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We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 °C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein. [ABSTRACT FROM AUTHOR]

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl- system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère-Blandin, C. (1999) Biochem. J. 343, 603–613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin. [ABSTRACT FROM AUTHOR]

Acylation with hydroxycinnamic acids stabilizes anthocyanins and makes their colour bluer (bathochromic shift). We purified to homogeneity one acytation enzyme, hydroxycinnamoyl-CoA:anthocyanidin 3,5-diglucoside 5-O-glucoside-6m-O-hydroxycinnamoyltransferase, from blue petals of Gentiana triflora. It is a single polypeptide protein of 52 kDa with a pI of 4.6. It catalyzes the transfer of the p-coumaric acid and caffeic acid from their CoA esters to the 5-glucosyl moiety of anthocyanidin 3,5-diglucosides but could not use malonyl-CoA as an acyl donor. Neither anthocyanidin 3-monoglucoside nor anthocyanins aromatically acylated at the 3-glucosyl moiety could be acylated by this enzyme. Aromatic acylation of anthocyanidin 3,5-diglucoside by this enzyme caused a bathochromic shift and increased pigment stability in noutral to weakly basic pH. Other unthocyanins froth the petals of G. triflora were isolated and their structures were determined by last-atom-bombardment MS and NMR. The biosynthetic pathway of gentiodelphin, a diacylated anthocyanin accumulating in G. triflora petals, is proposed on the basis of these results. [ABSTRACT FROM AUTHOR]

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulthydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine. [ABSTRACT FROM AUTHOR]

Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethytenglycol) precipitation and column chromatograpliy. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements: for optimum activity it requires 0.5-1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component: it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent Km and Vmax, for an intronless plant pre-tRNATyr are 10.3 nM and 1.12 fmol/min, respectively. [ABSTRACT FROM AUTHOR]

A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels alter reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/PK ratios of 0.6 and 1.0, respectively, These proteins had large potent microbicidal activities (0.018–018 μM) against different strains of gram negative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a ]9-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish. [ABSTRACT FROM AUTHOR]

Ribosomes purified from Escherichia coli were preincubated with AcPhe-tRNA and poly(U). Then purified components necessary for polypeptide synthesis were added. Incubation of the complete system led to a burst of elongation which lasted for nearly 10 s. During the initial burst approximately 10% of the ribosomes participated in the elongation of poly(Phe) at an average rate per ribosome close to eight peptide bonds/s. The missense error rate with leucine was 4 × 10-4 during the burst. Accordingly, the preincubated elongation system functions at a rate, as well as an accuracy, close to those of protein synthesis in vivo. [ABSTRACT FROM AUTHOR]

Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 6.5, DEAE-Sepharose at pH 8.9, Sephadex G-150, hydroxyapatite and hexyl-agarose. Both forms were finally separated by affinity chromatography on concanavalin-A—Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino-terminal amino acid sequence which was found to be the same (H2N-Asp-Arg-Val) as for angiotensin I and II. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be Km = 5.0 μM proangiotensin and V = 270 nmol of angiotensin I h-1 unit renin-1 for the concanavalin-A-binding form and Km = 5.6 μM proangiotensin and V = 250 nmol angiotensin I h-1 unit renin-1 for the prohormone which did not bind to concanavalin-A—Sepharose. The form of proangiotensin not bound to concanavalin-A—Sepharose was found to be more thermally labile (tm of 59.0 °C) than the form binding to concanavaiin A (tm of 61.5 °C, where tm = temperature at which 50% reactivity is lost). [ABSTRACT FROM AUTHOR]

The seeds of Vicia cracca are known to contain two lectins: an N-acetylgalactosamine-binding lectin, which reacts specifically with human erythrocytes of blood group A, and a mannose/glucose-binding lectin unspecific towards human blood groups. The second lectin is the object of the present work. Purification was achieved by affinity chromatography on Sephadex G-100. The native lectin has a molecular weight of 44000. It is composed of two small (Mr 5700) and two large subunits (Mr 17500). Binding of sugars to the lectin was assayed by equilibrium dialysis and spectrophotometrically; the association constants for glucose are 1.38 × 10³ l/mol and 2.5 × 10² 1/mol respectively. The primary structure of the small subunit was determined by analyses of the whole chain, of tryptic and CNBr peptides. It was shown to consist of 53 amino acid residues. The mannose/glucose-binding lectin from Vicia cracca is highly homologous to the lectins from Vicia sativa, Vicia faba, Pisum sativum and Lens culinaris. There is. however, only limited homology to the N-acetylgalactosamine-specific lectin from Vicia cracca. [ABSTRACT FROM AUTHOR]

Binding of added progesterone lo native uteroglobin requires the reduction of the disulfide bonds that hold together the two polypeptide chains of the protein. The hypothesis that in the native oxidized state of uteroglobin the steroid binding cavity is preformed and occupied by a progesterone molecule has been tested by several experimental means. The results demonstrate that progesterone does not interact with oxidized uteroglobin, and show that the majority of the oxidized uteroglobin molecules purified from pseudopregnant rabbits do not contain a progesterone molecule. Oxidation of reduced uteroglobin in the presence of saturating amounts of progesterone does not result in significant retention of the steroid inside the oxidized protein. [ABSTRACT FROM AUTHOR]

A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18500-19000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-α-toluenesulfonyl-L-arginine methyl ester, N-α-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-α-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl, and loses its activity in 5 min at 67 °C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl, and α1-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported. [ABSTRACT FROM AUTHOR]

Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1600 nmol min-1 mg protein -1 which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59000. The isoelectric point of the enzyme was estimated to be 6,7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein. Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 μM to 58 mu;M. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

Tryptophan 5-monooxygenase was purified approximately 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-1 mg-1 at 30 °C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAB-Sepharose CL-6B and designated as form I and form II based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 59000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylalanine hydroxylation but not an activity of tyrosine hydroxylation. [ABSTRACT FROM AUTHOR]

Reports on the purification of an alpha-amanitin-resistant DNA-dependent RNA polymerase II from the lower eukaryote Aspergillus nidulans to apparent homogeneity. Use of extraction of the enzyme at low salt concentration, polymin P fractionation, binding to ion-exchangers and density gradient centrifugation; Resolution of the subunit composition by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate.

A soluble, sodium-nitroprusside-stimulated guanylate cyclase has been purified from bovine lung by DEAEcellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72 000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145 000. The enzyme sedimented on sucrose gradients with an s20,w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150 000 and consists of two, presumably identical, subunits of Mr 72 000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 µmol cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 µM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 µmol min-1 mg-1. [ABSTRACT FROM AUTHOR]

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernatant. A comparison is made between these preparations with respect to yield and specific activity. EIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase has been purified 192-fold from Methylomonas M15, grown on methanol as sole source for carbon and energy. The purification procedure involved N-cetyl- N,N,N,-trimethylammonium bromide precipitation, ion-exchange chromatography and affinity chromatography on blue-dextran-Sepharose. The final enzyme preparations were homogeneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weight of the enzyme was calculated to be 108000 ± 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels indicates that the enzyme is a dimer composed of two probably identical subunits each having a molecular weight of 55000. The enzyme exhibits activity with either NAD or NADP as electron acceptor. Mg2+ or an other bivalent cation is not essential for enzyme activity. The Km values were found to be 0.2 mM for NAD, 0.014 mM for NADP, 0.29 mM for glucose 6-phosphate with NAD and 0.11 mM for glucose 6-phosphate with NADP as electron acceptor. The reduced coenzymes NADPH and NADH as well as ATP are powerful inhibitors of the enzyme. The intracellular levels of glucose 6-phosphate, ATP, NAD and NADP were measured and found to be approximately 2.5 mM, 1.5 mM, 1 mM and 2 mM respectively, during exponential growth. From 3 consideration of the substrate pool sizes and types of inhibitors, it is concluded th3.t this enzyme may function in two roles m the cell: NADH production for energetics and NADPH production for reductive biosynthesis. [ABSTRACT FROM AUTHOR]

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 1589]. Although no function has been assigned to δ protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of δ and σ proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified σ polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified δ protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of δ protein on poly(dA-dT) transcription was strongly dependent on the presence of KCI in the RNA synthesis reaction mixture. [ABSTRACT FROM AUTHOR]

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000, red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc elcctrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase. [ABSTRACT FROM AUTHOR]

A specific non-histone chromatin-associated protein, having a high content of both acidic and basic amino acids has been isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells. The protein has been prepared by the extraction of chromatin with 0.35 M sodium chloride and purified by chromatography on carboxymethyl-cellulose with a gradient of lithium chloride at pH 9.0. The amino acid sequence of the first 29 residues of the amino-terminal region has been determined using an automatic protein sequencer. The primary structure of this protein differs from that of any of the histones yet sequenced and therefore, cannol be a degradation product of any of them. Moreover, the N-terminal amino acid sequence shows considerable similarity to the HMG-1 and HMG-2 chromosomal proteins described by Goodwin et al. [Eur. J. Biochem. 38. t4-19 (1973)] whose N-terminal sequences were also determined in this laboratory. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly( ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine. and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture. [ABSTRACT FROM AUTHOR]

A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight-edge (transparent ruler) is presented. This method is then illustrated by an analysis of the wavy υ vs [S] curves and non-linear double-reciprocal plots reproducibly given by NAD-dependent 15-hydroxyprostaglandin dehydrogenase for which a new improved purification is described. It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead-end complexes. [ABSTRACT FROM AUTHOR]

Six neurotoxins were purified from Dendroaspis viridis venom using gel filtration and equilibrium chromatography. The amino acid sequences of two of these neurotoxins (72 and 73 residues, five disulphide bridges) have been determined using almost exclusively automated Edman degradation. These two sequences are very similar: the only difference lies in the presence of one extra glycine at the C-terminal end of one of them. There is a good homology with the sequences of toxins now isolated from other Elapidae and Hydrophidae venoms. [ABSTRACT FROM AUTHOR]

A factor, which makes a ternary complex with GTP and eukaryotic initiator tRNA (Met- tRNAi), has been purified 100-fold from developed cysts of Artemia salina. Some of the properties of the purified factor have been studied. Mg2+ appears to inhibit ternary complex formation. Little or no ternary complex is formed when 5 μM GTP is replaced by an identical concentra- tion of UTP, CTP or ATP. The analog, guanosine 5′-(β,γ-imino)triphosphate [GMP-P(NH)P] seems to be a much better substitute for GTP than guanosine 5′-(β,γ-methylene)triphosphate [GMP- P(CH2)P]. Since GMP-P(NH)P is as effective as GTP in ternary complex formation, it would appear that GTP plays the role of an allosteric effector in this step of eukaryotic polypeptide chain initiation. GDP inhibits both the rate and extent of ternary complex formation. The inhibition is largely reversed by adding a 5-fold molar excess of GTP over GDP. dGDP is slightly less inhibitory than GDP. UDP and CDP are much less inhibitory than GDP and very little inhibition is obtained with ADP. The preformed ternary complex is rapidly and completely destroyed in the presence of N-ethylmaleimide. The results suggest that free —SH groups of the factor may be essential for maintaining the integrity of the ternary complex. [ABSTRACT FROM AUTHOR]

An inducible membrane-bound L-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of L-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of 02 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 °C. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 °C which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandetic acid was calculated to be 1.8 × 10-4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents. [ABSTRACT FROM AUTHOR]

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 µmol to 82.7 µmol hydroxyproline mg protein-1 h-1 at 37 °C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61 000 and 64 000 as determined by sodium dodecylsulphate—polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera tohuman and chicl prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human and chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments. [ABSTRACT FROM AUTHOR]

1. A procedure has been described for the purification of the major isozyme of yeast phosphoglucomutase of highest known specific activity. 2. The native enzyme has a molecular weight of about 65 400 and was found to be homogeneous as judged by sucrose density gradient centrifugation, gel filtration, electrophoresis on acrylamide gel and ultracentrifugal analysis. In the presence of denaturing agents such as guanidine hydrochloride or sodium dodecyl sulfate, the enzyme dissociated into 32 000-molecular-weight subunits. 3. As isolated, the enzyme has one mole of phosphate bound per mole of enzyme. Preparations incubated with 1.0 mM EDTA in 10 mM citrate buffer, pH 5.5 and dialysed against 10 mM metal-free citrate buffer, pH 5.5, contain no intrinsically bound Zn2+ and were enzymatically inactive but fully active in the presence of 5 mM M2+ and 84% as active with 0.5 mM Zn2+. Simultaneous presence of both ions at these concentrations did not enhance activity. Enzyme was completely and irreversibly inactivated by preincubation with Be2+. Inactive enzyme had one mole of Be2+ bound per mole of enzyme. 4. Enzyme exhibited "ping-pong" kinetics rather than "random sequential". Km values for glucose 1-phosphate and for glucose 1,6-bisphosphate were calculated to be 2.34 × 10-5 M and 2.24 × 10-6 M, respectively. Rate of enzyme phosphate turnover was studied with rapid-mixing technique. The rates of 32P release from 32P-labeled enzyme and its appearance as glucose 6-[32P]phosphate were comparable and remained unaffected by addition of glucose 1,6-bisphosphate. [ABSTRACT FROM AUTHOR]

Inhibitors for yeast proteinase A have been purified from boiled extracts from baker's yeast. Upon SP-Sephadex C-25 column chromatography, four different peaks of proteinase A inhibitory activity appear. The inhibitors I2,A and I3,A have been further purified by QAE-Sephadex chromatography and preparative disc electrophoresis. Both inhibitors are homogeneous in dodecylsulfate-gel electrophoresis. From the electrophoretic mobilities in dodecylsulfate-polyacrylamide gels, a molecular weight 6100-,+200 was estimated for both inhibitors. In the absence of dodecylsulfate, both inhibitors showed one predominant band and a second faint, diffuse band. As a result of Sephadex G-75 gel filtration, a molecular weight of 23000-,+2000 was obtained for both inhibitors, indicating an aggregation of the monomers to a tetramer. Upon electrofocusing in acrylamide gels the isoelectric points of the inhibitors were found to be 5.7 for I2,A and 6.3 for I3,A. Both inhibitors inhibited proteinase A. neither proteinases B and C were inhibited from yeast or pepsin. Both inhibitors were inactivated after incubation with purified yeast proteinase B, but not with the yeast proteinases A or C. the inhibition of proteinase A at high inhibitor/proteinase ratios was more pronounced at pH 5 and 7 as compared to pH 3. An autocatalytic activation of mixture of inhibited proteinases A, B and C by addition or liberation of traces of active proteinase A or B is discussed. [ABSTRACT FROM AUTHOR]

Highly purified human chorionic gonadotrophin , assaying 15000 - 17000 IU/mg in three different bioassay systems, has been obtained from a crude commercial preparation. The purification method is a simple and rapid two-step procedure, based on preparative isoelectric focusing on sucrose density gradient (Ampholine pH range 3 - 10), followed by chromatography on Sephadex G-100. The purified gonadotrophin (mol. Wt 65000), obtained from an isoelectric pH of 4.3, moved as a single band in polyacrylamide gel electrophoresis at a pH 8.3 and showed a single precipitation line in immunoelectrophoresis. The immunological and biological activities of protein components obtained from various isoelectric zones during fractionation are reported. [ABSTRACT FROM AUTHOR]

The present investigation was designed to determine whether the rapid intracellular redistribution of lysosomes to a perinuclear position in reproductive target cells after administration of specific gonadal hormones in vivo was accompanied by evidence of penetration of the nucleoplasm by lysosomal marker enzymes. Nuclei were prepared by conventional isolation and purification procedures from preputial glands or uteri that were excised from gonadectomized rats within 2-15 min after intravenous injection of physiological doses of estradiol-17β, testosterone or saline control solutions. Control and experimental preparations were essentially "pure", as judged by routine phasecontrast microscopic criteria in general use. However, nuclei from target cells of animals given hormone contained substantial activities of representative lysosomal hydrolases, including acid phosphatase, β-glucuronidase and acid ribonuclease II. In contrast, the samples originating from the target tissue of saline-treated controls or from non-target tissues contained minimal concentrations of these enzyme activities. After further purification of the nuclei by removal of the outer nuclear membrane through brief exposure to very low concentrations of Triton X-100 in the cold, the resultant 'ultrapurified' nuclei retained significant concentrations of structurally latent lysosomal marker enzymes after hormonal pretreatment without evidence of appreciable contamination by enzymes of mitochondrial origin. Similar results were obtained with nuclei isolated by non-aqueous procedures. These combined observations appear to exclude adsorption artifacts. The total activities of lysosomal enzymes in the unfractionated homogenates and the Mg2+-dependent ribonuclease activity indigenous to the nuclear compartment were unaffected by prior hormone treatment. These results are consistent with the hypothesis that invasion of the nucleoplasm of specific target cells by lysosomal products may serve as a mechanism for triggering genic derepression in steroid hormone-induced growth. [ABSTRACT FROM AUTHOR]

The purification of ribose-5-phosphate isomerase from Candida utilis is described. The procedure used extends over six steps to a 27% yield of a homogeneous protein. The following properties have been elucidated: the enzyme has a molecular weight of 105000 and will dissociate into four subunits upon the addition of sodium dodecylsulfate. The specific activity is about 350 international units per mg protein. The pH optimum, Km for ribose-5-phosphate, amino acid composition and isoelectric point have been determined. The isomerase is extremely sensitive to organic mercurials. [ABSTRACT FROM AUTHOR]

Reports on the purification of murine liver homogentisate 1,2-dioxygenase (HGO). Determination of the molecular mass by gel filtration; Value of optimum pH for in vitro activity; Inhibition of HGO by incubation of the enzyme with iron.

Discusses the purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae. Catalysis of the freely reversible interchange of an acetyl group; Determination of values for substrates of the forward and reverse directions; Localization of the enzyme in the mitochondrial matrix.

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C,, Maxe, I., Franzén, A., Ljungh, Å., Heinegård, D. & Rubin, K. (1987) Lancet 11, 515]. We now report on the purification of a cellwall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell-wall components with capacity to inhibit binding of 125I-labeled BSP to staphylococcal ceils were solubilized with LiCl (1.0 M, pH 5.0). Preparative SDS/PAGE and protein-overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with Mr 75000-110000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono-Q anion-exchange column. Inhibitory activity was eluted at 0.6-0.8 M NaCl and could be further purified by affinity chromatography on BSP-Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity. Analysis by SDS/PAGE revealed a single Mr 97000 polypeptide in the eluate. The purified Mr 97000 protein bound BSP in protein-overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the Mr 97000 protein. Our data demonstrate the presence of a S. aureus cell-surface BSP-binding protein. This protein could be involved in bacterial tropism in osteomyelitis. [ABSTRACT FROM AUTHOR]

The elution of pristanoyl-CoA oxidase from butyl-Sepharose required unusually high concentrations of ethylene glycol, enabling the large-scale purification of this oxidase in a Single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P. P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G, P. (1991) J. Biol. Chem. 266, 24676-24683), migrated as a 513-kDa protein during native gel electrophoresist. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/ mol enzyme. Its amino acid composition was different from those of other known acyl-CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl-CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2-methylpalmitoyl-CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clew signal only in rat liver, possibly indicating that pristanoyl-CoA oxidase might be a rat-specific oxidase. [ABSTRACT FROM AUTHOR]

A protein with NAD-dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion-exchange chromatography (diethylaminoethyl-cellulose), affinity chromatography (5′-AMP-Sepharose) and gel-filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50–55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel-filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min-1 mg-1 using 250 Tris/HC1, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore, it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2, prostaglandin E2, prostaglandin F2α or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2. [ABSTRACT FROM AUTHOR]

The aerobic denitrifier Thiosphaera pantotropha is able to reduce simultaneously nitrous oxide and oxygen even after anaerobic growth [Bell, L. C. & Ferguson, S. J. (1991) Biochem J. 273, 423–427]. A nitrous oxide reductase was purified from anaerobically grown T. pantotropha cells. It is argued, on the basis of inhibitor sensitivities and from immunological evidence, that the same nitrous oxide reductase is involved in nitrous oxide reduction in aerobically grown cells. The purified nitrous oxide reductase was shown to have molecular properties very similar to nitrous oxide reductases previously isolated from anaerobically denitrifying bacteria. The visible absorption spectra of the T. pantotropha enzyme resemble those of the oxygen-affected form of nitrous oxide reductases from other organisms. It is thus concluded that the T. pantotropha nitrous oxide reductase is not peculiarly resistant to the structural changes caused by oxygen. The activity of the purified T. pantotropha nitrous oxide reductase was reconstituted in vitro using horse heart cytochrome c, T. pantotropha cytochrome c551 and T. pantotropha pseudoazurin as electron donors. It is suggested on this basis that either of the T. pantotropha electron-carrier proteins are possible physiological electron donors to T. pantotropha nitrous oxide reductase. Oxygen was shown not to inhibit the in-vitro reduction of nitrous oxide with horse heart ferrocytochrome c as electron donor to the reductase. [ABSTRACT FROM AUTHOR]

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 ± 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 ± 5 kDa. The enzyme had a pI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 H and pH 8,3 but the type of buffer used cted the results, The enzyme could utilize N4+ as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg2+, Km values for L-glutamine was determined to be 0.37 ± 0.05 mM, for chorismate 67 ± 3 μM, and for MgCl2 0.26 ± 0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20 μM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type. [ABSTRACT FROM AUTHOR]

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of catepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes. [ABSTRACT FROM AUTHOR]

Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 µmol · min-1 · mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN. [ABSTRACT FROM AUTHOR]

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits α, β, γ, δ and ε with molecular masses of 58000, 50000, 36000, 28000 and 13000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20–30 U/mg) was only observed in the presence of high concentrations of Ca2+ (to mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca2+-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed. [ABSTRACT FROM AUTHOR]

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (α subunit 26 kDa, β subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the β subunit. However, the amino acid sequence of the α subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes. [ABSTRACT FROM AUTHOR]

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/ mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 µM and a kcat/Km of 1.8 × 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyI-CoA above 10 µM. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase. [ABSTRACT FROM AUTHOR]

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in Iow quantity and consisted of, probably eight, apparently identical chains of Mr = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mrα = 63 000-65 000 and Mrβ = 61 000-62 000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: α, -(Val)-(Ser)-(Ala)-Leu; β, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by αchymotrypsin. The purification (2600-fold from the particulate fraction or 125000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 µmol cyclic AMP formed mg protein-1 min -1 at 30°C in the presence of manganese as cosubstrate. The colubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite_ half-lives of 27 min min, 50 min, and 160 min were obtained at 30°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 at 4°C. The purified enzyme enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicated that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 35 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem 257, 10818-10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported. [ABSTRACT FROM AUTHOR]

Studies the influence of metyrapone, chalcone epoxide, clotrimazole and benzil in the activity of microsomal epoxide hydrolase towards styrene oxide. Types of liver microsomes used; Epoxide hydrolase purification; Ability of the four effectors to activate the hydrolysis of styrene oxide by hydrolase in situ; Increase of epoxide hydrolase activity in liver microsomes; Association of the effect of modulators on hydrolase activity on enzyme protein interaction.

Reports on the purification and characterization of brain pyridoxal kinase from sheep brain. Use of ammonium sulphate fractionation, diethylaminoethanol-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration; Molecular mass of the native enzyme.

A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-l-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of high-mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1-deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 μM, 75 μM, and 140 μM, respectively. Man9 mannosidase cleaves three of the four αl,2-1inked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrotyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl α-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

A membrane-bound α-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on G DP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to α-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of βgalactoside α1 → 2-fucosyltransferase and N-acetylglucosaminide α1 → 4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above. [ABSTRACT FROM AUTHOR]