A neutral collagenase from human gastric mucosa

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins ²2-macroglobulin and a smaller component of mol.wt. approx. 40000; α1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.

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High Molecular Weight Forms of Antithrombin III Complexes in Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657310 ◽

1983 ◽

Vol 49 (01) ◽

pp. 032-036 ◽

Cited By ~ 6

Author(s):

E Marciniak ◽

G Gora-Maslak

Keyword(s):

Antithrombin Iii ◽

Serum Proteins ◽

Gel Filtration ◽

Factor Xa ◽

Molecular Size ◽

Molecular Forms ◽

Factor X ◽

Monomeric Complex ◽

At Iii ◽

Antibody Competition

SummaryA double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 ± 21 μg/ml. Complexes of AT III with thrombin or factor X a crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT III complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

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Fibrin and Fibrinogen Proteolysis Products: Comparison between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651859 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0524-0535 ◽

Cited By ~ 14

Author(s):

Norma Alkjaersig ◽

Andrew Davies ◽

Anthony Fletcher

Keyword(s):

Molecular Weight ◽

Analytical Methods ◽

Gel Filtration ◽

Molecular Size ◽

The Other ◽

Reaction Products ◽

Human Fibrinogen ◽

Polyacrylamide Electrophoresis ◽

Large Molecular Weight ◽

Stabilized Fibrin

SummaryThe proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the “double D” cross-linked form.

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Isolation and characterization of cathepsin D from human gastric mucosa

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19813302 ◽

1981 ◽

Vol 46 (13) ◽

pp. 3302-3313 ◽

Cited By ~ 14

Author(s):

Jan Pohl ◽

Ladislav Bureš ◽

Karel Slavík

Keyword(s):

Gastric Mucosa ◽

Cathepsin D ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Human Gastric Mucosa ◽

Ph Optimum ◽

Isolation And Characterization

The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.

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Studies on the Identity of a Vascular Permeability Factor of Renal Origin

Clinical Science ◽

10.1042/cs0380309 ◽

1970 ◽

Vol 38 (3) ◽

pp. 309-325 ◽

Cited By ~ 31

Author(s):

M. F. Cuthbert ◽

W. S. Peart

Keyword(s):

Blood Pressure ◽

Vascular Permeability ◽

Active Material ◽

Gel Filtration ◽

Molecular Size ◽

Vascular Permeability Factor ◽

Permeability Factor ◽

Pressor Activity ◽

Renal Cortical Tissue ◽

Ph Range

1. The administration of crude renal extract to bilaterally nephrectomized rats causes an increase in vascular permeability to plasma proteins. This is associated with a fall in plasma volume. The active material resembles the enzyme renin in pressor activity, heat-lability, pH range of activity and molecular size. 2. To test the possibility that renin might be responsible, renin was extracted from rat renal cortical tissue using methods similar to those used for the purification of pig renin: these involved saline extraction, protein precipitation, freeze drying, ion-exchange and gel-filtration. 3. The final preparation had a pressor activity some 300 times that of the initial saline extract and gel-filtration suggested that the molecular size of rat renin is 40 000–50 000. Assay for pressor and vascular permeability activity at selected stages in the purification showed that both activities ran parallel and could not be dissociated. These results provide strong evidence that the vascular permeability factor is in fact renin. 4. Increase in vascular permeability after injection of semi-purified material could still be demonstrated after bilateral adrenalectomy, though the effect was reduced. 5. In all experiments in which vascular permeability was increased and in which blood pressure was measured, a considerable and sustained rise in blood pressure occurred. It is possible that the increase in blood pressure is causally related to the increase in permeability.

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Purification and partial characterization of four proteins from human parotid saliva

Biochemical Journal ◽

10.1042/bj1230455 ◽

1971 ◽

Vol 123 (3) ◽

pp. 455-464 ◽

Cited By ~ 107

Author(s):

Anders Bennick ◽

George E. Connell

Keyword(s):

Serum Proteins ◽

Protein A ◽

Gel Filtration ◽

Binding Activity ◽

Significant Activity ◽

Parotid Saliva ◽

Polyacrylamide Electrophoresis ◽

Ph Range ◽

Human Parotid Saliva

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3–10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, β-glucuronidase, β-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.

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Fibrin and Fibrinogen Proteolysis Products: Comparison Between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis

10.1055/s-0039-1682462 ◽

1977 ◽

Author(s):

N. Alkjaersig ◽

A. Davies ◽

A. Fletcher

Keyword(s):

Molecular Weight ◽

Analytical Methods ◽

Gel Filtration ◽

Molecular Size ◽

The Other ◽

Reaction Products ◽

Human Fibrinogen ◽

Polyacrylamide Electrophoresis ◽

Large Molecular Weight ◽

Stabilized Fibrin

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS Polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS Polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400, 000 to 800, 000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and non-stabilized fibrin in that fragment D was present in the “double D” cross-linked form.

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The isolation and properties of a non-pepsin proteinase from human gastric mucosa

Biochemical Journal ◽

10.1042/bj1690617 ◽

1978 ◽

Vol 169 (3) ◽

pp. 617-624 ◽

Cited By ~ 22

Author(s):

N B Roberts ◽

W H Taylor

Keyword(s):

Bone Marrow ◽

Gastric Mucosa ◽

Gastric Adenocarcinoma ◽

Proteolytic Activity ◽

Structural Characteristics ◽

Human Gastric Mucosa ◽

Cathepsin E ◽

Activity Curve ◽

Rabbit Bone ◽

Ph Range

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.

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