scholarly journals Properties of pig synovial collagenase

1980 ◽  
Vol 189 (2) ◽  
pp. 349-357 ◽  
Author(s):  
J A Tyler ◽  
T E Cawston
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Type Ii ◽  
Polyacrylamide Gels ◽  
Optimum Ph ◽  
Detectable Difference

1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.

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 and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

1978 ◽  
Vol 173 (3) ◽  
pp. 759-765 ◽  
Author(s):  
J A Sharp ◽  
M R Edwards
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Transferase Activity ◽  
Coa Transferase

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide ‘mapping’ and N-terminal analysis.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

scholarly journals Purification and characterization of subcomponent C1q of the first component of mouse complement

1981 ◽  
Vol 193 (2) ◽  
pp. 621-629 ◽  
Author(s):  
K Yonemasu ◽  
T Sasaki
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weight Range ◽  
Covalently Linked

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 × 10(13)-4 × 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.

Purification of Human Renal Renin

1978 ◽  
Vol 55 (s4) ◽  
pp. 117s-119s
Author(s):  
Eve E. Slater ◽  
Robert C. Cohn ◽  
Victor J. Dzau ◽  
Edgar Haber
Keyword(s):  
Enzymatic Activity ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Single Band ◽  
Kidney Cortex ◽  
Human Renin ◽  
Cadaver Kidney

1. Human renal renin has been purified 200 000-fold from cadaver kidney cortex by a method which employs affinity chromatography on aminohexyl pepstatin. 2. The product of this purification has a specific activity of 400 Goldblatt units/mg when compared with Haas human renin standard. 3. This product appears as a single band on sodium dodecyl sulphate gel and polyacrylamide-disc gel electrophoresis. Renin enzymatic activity was recovered after elution from a polyacrylamide-disc gel run at pH 7·8. 4. Yield with this method was 1%.

scholarly journals Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

1983 ◽  
Vol 213 (1) ◽  
pp. 187-191 ◽  
Author(s):  
A Lewendon ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Step ◽  
Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

scholarly journals The purification of shikimate dehydrogenase from Escherichia coli

1985 ◽  
Vol 226 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Chaudhuri ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Shikimate Dehydrogenase ◽  
E Coli ◽  
Gel Permeation

A procedure was developed for the purification of shikimate dehydrogenase from Escherichia coli. Homogeneous enzyme with specific activity 1100 units/mg of protein was obtained in 21% overall yield. The subunit Mr estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 32 000. The native Mr, estimated by gel-permeation chromatography on a TSK G2000SW column, was also 32 000. E. coli shikimate dehydrogenase is therefore a monomeric NADP-linked dehydrogenase.

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