bluuebunny/crossref_metadata_2025 huggingface dataset:
The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat
Authors: Orpin C. G. , Knight M. , Evans W. C.
Published: August 2015

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1′-dimethyl-4,4′-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [14C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[14C]methylisonicotinate, and no [14C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-14C]isonicotinate released 95% of the radioactivity as 14CO2. The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO2 by enzyme systems requiring NAD+.
The microbial biosynthesis of methionine
Authors: Salem A. R. , Foster M. A.
Published: August 2015

1. The enzymes leading to the methylation of homocysteine have been examined in three micro-organisms: a cobalamin-producing bacterium, Bacillus megaterium; a yeast, Candida utilis; and a basidiomycete fungus, Coprinus lagopus. The yeast and the fungus contain negligible endogenous cobalamin. 2. Extracts of each organism catalyse C1-transfer from serine to homocysteine with a polyglutamate folate coenzyme. 3. The enzymes generating the methyl group of methionine from C-3 of serine have similar properties in each case, but different mechanisms of homocysteine transmethylation from 5-methyltetrahydrofolates were found. 4. B. megaterium contains an enzyme with properties suggestive of a vitamin B12-dependent homocysteine transmethylase, whereas Cand. utilis and Cop. lagopus transfer the methyl group by a reaction characteristic of the cobalamin-independent mechanism established for Escherichia coli. 5. The specificity of each transmethylase for a 5-methyltetrahydropteroylpolyglutamate is consistent with the results of analyses of endogenous folates in these organisms, which showed only conjugated forms. 6. None of the extracts catalysed methionine production from S-adenosylmethionine and homocysteine. 7. These results are compared with results now available for methionine synthesis in other organisms, which show a considerable diversity of mechanisms.
Relative changes in the function of muscle ribosomes and mitochondria during the early phase of steroid-induced catabolism
Authors: Bullock Gillian R. , Carter Elizabeth E. , Elliott P. , Peters R. F. , Simpson Phillida , White A. M.
Published: August 2015

1. Five glucocorticoids, when administered daily to rats for 5–7 days at a dosage of 5mg/kg, were in the following order of effectiveness with respect to their ability to decrease the weight gain of whole animals and the vastus lateralis, vastus medialis and gluteus medius muscles: corticosterone<prednisolone<triamcinolone <betamethasone<dexamethasone<triamcinolone acetonide. 2. The low catabolic potencies of corticosterone and prednisolone were reflected in the high doses (20mg/kg) required to decrease significantly the incorporating activity in vitro of ribosomes isolated from the muscles 6h later. However, triamcinolone was as effective as triamcinolone acetonide in causing a significant effect at 0.05mg/kg, although it had a far lower catabolic potency than the acetonide. 3. The relationship between decrease in ribosomal activity in vitro and weight loss was better understood by considering the duration of the effect after single doses of steroids. Ribosomes returned to normal function between 6 and 12h after the administration of corticosterone (20mg/kg), but still had decreased activity 12h after a similar dose of prednisolone. Normal ribosomal function was renewed between 12 and 18h after the administration of triamcinolone and betamethasone at 5mg/kg, but decreased activity persisted for more than 48h after similar doses of dexamethasone or triamcinolone acetonide. 4. There was no difference in the rate of incorporation of amino acids into nascent protein on polyribosomes from muscle of control animals and animals treated 6h previously with triamcinolone acetonide or dexamethasone. 5. The rate of weight gain of the heart was not affected by any of the steroids tested and heart ribosomes maintained normal activity although concentrations of steroids in this organ 5min after administration were 2–3 times that in skeletal muscle. 6. Mitochondria, isolated from the muscle of animals that had received triamcinolone acetonide (20mg/kg) 12h previously, were shown, by using a Coulter counter, to be enlarged. Nevertheless oxidation was only slightly uncoupled 12h after drug administration and they had normal function 6h later. Similar results with respect to mitochondrial function were obtained after the administration of dexamethasone (20mg/kg) and betamethasone (20mg/kg). 7. From these results it is concluded that the mitochondria are not functionally involved in the early phase of steroid-induced muscle catabolism. The observed decrease in the incorporating ability of muscle ribosomes in vitro appears to be much more closely linked to the initial catabolic event.
Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre
Authors: Kalogerakos T G , Oikonomakos N G , Dimitropoulos C G , Karni-katsadima I A , Evangelopoulos A E
Published: August 2015

Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 × 10(4) M-1 - min-1 and 2.15 × 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 × 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.
Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi
Authors: Matagne R F , Schlösser J P
Published: August 2015

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.
The equilibrium constant and the reversibility of the reaction catalysed by nicotinamide-adenine dinucleotide kinase from pigeon liver
Authors: Apps D K , Nairn A C
Published: August 2015

The reversibility of the NAD+ kinase reaction was established, and the kinetic parameters of the rate equation in the reverse direction were determined. The equilibrium constant of the reaction was determined by using the purified pigeon liver enzyme and radioactively labelled nicotinamide nucleotides. The relationship between kinetic parameters of the forward and reverse reactions is in reasonable agreement with the measured equilibrium constant. As expected from the proposed mechanism of action, the enzyme does not catalyse isotope exchange between NAD+ and NADP+ in the absence of ADP and ATP. Although homogeneous as judged by polyacrylamide-gel electrophoresis, the enzyme preparation exhibits ADP/ATP isotope-exchange activity which could not be separated from NAD+ kinase activity, but kinetic evidence suggests that this is probably due to a contaminant.
The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid Δ4-Δ5-isomerase component of cholesterol oxidase
Authors: Smith A G , Brooks C J W
Published: August 2015

1. 5-Cholesten-3-one was shown to be an intermediate in the conversion of cholesterol into 4-cholesten-3-one by Nocardia cholesterol oxidase. 2. The absence of a C-17 side chain from 5-androstene-3,17-dione slightly increased the Vmax. of the isomerase activity relative to 5-cholesten-3-one (1.7-fold), but greatly increased the Km. 3. Incubations of [4alpha-2H]-and [4beta-2H]-cholesterol with cholesterol oxidase showed that the 4beta-hydrogen atom can be transferred to the 6beta-position. However, incubations of cholesterol, 5-cholesten-3-one and 4-cholesten-3-one with the enzyme in 2H2O led to some incorporation of 2H into the 4-cholesten-3-one products, mostly at position 6beta. 4. Both the isomerase and the oxidase activities of cholesterol oxidase were inhibited by 5,10-seco-19-nor-5-cholestyne-3,10-dione.
The purification and properties of p-cresol-(acceptor) oxidoreductase (hydroxylating), a flavocytochrome from Pseudomonas putida
Authors: Hopper D J , Taylor D G
Published: August 2015

The enzyme that catalyses the hydroxylation of the methyl group of p-cresol was purified from Pseudomonas putida. It has mol.wt. 115000 and appears to contain two subunits of equal molecular weight. One subunit is a c-type cytochrome and the other is a flavoprotein. Reduction of the cytochrome occurred on addition of substrate. The same enzyme catalyses both p-cresol hydroxylation and the further oxidation of the product, 4-hydroxybenzyl alcohol. The stoicheiometry of acceptor reduced per molecule of substrate oxidized is that for two dehydrogenation reactions. The Km for p-cresol is 7.3 × 10(-6) M and that for 4-hydroxybenzyl alcohol is 47.6 × 10(-6) M. The enzyme, which is assayed with phenazine methosulphate as electron acceptor, was stimulated by particulate material, which probably contains the acceptor in vivo.
Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate
Authors: Briley P A , Eisenthal R , Harrison R , Smith G D
Published: August 2015

Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.
Free and lipid myo-inositol in tissues from rats with acute and less severe streptozotocin-induced diabetes
Authors: Palmano K P , Whiting P H , Hawthorne J N
Published: August 2015

Acute diabetes with ketosis was induced in rats by intraperitoneal streptozotocin and also a milder form of diabetes without ketosis by injecting less of the drug. The acutely diabetic rats were killed 72h after injection and the others after either 2 or 13 weeks. Free and lipid myo-inositol was then measured in various tissues and body fluids by g.l.c. of the trimethylsilyl ether. Serum inositol was increased in the acutely diabetic group, whereas liver inositol was decreased. Brain and kidney inositol concentrations were increased in the mildly diabetic animals at 13 weeks and there was a progressive decrease in sciatic-nerve inositol. Lipid inositol of sciatic nerve was decreased in the acutely diabetic group only. Brain lipid inositol concentration was decreased in mild diabetes at 13 weeks. Possible implications of these findings in relation to diabetic neuropathy was discussed.
Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin
Authors: Kazim A L , Atassi M Z
Published: August 2015

The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.
Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex
Authors: Grazi E , Blanzieri M
Published: August 2015

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.
Studies on C3 convertases. Inhibition of C5 convertase formation by peptides containing aromatic amino acids
Authors: Kossorotow A , Opitz W , Etschenberg E , Hadding U
Published: August 2015

The influence of various peptides containing the aromatic amino acids phenylalanine and tyrosine on the formation of the enzyme EAC1423 of the complement system from component C3 and enzyme EAC142 was investigated. Kinetic analysis of enzyme EAC1423 formation and studies on the binding of the C3b fragment of 125I-labelled component C3 to enzyme EAC142 both showed that binding of the C3b fragment of component C3 was decreased by the peptides. Kinetic studies on component-C3 turnover in the fluid phase of enzyme EAC142 failed to reveal effects of the peptides. However, an initial lag in component-C3 turnover occurred that at constant component-C3 concentration was inversely proportional to enzyme EAC142 concentration. This lag in enzyme EAC142 activity is considered as an indication that the interaction of enzyme EAC142 with component C3 possibly does not follow simple Michaelis-Menten kinetics, as was previously assumed. It is shown that the stages after enzyme EAC1423 formation are not influenced by the peptides, suggesting a high degree of specificity of the peptides for the inhibition of enzyme EAC1423 formation.
The histidine residues of phospholipase C from Bacillus cereus
Authors: Little C
Published: August 2015

The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.
Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands
Authors: Clarke C M , Knowles J R
Published: August 2015

The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.
Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand
Authors: Clarke C M , Knowles J R
Published: August 2015

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].
Nitrogenase of Klebsiella pneumoniae. Distinction between proton-reducing and acetylene-reducing forms of the enzyme: effect of temperature and component protein ratio on substrate-reduction kinetics
Authors: Thorneley R N F , Eady R R
Published: August 2015

Non-linear rates of acetylene reduction and concomitant H2 evolution were observed for the nitrogenase of Klebsiella pneumoniae at 10 degrees C. A lag phase of 1-4 min, dependent on the ratio of Mo-Fe protein to Fe protein present, occurred before linear rates of acetylene reduction were achieved. A complementary burst phase for concomitant H2 evolution in the presence of acetylene was also observed. When the proton was the only reducible substrate present, linear rates of H2 evolution were observed. N2 was a poor substrate under these conditions. Similar lag and burst phases occurred at 30 degrees C, but only when a large molar excess of Mo-Fe protein with respect to Fe protein was present. The results at 10 degrees C show that the binding of acetylene to the enzyme stimulates electron flow, but that these electrons, which initially reduce protons, can only reduce acetylene after a lag phase that cannot be accommodated in the turnover time calculated under steady-state conditions.
The reaction of Fe(III) myoglobin fluoride with reducing agents
Authors: Cox R P
Published: August 2015

The reaction of reducing agents with Fe(III) myoglobin fluoride from sperm whale was studied at alkaline pH values. The rate of reduction by dithionite was indistinguishable from the rate of ligand dissociation even when the values of the rate constants for both were only 1.0 X 10(-3)S-1 (at pH 10.6). Reduction by the reduced Methyl Viologen radical ion and reduced Safranine was faster than the rate of dissociation, providing evidence that these reductants can donate electrons to the iron centre via a pathway involving an (undetectable) liganded Fe(II) intermediate.
Inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by dicyclohexylcarbodi-imide
Authors: Feinstein D L , Fisher R J
Published: August 2015

The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.
Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum
Authors: Del Campo G , Puente J , Valenzuela M A , Traverso-Cori A , Cori O
Published: August 2015

A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5′-[alpha, beta-methylene]triphosphate and adenosine 5′-[beta, gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP.
Mixed-valence cytochrome oxidase-formate complex. A steady-state intermediate
Authors: Brittain T , Greenwood C , Johnson A
Published: August 2015

At neutral pH, formate binds to the haem a3 component of cytochrome c oxidase to give a complex that reacts differently from the non-liganded enzyme with reducing agents. Addition of sodium dithionite to the formate complex leads directly to the formation of the fully reduced species, whereas reduction with ascorbate/tetramethylenephenylene-diamine can lead to the production of a mixed-valence species. The stability of this mixed-valence form was studied, and the species appears to represent a ‘steady-state’ situation that is stable only in the presence of an excess of O2 and reducing equivalents. Characterization of the mixed-valence complex by electron paramagnetic resonance and magnetic circular dichroism reveals the presence of reduced low-spin haem a together with reduced detectable copper and high-spin ferric haem a3.
The separation and characterization of bronchial glycoproteins by density-gradient methods
Authors: Creeth J. Michael , Bhaskar K. Ramakrishnan , Horton John R. , Das Indrajit , Lopez-Vidriero Maria-Teresa , Reid Lynne
Published: August 2015

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.
Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein
Authors: Jones A J S , Rumsby M G
Published: August 2015

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.
Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy
Authors: Williams-Smith D L , Bray R C , Barber M J , Tsopanakis A D , Vincent S P
Published: August 2015

Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.
The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes
Authors: Okita T W , Volcani B E
Published: August 2015

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.
Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction
Authors: Bella A , Whitehead J S , Kim Y S
Published: August 2015

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.
Purification and properties of the hexosaminidase A-activating protein from human liver
Authors: Hechtman Peter , LeBlanc Dorothy
Published: August 2015

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of GM2 ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of GM2 ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the GM2 ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for GM2 ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-GM2 ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of GM2 ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme–activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.
Interaction of cartilage proteoglycans with hyaluronic acid. The role of the hyaluronic acid carboxyl groups
Authors: Christner J E , Brown M L , Dziewiatkowski D D
Published: August 2015

Hyaluronic acid-derived oligomers of five to fifteen repeat dissaccharides effectively bind to bovine nasal-cartilage proteoglycan and inhibit the interaction between proteoglycans and high-molecular-weight hyaluronic acid. If, however, the hyaluronic acid oligosaccharides are modified by reaction with diazomethane to form the carboxyl methyl esters of the glucuronic acid residues, their inhibitory activity is abolished. The binding capacity can be fully restored by saponification. The amide derivative, which is formed by condensation of the oligosaccharide carboxyl groups with glycine methyl ester, is also ineffective in blocking the proteoglycan-hyaluronic acid interaction. In this case, binding activity is not restored when the amidated oligomers are subjected to saponification to yield the free carboxylate groups on the glycine residues. Thus the displacement of the carboxylate groups on the polysaccharide chain by the interposition of a glycine residue blocks the interaction between the proteoglycans and the hyaluronic acid oligomers. When the oligosaccharide methyl ester is reduced with NaBH4, the resultant glucose-containing oligomers exhibit decreased binding to proteoglycans. Thus it appears that the hyaluronic acid carboxylate anion in a specific spatial orientation is required for hyaluronic acid-proteoglycan interaction.
Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K
Authors: Williamson J , Greene J , Chérif S , Milner-White E J
Published: August 2015

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3—MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.
The immunological similarity of rat glandular kallikreins
Authors: Proud D , Bailey G S , Nustad K , Gautvik K M
Published: August 2015

The immunological properties of the submandibular kallikrein, urinary kallikrein, pancreatic kallikrein and pancreatic prekallikrein of the rat were studied by immunodiffusion, immunoelectrophoresis and radioimmunoassay. Although they behaved differently electrophoretically, all the antigens showed identical immunological behaviour. The implications of this are discussed.
A simple and rapid method for isolation of reduced carbohydrate fragments from the linkage region of cartilage keratan sulphate
Authors: Kieras F J
Published: August 2015

The method described is a simple and rapid procedure for isolation in high yield of carbohydrate fragments containing terminal galactosaminitol derived from the polysaccharide-protein linkage region of cartilage keratan sulphate. It is based on the observation that reducing sugars bind tightly to Dowex-1 resin (hydroxide form), whereas reduced analogues (sugar alcohols) do not [H. Yamaguchi, S. Inamura & K. Makino (1976) J. Biochem. (Tokyo) 79, 299-303].
The variability of nitro group-protein interaction in the 2,4-dinitrophenyl-binding antibodies M315, M460 and X25 investigated by resonance Raman spectroscopy
Authors: Gettins P , Dwek R A , Perutz R N
Published: August 2015

Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab‘ and X25 Fab’. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N-C-C-2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab‘ and X25 Fab’ fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline.
Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas
Authors: Martínez M C , Avilés F X , Sansegundo B , Cuchillo C M
Published: August 2015

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.
A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure
Authors: Pearson J P , Allen A , Parry S
Published: August 2015

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 × 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 × 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.
Acetylcholinesterase: evidence that sodium ion binding at the anionic site causes inhibition of the second-order hydrolysis of acetylcholine and a decrease of its pKα as well as of deacetylation
Authors: Smissaert H R
Published: August 2015

For bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), the Michaelis parameters Vmax., and Km for the natural substrate acetylcholine were estimated as a function of pH and sodium chloride concentration by the pH-stat method. A single dissociation constant for Na+ binding (K = 7 × 10(-3) M) suffices to explain the salt dependence of Vmax./Km and of Km as well as the pH dependence of Vmax./Km and Vmax., Km being pH independent. This finding provides evidence for a specific effect of Na+, presumably by binding at the anionic subsite of the active centre. Na+ binding causes a 50-fold decrease in kcat./Km as well as a decrease of one unit in the pKa of both kcat./Km and kcat.. The intrinsic pKa in the absence of salt at 25 degrees C is about 7.5. Comparison of the degree of fit of the data to the Debeye-Huckel equation, in accordance with an alternative general salt effect, as well as published data for sodium and potassium chlorides also favour a specific salt effect.
Effect of partial hepatectomy on removal of O6-methylguanine from alkylated DNA by rat liver extracts
Authors: Pegg A E , Perry W , Bennett R A
Published: August 2015

1. The activity of an enzyme catalysing the loss of O6-methylguanine from methylated DNA was increasing during liver regeneration after partial hepatectomy. Activity was increased 3-fold by 24h and was maximal (6-fold increase) over the period 48-72h after operation. 2. This activity could also be induced by chronic treatment with dimethylnitrosamine, but the maximal response amounted to a 2-3-fold change (with the greater effect in male rats) after 4-6 weeks of exposure to daily doses of 2 mg of dimethylnitrosamine/kg. 3. Neither partial hepatectomy nor treatment with dimethylnitrosamine increased the activities of two other enzymes repairing alkylated DNA, DNA (7-methylguanine-)glycosylase and DNA (3-methyladenine-)glycosylase. 4. These results therefore indicate that there is a selective induction of the O6-methylguanine removal system during hepatocyte proliferation. Since this product is known to lead to mutations and its persistence in DNA throughout cell replication has been implicated in tumour initiation, this induction may play a role in resistance to carcinogenesis by alkylating agents.
Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture
Authors: Carlstedt I , Cöster L , Malmström A
Published: August 2015

35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.