Isolation and Properties of Elastase-Like Enzymes from Human Platelets and Pig Aorta

1975 ◽  
Author(s):  
W. Hornebeck ◽  
Y. Legrand ◽  
J. P. Caen ◽  
L. Robert
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
Proteolytic Activity ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Purification Procedure ◽  
Pancreatic Elastase

An elastase-like enzyme has been isolated from human platelets. Its purification using precipitations with ammonium sulphate, gel filtration and affinity chromatography on Agarose-elastin, is described. The acrylamide gel of the affinity peak reveals only one band corresponding to a molecular weight of about 25,000 daltons. The amino acid composition is similar to pancreatic elastase. Using the same kind of purification procedure an aortic elastase-like enzyme has also been isolated and characterized. These two enzymes possess comparable proteolytic activity on various synthetic and natural substrates considered as specific for elastases. The ratio of their activity on these substrates differs however from that of pancreatic elastase. The inhibitory effect of α1, antitrypsine and α2 macroglobuline were also studied and shown to differ quantitatively from those on pancreatic elastase. These elastase like enzymes may be responsible for the degradation of elastin occuring in ageing and arteriosclerosis.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Isolation of human β1A-globulin by modern techniques

1969 ◽  
Vol 115 (5) ◽  
pp. 897-902 ◽  
Author(s):  
P. A. Charlwood
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Ammonium Sulphate ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Reversible Aggregation ◽  
Satisfactory Quality

1. Human β1A-globulin was isolated from serum by precipitation with ammonium sulphate, gel filtration and electrophoresis in polyacrylamide gel. 2. The product was found by ultracentrifugation, analytical electrophoresis in polyacrylamide gel and two-dimensional immunoelectrophoresis to be of satisfactory quality for further study. 3. The amino acid composition of β1A-globulin was determined. 4. In ordinary dilute buffers near neutrality, β1A-globulin had S020,w 6·42s and M 131 000, but some reversible aggregation occurred at lower pH. In neutral 6m-guanidine hydrochloride the molecular weight was not measurably different from that in dilute buffer.

scholarly journals The purification in high yield and characterization of rat hepatic glucokinase

1976 ◽  
Vol 153 (2) ◽  
pp. 363-373 ◽  
Author(s):  
M J Holroyde ◽  
M B Allen ◽  
A C Storer ◽  
A S Warsy ◽  
J M E Chesher ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
Specific Activity ◽  
Sedimentation Equilibrium ◽  
High Yield ◽  
Equilibrium Studies ◽  
Enzyme Specific Activity

A new improved procedure for the purification of rat hepatic glucokinase (ATP-d-glucose 6-phosphotransferase, EC 2.7.1.2) is given. A key step is affinity chromatography on Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-d-glucopyranose. A homogeneous enzyme, specific activity 150 units/mg of protein, is obtained in about 40% yield. The molecular weight of the pure enzyme was determined by several procedures. In particular, sedimentation-equilibrium studies under a variety of conditions indicate a molecular weight of 48000 and no evidence for dimerization; reports in the literature of other values are discussed in the light of this evidence on the pure enzyme. The amino acid composition suggests that hepatic glucokinase is closely related to rat brain hexokinase and also the wheat “light” hexokinases.

Structural study on liver gelactin

1984 ◽  
Vol 62 (11) ◽  
pp. 1072-1075 ◽  
Author(s):  
Julian Gruda ◽  
Hélène-Marie Thérien
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Structural Study ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Gel Filtration ◽  
Rabbit Liver

Electron microscopy, ultracentrifugation, gel filtration, and isoelectric focusing were carried out with gelactin, an actin-gelling protein from rabbit liver. Gelactin is a dimeric acidic protein (isoelectric point (pI) = 5.45), with a molecular weight of 190 000, a Svedberg constant of 6.25, and a Stoke's radius and length of 7.0 and 28 nm, respectively. While different from α-actinin by pI and amino acid composition, gelactin belongs by its dimensions to the class of α-actinins.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

Sign in / Sign up

Export Citation Format

Share Document