bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro
Authors: Snary D. , Allen A.
Published: August 2015

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-14C]-glucose and from [G-3H]threonine or [G-3H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with 14C in the carbohydrate and 3H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.
General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide
Authors: Mosbach K. , Guilford H. , Ohlsson R. , Scott M.
Published: August 2015

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.
Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat
Authors: McGee Margaret M. , Greengard Olga , Knox W. Eugene
Published: August 2015

The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13–15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.
The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues
Authors: Etherington D. J.
Published: August 2015

1. An enzyme present in rat liver extracts degraded insoluble collagen maximally at pH3.5. Collagenolytic activity was more abundant in kidney, spleen and bone marrow and was also present in decreasing concentrations in ileum, lung, heart, skin and muscle. 2. The crude collagenolytic cathepsin was activated by cysteine and dithiothreitol, but not by 2-mercaptoethanol. Iodoacetamide, p-chloromercuribenzoate and 7-amino-1-chloro-3-l-tosylamidoheptan-2-one hydrochloride inhibited the enzyme. Zn2+, Fe3+ and Hg2+ ions were strongly inhibitory, but Ca2+, Co2+, Mg2+ and Fe2+ ions had little or no effect. EDTA was an activator of the enzyme. Inhibitors of cathepsin B were found to enhance collagenolysis, but phenylpyruvic acid, a cathepsin D inhibitor, inhibited the enzyme. Di-isopropyl phosphorofluoridate had no effect. 3. Collagenolysis at pH3.5 and 28°C was restricted to cleavage of the telopeptide region in insoluble collagen, and the material that was solubilized consisted mostly of α-chains. 4. The collagenolytic cathepsin was separated from cathepsins B2 and D by fractionation on Sephadex G-100 and a partial separation from cathepsin B1 was obtained by chromatography on DEAE-Sephadex. 5. The function of the collagenolytic cathepsin in the catabolism of collagen is discussed in relation to the action of the other lysosomal proteinases and the neutral collagenase.
Effect of compounds of the urea–guanidinium class on renaturation and thermal stability of acid-soluble collagen
Authors: Russell A. E. , Cooper D. R.
Published: August 2015

The effects of guanidinium salts in decreasing the renaturation rate and lowering the thermal stability of acid-soluble calf-skin collagen have been compared with those of formamide and urea. With the exception of guanidinium sulphate at higher concentrations, no qualitative differences were apparent in the effects of these perturbants, which thus differed only in molar activity. Activity variation in the guanidinium salts reflected a net effect resulting from additivity of cation and anion contributions. As observed in other protein systems, lyotropic activity increased in the series formamide<urea<guanidinium ion, and in the guanidinium salts in the anion order fluoride<sulphate<chloride<bromide<nitrate<iodide. Low activities of guanidinium fluoride and sulphate were attributable to counter-effects of the anions, which acted as structural stabilizers. Changes in renaturation kinetics induced by either temperature or added perturbants appeared to conform with the Flory–Weaver model for the collagen transition. Additivity and non-specificity of the observed effects are discussed with particular reference to a common mechanism involving weak, non-saturated binding of perturbants at protein peptide groups.
Intracellular distribution of serum albumin and its possible precursors in rat liver
Authors: Geller D. M. , Judah J. D. , Nicholls Marion R.
Published: August 2015

1. The fractionation of intracellular albumin labelled with radioactive l-leucine was studied in rat liver by means of isoelectric focusing. 2. Isoelectric fractionation was compared with ion-exchange chromatography for purification of radioactive intracellular albumin obtained by antibody precipitation. Similar results were obtained with both methods of separation. Purified albumin contains only a minor amount of the radioactivity. The remainder is associated with albumin-like protein(s). 3. The albumin-like protein has the properties of a precursor of plasma albumin. 4. The distribution and turnover of radioactive albumin in rough and smooth microsomal fractions and in a Golgi-rich fraction were studied. 5. It is concluded that newly synthesized albumin, as such, appears only momentarily if at all in any intracellular structure before its appearance in the plasma. 6. It is also concluded that the rate-limiting step in the secretion of plasma albumin is the conversion of precursor(s) into albumin. We can find no evidence to suggest that there is any significant transport of albumin, as such, during the course of secretion.
Oxidation of p-dimethylaminomethylbenzylamine by pig kidney diamine oxidase. A new method for spectrophotometric assay
Authors: Bardsley W. G. , Crabbe M. J. C. , Shindler J. S. , Ashford J. S.
Published: August 2015

1. The oxidation of some para-substituted benzylamines by diamine oxidase produces the corresponding aldehydes. This was studied to develop a spectrophotometric method for following the enzyme reaction, as the aldehydes produced absorb strongly at 250nm where the substrates are almost optically transparent. 2. p-Dimethylaminomethylbenzylamine was the most useful substrate and full details of its preparation are given. The synthesis of its related oxidation product, p-dimethylaminomethylbenzaldehyde, is also described. 3. The effects of variations in pH, ionic strength, temperature and oxygenation on the reaction are described and the usefulness of the method is illustrated by several applications and assessed by comparison with the standard spectrophotometric assay.
The amino acid sequence of rabbit slow-muscle troponin I.
Authors: Grand Roger J. A. , Wilkinson J. Michael
Published: August 2015

Troponin I was isolated from six red muscles in the hind leg of the rabbit. Soleus, semi-tendinosus, vastus intermedius and adductor longus muscles contained primarily slow-muscle troponin I, vastus lateralis contained fast-muscle troponin I and quadratus femoris contained a mixture of the two. The complete amino acid sequence of the troponin I from slow muscle was determined. Seven CNBr fragments were isolated and sequenced by using the dansyl–Edman technique after digestion with proteolytic enzymes. The CNBr fragments were ordered by isolation of tryptic peptides containing carboxy[14C]methyl-methionine. Direct evidence for the conjunction of residues 8 and 9 has not been obtained, and one of the carboxyl groups between residues 71 and 79 may carry an amide group. Slow-muscle troponin I is a single polypeptide chain of 184 residues with a mol.wt. of 21146. It has a net overall positive charge of 18 at pH7, and an absorption coefficient, A1%,1cm280, of 5.43. The protein was isolated with both a blocked and an unblocked N-terminus, although the nature of the blocking group was not determined. Proline was found to be the N-terminal amino acid. Two forms of the protein could also be distinguished by the presence of an extra two residues at the C-terminus. Comparison of sequences of troponin I from rabbit slow, fast and cardiac muscle shows that homology is most marked in the C-terminal half of the molecules. Towards the N-terminus the homology becomes much less marked. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50079 (32 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained in the terms given in Biochem. J. (1977), 161, 1.
Methylated nucleotide sequences in HeLa-cell ribosomal ribonucleic acid. Correlation between the results from ‘fingerprinting’ hydrolysates obtained by digestion with T1 ribonuclease and with T1 plus pancreatic ribonuclease
Authors: Maden B E H , Khan M S N
Published: August 2015

The methylated nucleotide sequences in HeLa-cell rRNA were previously characterized after enzymic digestion of the rRNA by T1 ribonuclease alone or by combined T1 plus pancreatic ribonucleases. For any methylated product occurring in a T1-ribonuclease digest there must be one or more corresponding products in a combined T1-plus-pancreatic-ribonuclease digest. Here we correlate fully the inter-relationship between the methylated products occurring in the two digestion systems. The analysis has led to the resolution of some previous uncertainties and has permitted an almost complete qualitative and quantitative description of the methylated components in HeLa-cell rRNA. The data are compared with those reported by other authors for HeLa-cell rRNA.
Correction of partial amino acid sequence of erabutoxins
Authors: Maeda N , Tamiya N
Published: August 2015

The amino acid sequences of erabutoxins a and b were re-examined. The previously reported sequence of Ser-Glu at positions 21 and 22 of erabutoxins was corrected to Glu-Ser.
Lysostaphin endopeptidase-catalysed transpeptidation reactions of the imino-transfer type
Authors: Sloan G L , Smith E C , Lancaster J H
Published: August 2015

The glycylglycine endopeptidase in lysostaphin has been found capable of catalysing both hydrolysis and transpeptidation reactions when acting on glycyl peptides. The ability of the enzyme to utilize dansyldiglycine (5-dimethylaminoaphthalene-1-sulphonylglycylglycine) as an acceptor molecule in transpeptidation reactions, although it is incapable of hydrolysing the peptide bond in this compound, indicates the enzyme must be capable of forming the equivalent of an imino-enzyme intermediate during the catalytic process.
The effect of matrix on the binding of albumin to immobilized Cibacron Blue
Authors: Angal S , Dean P D G
Published: August 2015

With Cibacron Blue as a ligand, the suitability of Sepharose, Sephacryl, cellulose and Ultrogel as column supports for the affinity chromatography of human serum albumin was examined. Sepharose and Sephacryl were the most effective matrices. Albumin was readily removed from such columns by using an electrophoretic desorption technique.
Hydroxylation of aromatic compounds by reduced nicotinamide-adenine dinucleotide and phenazine methosulphate requires hydrogen peroxide and hydroxyl radicals, but not superoxide
Authors: Halliwell B
Published: August 2015

1. A mixture of NADH and phenazine methosulphate hydroxylates aromatic compounds at acidic pH values. 2. Hydroxylation is inhibited by catalase and by scavengers of the hydroxyl radical (-OH) but not by superoxide dismutase. 3. It is concluded that neither O2 leads to nor HO2- is sufficiently reactive to hydroxylate aromatic rings.
Identification and partial characterization of an inhibitor of collagenase from rabbit bone
Authors: Sellers Anthony , Reynolds John J.
Published: August 2015

Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.
Evidence for the role of vitamin B-6 as a cofactor of lysyl oxidase
Authors: Murray J C , Levene C I
Published: August 2015

At 24 h after injection of 16-day chick embryos with [C-3H]pyridoxine hydrochloride, some of this label appears in the epiphysial cartilage. Over 35% of this radioactivity appears in the form of [G-3H]pyridoxal and a further 30% as other vitamin B-6 compounds. Partial purification of lysyl oxidase from the labelled epiphysial cartilage reveals a single peak of radioactivity coinciding with a single peak of enzyme activity. On dialysis against phosphate-buffered saline, 75% of this radioactivity is found to be non-diffusible. After incubation with isonicotinic acid hydrazide, a carbonyl reagent that appears to inhibit lysyl oxidase both in vivo and in vitro, a further 70% of the radioactivity is lost, with a roughly corresponding loss of enzyme activity. It is suggested that a form of vitamin B-6 is required as a cofactor of lysyl oxidase, and that this may have important implications in terms of connective-tissue metabolism.
A model for the allosteric regulation of pH-sensitive enzymes
Authors: Shindler J S , Tipton K F
Published: August 2015

1. In an enzyme that has two independent binding sites for a ligand, any inhibitor that binds solely to the free enzyme will give rise to positive co-operativity. 2. A model is considered for the allosteric control of enzymes by effectors in which their effects are mediated by ligand-induced perturbations of the ionization constants of a group or groups involved in the binding of substrate to the active site. 3. The model described offers a plausible explanation for the observation that the sigmoidal initial-rate curves reported for some regulatory enzymes are not expressed at all pH values where the enzyme is catalytically active.
Purification, biochemical characterization and serological analysis of cellular deoxyribonucleic acid polymerases and a reverse transcriptase from spleen of a patient with myelofibrotic syndrome
Authors: Chandra P , Steel L K
Published: August 2015

The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and template-primers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation-velocity measurements of the purified enzymes gave values of 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.
Conjugated and unconjugated bilirubins in bile of humans and rhesus monkeys. Structure of adult human and rhesus-monkey bilirubins compared with dog bilirubins
Authors: Blumenthal S G , Taggart D B , Ikeda R , Ruebner B H , Bergstrom D E
Published: August 2015

1. Bilirubin-IXalpha, -IXalpha diglucuronide, -IXalpha monoglucuronide, -IXalpha monoglucoside -IXalpha monoxyloside, a bilirubin-IXalpha diconjugate containing glucose and another unknown compound, and bilirubin-IXbeta are present in gall-bladder bile of adult human, rhesus monkey and dog. Dog bile normally also contains other bilirubin-IXalpha diconjugates, i.e. compounds containing two conjugating sugars such as glucuronic acid and glucose, glucuronic acid and xylose and glucose xylose. 2. Azopigments alphaF, alphaO, alpha2, alpha3, betax and delta derived from human and rhesus-monkey bilirubins are identical in their chemical composition with those obtained from the dog. 3. Azopigments alphaF and betax found in diazotized biles of adult humans, rhesus monkeys and dogs are products of unconjugated bilirubin-IXbeta. 4. Technical modifications of previously published procedures [Heirwegh, Fevery, Michiels, Van Hees & Compernolle, (1975) Biochem. J. 145, 185-199] were introduced which make it possible to separate the bilirubins, diazotize the separated bilirubins, extract the azopigments and chromatograph them in one working day (6-8h).
The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis
Authors: Roughley P J , Barrett A J
Published: August 2015

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.
The interaction of protein A and Fc fragment of rabbit immunoglobulin G as probed by complement-fixation and nuclear-magnetic-resonance studies
Authors: Wright C , Willan K J , Sjödahl J , Burton D R , Dwek R A
Published: August 2015

Protein-A-Fc-fragment complexes were observed in sedimentation-velocity experiments by ultracentrifugation. The interaction was studied by protein-fluorescence-quenching titrations of the Fc fragment with protein A, allowing the dissociation constant to be determined under a variety of conditions. The first component of the complement pathway, C1, is activated by complexes of protein A with rabbit IgG (immunoglobulin G), and the structural basis for this interaction was studied by using n.m.r. (nuclear magnetic resonance). The four Fc-fragment binding sites on protein A were shown to contain aromatic amino acids, and to be connected by mobile hydrophilic regions. Neither n.m.r. nor proton-relaxation-enhancement studies show evidence of a large conformational change of the Fc fragment on binding protein A, and this suggests that the cross-linking of the Fc fragments may be primarily responsible for the activation of component C1. This is supported by the inability of a univalent tryptic fragment of protein A to activate complement fixation by rabbit IgG.
Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes
Authors: Savage B , Spencer N
Published: August 2015

1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5′-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.
Preparation and characterization of substrates suitable for the study of stereospecific secondary alkylsulphohydrolases of detergent-degrading micro-organisms
Authors: Matcham G W J , Dodgson K S
Published: August 2015

During the course of the purification of novel stereospecific secondary aklylsulphohydrolases present in certain detergent-degrading micro-organisms, it became apparent that substrates prepared by sulphating secondary alcohols with H2SO4 are heterogeneous. Apart from the racemization that occurs if resolved alcohols are sulphated, evidence is provided to show that other isomers are produced in which the position of the ester sulphate group on the alkyl chain has been altered. These changes can be avoided if pyridine/SO3 reagent (prepared with SO3) is substituted as sulphating agent. Experiments in which secondary alkyl sulphates prepared by both methods were tested as potential substrates for the two secondary alkylsulphohydrolase enzymes of Comamonas terrigena have provided initial information about the specificity of the enzymes.
The use of wheat-germ lectin-Sepharose for the purification of human haemopexin
Authors: Vretblad P , Hjorth R
Published: August 2015

Haemopexin was prepared in 37% yield from normal human serum by a simple procedure involving fractional poly(ethylene glycol) precipitation and subsequent chromatography on DEAE-Sepharose CL-6B. One peak from the ion exchanger consisted of only haemopexin and transferrin. These proteins were separated by chromatography on wheat-germ lectin-Sepharose 6MB. Haemopexin was selectively bound and was subsequently desorbed by N-acetyl-D-glucosamine. No impurities could be detected in the final preparation by immunoelectrophoresis or by immunodiffusion against a range of antisera. The protein gave two partially separated bands in polyacrylamide-gradient-gel electrophoresis, corresponding to apohaemopexin and haem-haemopexin complex.
The active site of β-glucosidase from Botryodiplodia theobromae. Effects of pH and dioxan on enzyme-catalysed reactions
Authors: Umezurike G M
Published: August 2015

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat in the absence or presence of added dioxan was found to be dependent on the ionization of two groups, which appeared to be a carboxyl group and an imidazole group. 2. Dioxan increased the Michaelis constant, Km, but decreased the maximum velocity, V.
The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values
Authors: Brocklehurst K , Dixon H B F
Published: August 2015

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.
Fragmentation of a mollusc haemocyanin with plasmin and immunological identification of the fragments
Authors: Gullick W J , Head E J , Wood E J
Published: August 2015

Haemocyanin from the gastropod mollusc Lymnaea stagnalis (pond snail) was partially digested with plasmin under a variety of experimental conditions and the products of digestion analysed by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Fragments were obtained corresponding to one, two, three, four and five oxygen-binding domains, one domain having a mol.wt. of approx. 50000 and containing 2 ions of Cu. The fragments obtained after extensive digestion were non-identical immunologically, and summation of their molecular weights allowed a minimal mol.wt. of 413000 to be calculated for the original, undigested, eight-domain polypeptide chain. The use of mild-digestion conditions allowed the time course and sequence of the digestion to be monitored. An initial cleavage gave a three-domain and a five-domain fragment. The three-domain fragment was resistant to further digestion. The five-domain fragment could be digested further to give, successively a four-domain, a three-domain, and finally a two-domain fragment, single-domain units being cleaved. These data form the basis for a proposed sequence for the different domains in the original chain.
Molecular and immunological comparison of human dihydropteridine reductase in liver, cultured fibroblasts and continuous lymphoid cells
Authors: Firgaira F A , Choo K H , Cotton R G , Danks D M
Published: August 2015

An antiserum was raised in a rabbit against highly purified human liver dihydropteridine reductase (EC 1.6.99.7). Dihydropteridine reductase from human liver, in human cultured fibroblasts and in continuous lymphoid cells all showed identical antigenic properties. The structural characteristics of the reductase from these three sources were further compared by the use of high-precision two-dimensional polyacrylamide-gel electrophoresis. The enzyme from radiolabelled fibroblasts and continuous lymphoid cells was isolated by immunoprecipitation or by affinity chromatography and compared with the purified liver enzyme. Two major polypeptide species were resolved, and polypeptides from all three sources co-migrated identically. Indirect evidence is presented indicating that one of the polypeptide species may have been derived from the other via a post-translational modification. These results support the concept that the same structural gene(s) encodes for dihydropteridine reductase in human liver, fibroblasts and lymphocytes.
Calcium-ion-binding activity in human small-intestinal mucosal cytosol Purification of two proteins and interrelationship of calcium-binding fractions
Authors: Davie M
Published: August 2015

Ca2+-binding activity was investigated in human small-intestinal mucosal cytosol. Binding was detected in fractions with molecular weights of 28000 and about 900000, as determined by gel filtration. No binding was found at molecular weight 12000-13000 (the molecular weight of calcium-binding protein in lower mammalian species) until the cytosol had been subjected to a hollow-fibre-filtration step. The appearance of Ca2+-binding at molecular weight 12000-13000 was associated with a decline in the 28000-mol.wt. calcium-binding fraction. The 12000-13000-mol.wt. fraction contained two distinct calcium-binding proteins. One of these proteins had properties similar to those of pig calcium-binding protein. Antiserum to this protein reacted against the 28000-mol.wt calcium-binding fraction in cytosol from human small-intestinal mucosa and from human kidney. An immunoassay method for one of the calcium-binding proteins was established. In normal duodenal mucosa the concentration was 915 micrograms/g and in the ileum it was 443 micrograms/g of mucosa. A subject with hypercalcaemic sarcoidosis had 1200 micrograms/g of mucosa in the jejunum, and a subject with an undetectable concentration of plasma 25-hydroxycholecalciferol had concentrations of calcium-binding protein in the mucosa similar to those found in normal subjects.
The interaction of yeast hexokinase with Procion Green H-4G
Authors: Clonis Y D , Goldfinch M J , Lowe C R
Published: August 2015

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G. Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. 2. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose, but not by D-galactose. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.
Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region
Authors: Scott J E , Orford C R
Published: August 2015

Rat tail tendon was stained with a cationic phthalocyanin dye, Cupromeronic Blue, in a ‘critical-electrolyte-concentration’ method [Scott (1980) Biochem. J. 187, 887-891] specifically to demonstrate proteoglycan by electron microscopy. Hyaluronidase digestion in the presence of proteinase inhibitors corroborated the results. Collagen was stained with uranyl acetate and/or phosphotungstic acid to demonstrate the banding pattern a-e in the D period. Proteoglycan was distributed about the collagen fibrils in an orthogonal array, the transverse elements of which were located almost exclusively at the d band, in the gap zone. The proteoglycan may inhibit (1) fibril radial growth by accretion of collagen molecules or fibril fusion, through interference with cross-linking, and (2) calcification by occupying the holes in the gap region later to be filled with hydroxyapatite.
Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from α-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity
Authors: Magee A I , Grant D A , Hermon-Taylor J , Offord R E
Published: August 2015

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.
Polymorphism of sarcoplasmic-reticulum adenosine triphosphatase of rabbit skeletal muscle
Authors: Damiani E , Betto R , Salvatori S , Volpe P , Salviati G , Margreth A
Published: August 2015

Antibody was raised in chickens against purified sarcoplasmic-reticulum Ca2+-activated ATPase (Ca2+-ATPase). The immunological relationship between the Ca2+-ATPase of fast-muscle and slow-muscle sarcoplasmic reticulum was investigated by a one-step and a two-step competitive enzyme-linked immunosorbent assay (ELISA). The results show marked antigenic differences between the membrane-bound Ca2+-ATPase of the sarcoplasmic-reticulum vesicles from fast muscle and slow muscle, beside differences in the membrane content of ATPase protein.
Specificity of mammalian spermidine synthase and spermine synthase
Authors: Pegg A E , Shuttleworth K , Hibasami H
Published: August 2015

1. The specificity of rat prostatic spermidine synthase and spermine synthase with respect to the amine acceptor of the propylamine group was studied. 2. Spermidine synthase could use cadaverine (1,5-diaminopentane) instead of putrescine, but the Km for cadaverine was much greater and the rate with 1mM-cadaverine was only 10% of that with putrescine. 1,3-Diaminopropane was even less active (2% of the rate with putrescine) and no other compound tested (including longer alpha, omega-diamines, spermidine and its homologues and monoacetyl derivatives) was active. 3. Spermine synthase was equally specific. The only compounds tested that showed any activity were 1,8-diamino-octane, sym-homospermidine, sym-norspermidine and N-(3-aminopropyl)-cadaverine, which at 1mM gave rates 2, 17, 3 and 4% of the rate with spermidine respectively. 4. The formation of polyamine derivatives of cadaverine and to a very small extent of 1,3-diaminopropane was confirmed by exposing transformed mouse fibroblasts to these diamines when synthesis of putrescine was prevented by alpha-difluoromethylornithine. Under these conditions the cells accumulated significant amounts of N-(3-aminopropyl)cadaverine and NN'-bis(3-aminopropyl)cadaverine when exposed to cadaverine and small amounts of sym-norspermidine and sym-norspermine when exposed to 1,3-diaminopropane.
Purification and structural characterization of a cartilage matrix protein
Authors: Paulsson M , Heinegård D
Published: August 2015

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.
Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain
Authors: Rivett A J , Smith I L , Tipton K F
Published: August 2015

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.
A study of the structures of the YaYa and YaYc glutathione S-transferases from rat liver cytosol Evidence that the Ya monomer is responsible for lithocholate-binding activity
Authors: Hayes J D , Strange R C , Percy-Robb I W
Published: August 2015

The two dimeric lithocholic acid-binding proteins previously identified as ligandin (YaYa) and glutathione S-transferase B (YaYc) were isolated from rat liver cytosol. These proteins have molecular weights of 44000 and 47000 respectively. The recovery of these two proteins from liver was not affected by the addition of the proteinase inhibitor Trasylol. No spontaneous interconversion between these two proteins was observed on storage. YaYa and YaYc proteins yielded peptides of identical molecular weight after limited digestion with Staphylococcus aureus V8 proteinase. Analytical and preparative tryptic-digest peptide ‘maps’ showed that all the soluble peptides obtained from YaYa protein were also recovered from YaYc protein. Approximately six extra soluble peptides, which were not recovered from YaYa protein, were obtained from the tryptic digest of YaYc protein. Subdigests of the insoluble tryptic-digest ‘cores’ also resulted in the recovery of identical peptides from both proteins. Evidence is presented that the Ya subunit possessed by both proteins is identical; glutathione S transferase B is a hybrid of ligandin and glutathione S-transferase AA. The Ya monomer is responsible for lithocholate binding.
Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the α-chain of human haemoglobin
Authors: Kazim A L , Atassi M Z
Published: August 2015

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.
Subunit interactions in tyrosinase from frog epidermis in immobilized enzyme systems
Authors: Iborra J L , Ferragut J A , Lozano J A
Published: August 2015

1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.
Differences in the interaction of the catalytic groups of the active centres of actinidin and papain Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes
Authors: Brocklehurst K , Baines B S , Malthouse J P
Published: August 2015

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.
A variant of human transferrin with abnormal properties
Authors: Evans R W , Williams J , Moreton K
Published: August 2015

Screening of human serum samples by polyacrylamide-gel electrophoresis in the presence of 6 M-urea revealed an individual who is heterozygous for a variant transferrin. The variant transferrin is able to bind two atoms of iron, but the iron in the C-terminal binding site is bound abnormally, as judged by its spectral properties, and is dissociated from the protein on electrophoresis in the presence of 6 M-urea. The iron-free C-terminal domain of the variant protein is less stable than normal to thermal and urea denaturation. Structural changes in the variant protein have not yet been characterized.