bluuebunny/crossref_metadata_2025 huggingface dataset:
A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase
Authors: LiMuti C , Bell J E
Published: August 2015

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.
Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.
Authors: Matthews I T W , Decker R S , Hornebeck W , Knight C G
Published: August 2015

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3′-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.
Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth
Authors: Prehm P
Published: August 2015

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5′-nucleotidase are suggested as modulators of hyaluronate synthesis.
The complete amino acid sequence of yeast phosphoglycerate kinase
Authors: Perkins R E , Conroy S C , Dunbar B , Fothergill L A , Tuite M F , Dobson M J , Kingsman S M , Kingsman A J
Published: August 2015

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.
Use of procainamide gels in the purification of human and horse serum cholinesterases
Authors: Ralston J S , Main A R , Kilpatrick B F , Chasson A L
Published: August 2015

Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase.
Structural glycoproteins from rabbit aortic media
Authors: Moczar M , Phan-Dinh-Tuy B , Moczar E , Robert L
Published: August 2015

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.
The properties of potato (Solanum tuberosum) lectin after deglycosylation by trifluoromethanesulphonic acid
Authors: Desai N N , Allen A K , Neuberger A
Published: August 2015

Potato (Solanum tuberosum) lectin, which is a very highly glycosylated glycoprotein, has been completely deglycosylated by use of the trifluoromethanesulphonic acid reagent described by Edge, Faltnek, Hof, Reichert & Weber [(1981) Analyt. Biochem. 118, 131-137]. This shows that both hydroxyproline-arabinofuranoside and serine-galactopyranoside linkages are hydrolysed. The deglycosylated lectin is still functional and cross-reacts with one component of an anti-(potato lectin) antiserum.
Determination of Km and V of an enzyme for an unstable substrate and its application to the oxidation of Nτ-methylimidazol-3-ylacetaldehyde by aldehyde dehydrogenase
Authors: Gitomer W L , Tipton K F
Published: August 2015

The method of Storer & Cornish-Bowden [(1974) Biochem. J. 141, 205-209] for determining the lag time in coupled enzyme assays was adapted to enable the kinetic parameters of the second (coupling) enzyme for the intermediate to be calculated. The validity and accuracy of this method of progress-curve analysis was established by comparing the Km value of glucose 6-phosphate dehydrogenase for glucose 6-phosphate generated in situ by the action of glucose phosphate isomerase on fructose 6-phosphate with that determined from initial-rate measurements. The method was applied to the determination of the Km value of ox liver cytoplasmic aldehyde dehydrogenase for N tau-methylimidazol-3-ylacetaldehyde that was generated in situ by the action of plasma amine oxidase on N tau-methylhistamine.
Purification and kinetic properties of two soluble forms of calmodulin-dependent cyclic nucleotide phosphodiesterase from rat pancreas
Authors: Vandermeers A , Vandermeers-Piret M C , Rathe J , Christophe J
Published: August 2015

The calmodulin-dependent cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase (EC 3.1.4.17) activity of rat pancreas was purified 280-fold by affinity chromatography on calmodulin-Sepharose 4B. It then accounted for 15% of the total cytosol cyclic GMP nucleotide phosphodiesterase activity, in the presence of Ca2+, and represented a minor component of proteins specifically adsorbed by the column. This activity was resolved on a DEAE-Sephacel column into two fractions, termed PI and PII, on the basis of their order of emergence. After this step, PI and PII were purified 5650- and 3700-fold respectively. The molecular weight of PI was 175 000 and that of PII was 116 000, by polyacrylamide-gradient-gel electrophoresis. Both forms of phosphodiesterase could hydrolyse cyclic AMP and cyclic GMP, although PII displayed a higher affinity toward cyclic GMP than toward cyclic AMP. PI and PII exhibited negative homotropic kinetics in the absence of calmodulin. Upon addition of calmodulin, both enzymes displayed Michaelis-Menten kinetics and a 5-9-fold increase in maximal velocity, at physiological concentrations of cyclic GMP and cyclic AMP. When a pancreatic extract freshly purified by affinity chromatography was immediately analysed by high-performance gel-permeation chromatography on a TSK gel G3000 SW column, PII represented as much as 78% of the eluted activity. This percentage decreased to 52% when the sample was stored at 0 degrees C for 20 h before analysis, suggesting that PII, possibly predominant in vivo, was converted into the heavier PI form upon storage.
K
m and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one with dihydropteridine reductase
Authors: Armarego W L F , Randles D , Taguchi H
Published: August 2015

The Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one were determined for dihydropteridine reductase (EC 1.6.99.10) from two sources. The parameters of the pterin are of the same order as those of the most effective substrates of dihydropteridine reductase. The Km values of the pterin are one order of magnitude smaller than those of the pyrimidinone, although the kcat. values are of the same order.
Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol)
Authors: Kirsten H , Christen P
Published: August 2015

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.
Mechanism of inactivation of TEM-1 β-lactamase by 6-acetylmethylenepenicillanic acid
Authors: Arisawa M , Adam S
Published: August 2015

The interaction between 6-acetylmethylenepenicillanic acid (compound Ro 15-1903; AMPA) and TEM-1 beta-lactamase was investigated in order to elucidate the mechanism of action of AMPA. Formation of the enzyme-inhibitor complex (EA) was accompanied by a shift of the absorbance maximum from 292 nm to 303 nm and an increase in the absorption. Regeneration of activity was very slow and incomplete, reaching about one-third of the initial activity after 48 h at 37 degrees C. This behaviour indicated a branched pathway of the decay of the inactivated enzyme. Kinetic and isoelectric-focusing experiments proved this assumption. The first-order constant of regeneration of active enzyme was 6 × 10(-6)-10 × 10(-6) s-1, whereas the rate constant leading to inactive enzyme (EA') was 10 × 10(-6)-15 × 10(-6) s-1 at pH 7.0. Both constants became larger at higher pH. Inactive enzyme (EA') consisted of two major species, with pI 5.36 (EA'1) and 5.30 (EA'2). The former increased at the beginning of incubation but decreased after prolonged incubation. From consideration of these results and previous data [Arisawa & Then (1983) Biochem. J. 209, 609-615], a likely mechanism of inactivation of TEM-1 beta-lactamase by AMPA is discussed.
Effect of dietary lipid on synaptosomal acetylcholinesterase activity
Authors: Foot M , Cruz T F , Clandinin M T
Published: August 2015

The effect of dietary lipid on the thermotropic properties of acetylcholinesterase activity was examined in rat synaptosomal membrane preparations after feeding diets containing soya-bean oil, sunflower oil or soya-bean phosphatidylcholine as the dietary fats. Arrhenius plots and energies of activation were altered by the duration of feeding as a function of time, as well as by the composition of diet fat fed. Animals fed sunflower oil had the highest maximal velocity for acetylcholinesterase activity. The observations of this study suggest that dietary fat is an important determinant of the physicokinetic properties of lipid-dependent functions in brain synaptosomal membranes.
Purification of human renin by affinity chromatography using a new peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-Leu-R-Leu-Val-Tyr)
Authors: McIntyre G D , Leckie B , Hallett A , Szelke M
Published: August 2015

A new affinity column for renin was prepared by coupling the isosteric peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr, where R is a reduced isosteric bond, -CH2-NH-), to activated 6-aminohexanoic acid-Sepharose 4B. Chromatography of a crude extract of human kidney cortex on this material resulted in a 5500-fold purification of renin in 76% yield. The purified enzyme (specific activity 871 units/mg) was free of non-specific acid-proteinase activity and was stable at pH 6.8 and −20 degrees C over a period of several weeks.
The purification of the hepatic glutathione S-transferases of rainbow trout by glutathione affinity chromatography alters their isoelectric behaviour
Authors: Ramage P I N , Nimmo I A
Published: August 2015

1. The basic glutathione S-transferases from rainbow-trout liver were more stable than the acidic ones. 2. The apparent pI values of these enzymes were lowered when they were eluted from a glutathione affinity column by reduced glutathione at pH 8.85. 3. The pI effect was not a function of the high pH alone, was diminished under conditions less favourable to glutathione oxidation, and did not occur when S-hexylglutathione affinity chromatography was used instead.
Nitrogenase of Klebsiella pneumoniae nifV mutants. Investigation of the novel carbon monoxide-sensitivity of hydrogen evolution by the mutant enzyme
Authors: McLean P A , Smith B E , Dixon R A
Published: August 2015

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.
Purification and characterization of the mouse thymocyte Thy-1 glycoprotein
Authors: Carlsson S R , Stigbrand T I
Published: August 2015

The thymocyte Thy-1 glycoprotein was purified from mouse strain NMRI (Thy 1.2) thymus. Crude membranes were prepared in Tween 20 and solubilized in deoxycholate. The glycoproteins were isolated by affinity chromatography on concanavalin A-Sepharose, and Thy-1 was further purified by two gel-filtration cycles on Sephacryl S-200. The concentration of Thy-1 in fractions obtained during purification was measured by a solid-phase radioimmunoassay with pure mouse brain Thy-1 as standard. Analysis of the purified preparation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed at least five distinct bands in the apparent-Mr region 25000-30000, the polymorphism probably being due to carbohydrate heterogeneity. Amino-acid-analysis data were compatible with the previously published sequence of mouse brain Thy-1. Sugar content was determined at 31% (w/w), and the carbohydrate composition indicates the presence of ‘complex-type’ oligosaccharide chains. The mean Mr of mouse thymocyte Thy-1 was calculated to be 18 100.
Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis
Authors: Rose K , De Pury H , Offord R E
Published: August 2015

Reaction conditions are described that permit the enzyme-assisted semi-synthetic replacement of residue B30 of pig insulin (or of analogue) to proceed in very high yield in 2 h or less. Immobilized trypsin may be used as catalyst, and excess amino acid ester may be recycled after a simple extraction. Alanine-B30 may be replaced by a variety of nucleophiles, including threonine O-t-butyl ether t-butyl ester, in which case the yield of crude product is about 99%. De-protection of the B30 threonyl ester analogue of insulin thus formed then affords human insulin in an overall yield of about 92%, based on pig starting material. The product has full biological potency, as determined by depression of blood glucose concentration in rats, and showed the expected behaviour on radioimmunoassay.
The pH-dependence of class B and class C β-lactamases
Authors: Bicknell R , Knott-Hunziker V , Waley S G
Published: August 2015

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.
Segmental motion and rotational diffusion of the Ca2+-translocating adenosine triphosphatase of sarcoplasmic reticulum, measured by time-resolved phosphorescence depolarization
Authors: Speirs A , Moore C H , Boxer D H , Garland P B
Published: August 2015

We studied the rotational mobility of the Ca2+ + Mg2+-activated ATPase in skeletal-muscle sarcoplasmic-reticulum vesicles, using time-resolved measurements of the depolarization of laser-flash-excited phosphorescence of the extrinsic triplet probe erythrosin. Our results are in general agreement with those of others [Bürkli & Cherry (1981) Biochemistry 20, 138-145] obtained by linear dichroism methods. In addition, we directly observed fast depolarization in the 1-5 microseconds time range that can be attributed to limited motion of part of the protein (segmental motion). Temperature-dependent changes in phosphorescence anisotropy indicated the onset of a conformational change in structure of the Ca2+ + Mg2+-activated ATPase at 11-13 degrees C. We also describe the synthesis of 5-iodoacetamidoerythrosin.
The purification and characterization of a Thy-1-like glycoprotein from chicken brain
Authors: Rostas J A P , Shevenan T A , Sinclair C M , Jeffrey P L
Published: August 2015

We have purified from chicken forebrain a membrane glycoprotein that is enriched in purified synaptic membranes and has an apparent mol.wt. of 22 800 in 15% sodium dodecyl sulphate/polyacrylamide gels. This molecule was compared with rat and human brain Thy-1 glycoproteins purified by the same procedure in order to determine whether it could be a homologue of Thy-1. Although polyvalent heterologous antisera raised against the rat and chicken molecules showed no immunological cross-reactivity with the other glycoprotein, a great deal of physical and chemical similarity was demonstrated between the chicken glycoprotein and rat Thy-1. Their apparent molecular weights, subcellular localization and amino acid and amino sugar compositions are very similar. C.d. spectra show that both molecules contain predominantly a beta-sheet and structure with no detectable alpha-helix. Electrophoretic analysis of the CNBr-cleaved molecules under reducing and non-reducing conditions shows that both molecules contain intramolecular disulphide bridges. Taken together these results suggest that the chicken brain glycoprotein is an immunologically distinct homologue of the mammalian Thy-1 glycoproteins.
Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts
Authors: Turakainen H
Published: August 2015

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.
Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure
Authors: Lambert N , Freedman R B
Published: August 2015

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.
Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction
Authors: Lambert N , Freedman R B
Published: August 2015

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of ‘scrambled’ ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.
Identification of protein disulphide-isomerase as a major acidic polypeptide in rat liver microsomal membranes
Authors: Mills E N C , Lambert N , Freedman R B
Published: August 2015

Protein disulphide-isomerase was purified to homogeneity from rat liver by a rapid high-yielding procedure. Structural properties of the pure enzyme were very similar to those of the bovine liver enzyme purified by the same method. The purified rat liver enzyme was subjected to two-dimensional gel electrophoresis in the presence and in the absence of microsomal membranes, and shown to co-electrophorese with a major acidic polypeptide clearly identifiable in the two-dimensional electrophoretic profile of microsomal membranes. This identification was confirmed by peptide ‘mapping’ of the pure enzyme and of the defined spot from a two-dimensional electrophoresis gel.
Comparative rates of transfer of N-acetylneuraminic acid to acceptors bearing one or more Gal(β 1-4)GlcNAc terminus by the Gal(β 1-4)GlcNAc(NeuAc-Gal) (α 2-6)-sialyltransferase from embryonic chicken liver. Utilization of oligosaccharides as acceptors in sialyltransferase assays
Authors: Bendiak B , Cook G M W
Published: August 2015

Using a number of branched and unbranched oligosaccharides, glycoproteins and artificial glycoproteins bearing Gal(beta 1-4)GlcNAc-R termini as acceptors (where R represents H, oligosaccharide, oligosaccharide-protein or fatty acid-protein), the comparative rates of transfer of NeuAc by the Gal(beta 1-4)GlcNAc(NeuAc-Gal) (alpha 2-6)-sialyltransferase of embryonic chicken liver were determined. Acceptor substrates were utilized at levels approximating physiological, near the Km value of the best acceptor, desialylated alpha 1 acid glycoprotein. The sialyltransferase has a marked preference for multi-branched acceptors. From the specificity data, it is concluded that the enzyme binds at least two Gal(beta 1-4)GlcNAc termini of an acceptor molecule, and that the relative orientation of the branches is an important factor determining the rate of catalysis by the enzyme. The use of oligosaccharides as acceptors to study sialyltransferase catalyses is emphasized. Results are discussed in the context of the mode of assembly of sialoside termini of known glycoprotein structures in vivo.
Identification of ecdysonoic acid and 20-hydroxyecdysonoic acid isolated from developing eggs of Schistocerca gregaria and pupae of Spodoptera littoralis
Authors: Isaac R E , Milner N P , Rees H H
Published: August 2015

Ecdysonoic acid and 20-hydroxyecdysonoic acid have been purified from developing eggs of the desert locust, Schistocerca gregaria, by high performance liquid chromatography (h.p.l.c.), and their structures were determined by p.m.r. spectroscopy and fast atom bombardment mass spectrometry of the free and methyl ester derivatives. 20-Hydroxyecdysonoic acid was also characterized from Spodoptera littoralis pupae. The occurrence of both 20-hydroxyecdysonoic acid and ecdysonoic acid in Sp. littoralis pupae was also established by h.p.l.c. comparison of the 3H-labelled acids formed from [3H]ecdysone and of their methyl esters with the corresponding substances from Sch. gregaria. The significance of ecdysteroid acids as products of ecdysteroid inactivation is discussed.
A recording viscometer for assaying mammalian collagenase
Authors: Macartney H W , Bates S R E , Blumson N L , Nelson D , Jamison D , Elmore D T
Published: August 2015

A recording viscometer for monitoring the action of mammalian collagenase on soluble collagen is described. For this system, where only one peptide bond is cleaved per subunit, it is shown theoretically that the decrease in viscosity is proportional to the fraction of molecules cleaved. Experimental confirmation was obtained by parallel monitoring of hydrolysis by using the fluorescamine assay of liberated amino groups. The initial velocity of reaction is proportional to substrate concentration and enzyme concentration.
Antibodies to bovine liver branched-chain 2-oxo acid dehydrogenase cross-react with this enzyme complex from other tissues and species
Authors: Heffelfinger S C , Sewell E T , Danner D J
Published: August 2015

Antibodies were produced in rabbits against bovine liver branched-chain 2-oxo acid dehydrogenase (BCOAD) for three reasons. First, since activity of BCOAD varies among tissues within a species, it is necessary to ascertain whether isoenzymes exist within the different tissues. Second, it is of great interest to determine the structural similarity of BCOAD among mammalian species. Third, heritable defects of this multienzyme complex are known in humans, and techniques for studying BCOAD subunit composition in cells expressing the defect include the use of specific antibodies. Antibodies produced against BCOAD inhibit complex activity and cross-react with BCOAD from various tissues and species. By using rocket immunoelectrophoresis, the complex can be quantified in mitochondrial extracts. Each subunit is immunogenic, making the detection of subunit variants possible by the combined techniques of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and Western blotting. Finally, antibodies produced against pig heart lipoamide dehydrogenase cross-react with BCOAD, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, giving further evidence for the commonality of this subunit among these three mitochondrial complexes.
Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin
Authors: Danielsson A , Björk I
Published: August 2015

Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 × 10(-6)-3.4 × 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Furthermore, the results indicate that the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.
Purification of hydrogenases by affinity chromatography on Procion Red-agarose
Authors: Schneider K , Pinkwart M , Jochim K
Published: August 2015

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.
Miltenberger Class I and II erythrocytes carry a variant of glycophorin A
Authors: Blanchard D , Asseraf A , Prigent M J , Cartron J P
Published: August 2015

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.
Regulation of surface-membrane enzymes by lipid ordering. A model based on allosteric transition theory
Authors: Blanquet P R
Published: August 2015

This paper shows for the first time that effects of lipid ordering on the activity of surface-membrane enzymes can be interpreted in terms of the two-state allosteric transition theory. In this mathematical modelling, the conversion of an active molecule into a catalytically inactivated one of a given enzyme can be directly triggered by a change in the order parameter of lipids. Depending on the lipid order, the tendency for this conversion is either independent of the state of other enzyme molecules or co-operatively aided through intermolecular interactions that in turn depend on lipid ordering. The theory so developed is then applied to (Na+ + K+)-dependent adenosine triphosphatase (EC 3.6.1.3). On comparison of the relevant theoretical expression and published measurements, the fit to experimental results is shown to support the model. Furthermore, the model accounts for much of the data in the literature of the behaviour of this enzyme.
Comparison of inhibitors of S-adenosylmethionine decarboxylase from different species
Authors: Pegg A E , Jacobs G
Published: August 2015

S-Adenosyl-L-methionine decarboxylases were purified from rat ventral prostate, yeast (Saccharomyces cerevisiae), slime mould (Physarum polycephalum) and bacteria (Escherichia coli) and tested for inhibition by a variety of nucleosides related to S-adenosylmethionine and by methyl- and ethyl-glyoxal bis(guanylhydrazone). Although the enzymes from these different sources are markedly different with respect to activation by cations, the inhibition by nucleosides was quite similar. Very little inhibition was seen when analogues of S-adenosylmethionine with a different base were tested or when the ribose ring was opened or the positive charge on the sulphur atom was not present. Some derivatives in which the amino acid portion of the molecule was altered were more potent inhibitors, but again there was little difference between the enzymes from different sources. 5′-(Dimethylsulphonio)-5′-deoxyadenosine and S-adenosyl-3-methylthiopropylamine were the most inhibitory substances and had similar Ki values, suggesting that the aminopropyl group does not contribute significantly to the binding. All of the S-adenosylmethionine decarboxylases were strongly competitively inhibited by methylglyoxal bis(guanylhydrazone) and even more powerfully by its ethyl analogue, although the putrescine-activated enzymes from prostate and yeast were more sensitive than the bacterial and slime-mould enzymes. All of the S-adenosylmethionine decarboxylases tested bound to a column of methylglyoxal bis(guanylhydrazone) linked to Sepharose and were not eluted by 0.5 M-NaCl, but could be released by 1 mM concentrations of the drug, providing a rapid and efficient method for their purification.
Characterization of dolichol and dolichyl phosphate phosphatase from soya beans (Glycine max)
Authors: Ravi K , Rip J W , Carroll K K
Published: August 2015

A series of polyprenols, ranging in length from 15 to 22 isoprene units, has been isolated from soya beans (Glycine max) and purified by high-pressure liquid chromatography. N.m.r., i.r. and mass spectra of the compounds indicated that they are alpha-saturated polyprenols of the dolichol type. The amount present in dry seeds was about 9 mg/100 g, whereas dolichyl phosphate (Dol-P) was present only in trace amounts. Dol-P phosphatase activity was detected in the microsomal fraction of 5-day-old germinating soya-bean cotyledons. The Dol-P phosphatase activity was linear with respect to time and protein concentration and exhibited a broad pH optimum (pH 7-9). Triton X-100 was necessary for significant enzyme activity. Enzyme activity was slightly enhanced by EDTA, whereas dithiothreitol was without effect. An apparent Km of 5 microM was determined for Dol-P. Bivalent metal ions were not required for enzyme activity. A number of phosphorylated compounds tested as enzyme substrates (including a number of nucleoside phosphates, glucose 6-phosphate, sodium β 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.

-glycerophosphate and Na4P2O7) did not compete with [1-3H]Dol-P as substrate. A number of phospholipids were also tested for their ability to act as Dol-P phosphatase substrates. At 1 mM concentration, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and lysophosphatidic acid each inhibited enzymic activity. However, at 0.1 mM concentration, phosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas phosphatidic acid and lysophosphatidic acid were still inhibitory. Phosphatidic acid showed competitive inhibition.
A new rapid method for the identification of reducible collagen cross-links in small tissue samples
Authors: Smolenski K A , Avery N C , Light N D
Published: August 2015

A normal-phase high-pressure liquid-chromatography system was used with amino-propyl-bonded silica as the column packing in order to resolve amino acids, reduced amino acid derivatives and reduced cross-links of collagen. The method utilized a solvent system comprising acetonitrile and 10 mM-potassium phosphate buffer, pH 4.3, as described by Schuster [(1980) Anal. Chem. 52, 617-620]. With modifications to the original gradient elution it was possible to resolve fully the radiolabelled components of reduced collagen in one run of less than 80 min. The method is very sensitive, and small biopsy samples can readily be investigated. Although solute retention times gradually decreased with repeated use, little loss of resolution of the reducible cross-links of collagen occurred during a 30-day trial period.
A study of the biosynthesis of the tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine in a β-lactam-negative mutant of Cephalosporium acremonium
Authors: Adlington R M , Baldwin J E , Lopez-Nieto M , Murphy J A , Patel N
Published: August 2015

A cell-free extract of Cephalosporium acremonium (Takeda N-2) was obtained that synthesized the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and also the dipeptide delta-(L-alpha-aminoadipyl)-L-cysteine from the corresponding L-amino acids.
The denaturation of rabbit muscle phosphorylase b by guanidinium chloride
Authors: Price N C , Stevens E
Published: August 2015

The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.