scholarly journals Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle

1988 ◽  
Vol 251 (1) ◽  
pp. 129-134 ◽  
Author(s):  
K Yamaguchi ◽  
M Hirata ◽  
H Kuriyama
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Cytosol Fraction ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Ph Range

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

scholarly journals Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

1989 ◽  
Vol 261 (2) ◽  
pp. 483-488 ◽  
Author(s):  
K Takazawa ◽  
H Passareiro ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Polyacrylamide Gel ◽  
Maximal Velocity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Maximal Activation

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

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

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver

1981 ◽  
Vol 193 (2) ◽  
pp. 503-512 ◽  
Author(s):  
E O Kajander ◽  
A M Raina
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Adenosine Kinase ◽  
Native Enzyme ◽  
Apparent Homogeneity ◽  
A Subunit ◽  
Adenosine Derivatives ◽  
Pore Gradient

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

scholarly journals Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus

1979 ◽  
Vol 177 (1) ◽  
pp. 49-62 ◽  
Author(s):  
C M Clarke ◽  
B S Hartley
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Dna Cleavage ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Activity Restriction

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).

Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

1974 ◽  
Vol 52 (7) ◽  
pp. 620-630
Author(s):  
André Lemay ◽  
Fernand Labrie
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Apparent Molecular Weight ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Protein Components ◽  
Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

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