. This is discussed. 8...
Synthesis of δ-aminolaevulinate synthase by isolated liver polyribosomes
Authors: Whiting M J
Published: August 2015

1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.
Properties and developmental regulation of an α-d-galactosidase from Dictyostelium discoideum
Authors: Kilpatrick D C , Stirling J L
Published: August 2015

An alpha-D-galactosidase was detected in cells of the cellular slime mould, Dictyostelium discoideum, at all stages of development. Its specific activity was highest during early development (interphase), and this accumulation of enzyme appears to require protein synthesis de novo. Its subcellular distribution differs from that of other D. discoideum glycosidases, since most activity was recovered in the soluble fraction. No evidence was obtained for more than one isoenzymic form after subjection of extracts to electrophoresis and various chromatographic procedures. It is excreted from the cell during development, but no evidence was found for an extracellular function for the enzyme.
Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver
Authors: Pösö H , Jänne J
Published: August 2015

Repeated injections of 1,3-diaminopropane into rats after partial hepatectomy caused a repression-type inhibiton of liver ornithine decarboxylase (EC 4.1.1.17) and totally prevented the marked increases in liver putrescine and spermidine concentrations that normally occur in response to partial hepatectomy. The inhibition of polyamine synthesis by diaminopropane was accompanied by a profound decrease (about 80%) in the synthesis of DNA in the regenerating rat liver without any changes in the synthesis of RNA and total liver protein.
Loss of a homologous group of proteins in a dominantly inherited ectodermal malformation
Authors: Tenenhouse H S , Gold R J M
Published: August 2015

Hair from mice bearing the dominantly inherited Naked trait (NN) and from normal (NN) mice of the same inbred strain was separated into its major protein components by standard techniques. The relative amounts of proteins in these components were then determined by a regression method from the amino acid composition of the hair samples and of the fractions into which they had been separated. The results indicated that the amount of soluble fibril in Naked-mouse hair is decreased. Polyacrylamide-gel electrophoresis of this fraction prepared from the hair of both normal and Naked mice revealed that all protein bands present in the normal are also present in the Naked mice. However, a densitometric scan of the gels at 280 nm showed that the soluble fibril fraction from Naked-mouse hair is deficient in several proteins which, on amino acid analysis, were found to contain 31% glycine and 10% tyrosine. Gel filtration of S-carboxymethylkerateine prepared from normal and mutant hair showed that the mutant hair is deficient in a heterogeneous, low-molecular-weight fraction also rich in glycine and tyrosine. Our present data do not reveal the mechanism whereby a single gene locus modulates the production of several different proteins.
Unmasking of histone amino groups in chromatin at high pH
Authors: Malchy B L , Kaplan H
Published: August 2015

The reactivity of the amino groups of histones in chromatin towards acetic anhydride was determined as a function of pH. In the pH range 7-10 the vast majority of amino groups in all five histones are buried. However, at higher pH values some of the histone amino groups become exposed, and the higher the lysine:arginine ratio for the histone the greater was the degree of unmasking observed. At pH 11.8 histone I appears to be completely dissociated, histones IIB1 and IIb2 have approx. 55% of the amino groups unmasked, and histones III and IV have approx. 25% of the amino groups unmasked.
Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe
Authors: Malthouse J P , Brocklehurst K
Published: August 2015

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain
Authors: Shipton M , Stuchbury T , Brocklehurst K
Published: August 2015

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25°C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M-1·s-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M-1·s-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.
Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver
Authors: Hawkins H C , Freedman R B
Published: August 2015

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.
Determination of the number and relative position of tryptophan residues in various albumins
Authors: Feldhoff R C , Peters T
Published: August 2015

A technique is described by which both the numbers of tryptophan residues and their approximate locations in the peptide chain of a protein can be determined by cleavage with N-bromosuccinimide followed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The number of new peptide bands appearing in the gel is a function of the number of tryptophan residues, and the relative migration of the bands permits calculation of peptide molecular weights and an estimation of the positions of the tryptophan residues in the peptide chain. The technique uses a sample of about 0.5 mg and is suitable for any protein that contains a small number of tryptophan residues. These are the very specimens that are difficult to assay accurately for tryptophan by spectrophotometric or colorimetric methods. Tryptophan residues which are within about 20 residues of the ends of the peptide chain or of each other would not be detected. The specificity of the cleavage with N-bromosuccinimide was ascertained by utilizing human serum albumin, which is known to have a single tryptophan residue at position 214. The technique was then applied to a comparative study of the numbers and locations of tryptophans in the serum albumins of 16 species, namely 11 mammals, three birds and two amphibians. The number of tryptophan residues were confirmed by an independent colorimetric method. All of the mammalian albumins contained a tryptophan residue near position 213. The three avian albumins examined have no tryptophan. Frog and toad albumins contained two tryptophan residues, which appear to be situated at different positions from those in mammalian albumins.
Purification of an exo-β-D-glucanase from cell-free extracts of Candida utilis
Authors: Notario V , Villa T G , Villanueva J R
Published: August 2015

β-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific β-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-β-linkages by an exo-splitting mechanism. Glucono-δ-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.
Kinetic studies on [methionine sulphoxide] cytochrome c
Authors: Brittain T , Greenwood C
Published: August 2015

A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 × 10(3)M-1-S-1 and k+2 = 5.80 × 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 × 10(3)M-1-S-1 and k+2 = 6.0 × 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 × 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.
The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum
Authors: Yates D W , Duance V C
Published: August 2015

The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.
Adenosine diphosphate binding to sodium-plus-potassium ion-dependent adenosine triphosphatase. The role of lipid in the nucleotide-potassium ion interplay
Authors: Jensen J , Ottolenghi P
Published: August 2015

Delipidated dogfish rectal-gland Na++K+-ATPase (Na++K+-dependent adenosine triphosphatase), almost devoid of hydrolytic activity, is able to bind about 2nmol of ADP/mg of protein. The “affinity” of delipidated enzyme for ADP is not affected by K+ in concentrations that greatly decrease the “affinity” of native Na++K+-ATPase. The K+-sensitivity of the ADP binding is in part restored by relipidation with dioleoyl phosphatidylcholine.
The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic β-hydroxy-β-methylglutaryl-coenzyme A reductase and cholesterol 7α-hydroxylase
Authors: Mitropoulos K A , Balasubramaniam S
Published: August 2015

The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.
Isolation of pure cholinergic nerve endings from the electric organ of Torpedo marmorata
Authors: Israël M , Manaranche R , Mastour-Frachon P , Morel N
Published: August 2015

A rapid method for the preparation of highly purified cholinergic nerve endings from the electric organ of Torpedo is described. The endings retain their cytoplasmic components, as shown by biochemical and morphological observations. The homogeneity of these synaptosomes make them a useful tool for further studies.
Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity
Authors: Avila J L , Convit J
Published: August 2015

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid β-glycerophosphatase, α-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.
Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts
Authors: Suzuki T , Takahashi E
Published: August 2015

1. The tRNA methyltransferase activity in vitro of leaves, cotyledons and roots of 85-day-old tea seedlings was studied. 2. The activity of extracts prepared from tea leaves with Polycar AT (insoluble polyvinylpyrrolidine) had optimum pH7.7 and was greatly influenced by thiol compounds, but only slightly by metal ions and ammonium acetate. 3. The activities of extracts, expressed per mg of protein, were as follows: roots greater than leaves greater than cotyledons. The only methylated base isolated after incubation with these preparations was 1-methyladenine. 4. The results did not support the view of involvement of methylation of nucleic acids in caffeine biosynthesis in tea plants. In contrast, it is suggested that theophylline is synthesized from the specific methylated precursor in nucleic acids, namely 1-methyladenylic acid, via 1-methylxanthine.
Effect of ornithine and lactate on urea synthesis in isolated hepatocytes
Authors: Briggs S , Freedland R A
Published: August 2015

1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline.
Developmental aspects of glutathione S-transferase B (ligandin) in rat liver
Authors: Hales B F , Neims A H
Published: August 2015

The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood.
Glycogen metabolism in the liver of the developing rat
Authors: Watts C , Gain E R
Published: August 2015

1. The total activity of glycogen synthease increased 20-fold from day 17 of gestation to birth at day 22, with a further increase of 18% in the 24h after birth. Active synthase (I) rose 45-fold to a maximum at day 21, fell 40% before birth, and then increased by a similar amount 24h after birth. The fraction of synthase in the active form correlated very well with the deposition of glycogen in the liver. 2. Total phosphorylase had a similar developmental pattern of total synthease with an 18-fold increase from day 17 to day 22. The appearance of active phosphorylase showed a lag-period compared with total phosphorylase and did not increase significantly until day 20. The fraction of phosphorylase in the active form did not correlate at all with glycogen deposition or mobilization. 3. There was a close relationshp between the ratio of phosphorylase a/synthase I and the glycogen content of the liver. An increase or decrease in this ratio would result in glycogenolysis of glycogenesis respectively. 4. It is postulated that a cycle between the two enzymes under basal conditions could exist which permits a continuous turnover of glycogen. Such a system would explain why active phosphorylase is always seen, even under conditions of net glycogen synthesis. The differences in hormone sensitivity of synthase and phosphorylase would also be accounted for as only one enzyme would have to respond acutely to hormonal influences.
The electrophoretic properties and aggregation of mouse lymphoma cells, chinese-hamster fibroblasts and a somatic-cell hybrid
Authors: Greig R G , Jones M N , Ayad S R
Published: August 2015

1. The electrophoretic mobilities of a mouse lymphoma cell, a Chinese-hamster fibroblast and a somatic-cell hybrid (also fibroblastic), produced by fusion of the hamster cell and a mouse lymphoma cell, were measured at 25 degrees C over a range of pH, concentration of Ca2+ ions and concentration of La3+ ions. 2. All the cells have pI at pH3.5. 3. Ca2+ ions decrease the mobilities and zeta potentials of the cells to zero in the range 1-100mM. 4. La3+ ions lower the mobilities and zeta potentials in the range 10 muM-1 mM, and the cells become positively charged above 1 mM. 5. The data are consistent with specific adsorption of La3+ ions on approx. 2 × 10(14) sites/m2 of cell surface with a free energy of approx. −37kJ/mol. 6. The effects of Ca2+, La3+ and ionic strength on the extent of aggregation of the cells and of neuraminidase-treated cells were studied. 7. Ca2+ ions do not markedly increase aggregation, whereas La3+ ions gave rise to extensive aggregation in the range 10 muM-1 mM, corresponding to the region of La3+ adsorption. 8. Both fibroblastic cell lines are aggregated at high ionic strength. 9. The fibroblastic cells have larger amounts of trypsin-sensitive carbohydrate than does the lymphoma cell; the possible role of this material in cellular aggregation is discussed.
Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression
Authors: Lloyd D , Edwards S W
Published: August 2015

1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN′-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN′-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.
Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions
Authors: Nishiki K , Jamieson D , Oshino N , Chance B
Published: August 2015

1. In the lung and liver of tocopherol-deficient rats, the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase were increased substantially, suggesting an important role for both enzymes in protecting the organ against the deleterious effects of lipid peroxides. 2. Facilitation of the glutathione peroxidase reaction by infusing t-butyl hydroperoxide caused the oxidation of nicotinamide nucleotides and glutathione, resulting in a concomitant increase in the rate of release of oxidized glutathione into the perfusate. Thus the rate of production of lipid peroxide and H2O2 in the perfused organ could be compared by simultaneous measurement of the rate of glutathione release and the turnover number of the catalase reaction. 3. On hyperbaric oxygenation at 4 × 10(5)Pa, H2O2 production, estimated from the turnover of the catalase reaction, was increased slightly in the liver, and glutathione release was increased slightly, in both lung and liver. 4. Tocopherol deficiency caused a marked increase in lipid-peroxide formation as indicated by a corresponding increase in glutathione release under hyperbaric oxygenation, with a further enhancement when the tocopherol-deficient rats were also starved. 5. The study demonstrates that the primary response to hyperbaric oxygenation is an elevation of the rate of lipid peroxidation rather than of the rate of formation of H2O2 or superoxide.