scholarly journals The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor

1983 ◽  
Vol 211 (2) ◽  
pp. 313-318 ◽  
Author(s):  
T E Cawston ◽  
G Murphy ◽  
E Mercer ◽  
W A Galloway ◽  
B L Hazleman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Concanavalin A ◽  
Culture Medium ◽  
Enzyme Inhibitor ◽  
Inhibitor Complex ◽  
Metalloproteinase Inhibitor ◽  
Rabbit Bone

1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 × 10(-10) M. 2. The molecular weight of the enzyme-inhibitor complex was found to be 54 000, but no enzyme activity could be recovered from the complex after treatment with either mercurials or proteinases. The complex thus differed from latent collagenase in terms of size, susceptibility to mercurials and behaviour on concanavalin A-Sepharose. 3. The interaction of the purified components was compared with that of crude collagenase and crude inhibitor in culture medium. Mercurial treatment partially reversed the inhibition in the crude system, but not when the purified components were used. 4. The significance of the results is discussed in relation to the extracellular control of the activity of collagenase.

scholarly journals The release of collagenase as an inactive proenzyme by bone explants in culture

1972 ◽  
Vol 126 (2) ◽  
pp. 275-289 ◽  
Author(s):  
G. Vaes
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Inhibitor Complex ◽  
Ph Values ◽  
Liver Lysosomes ◽  
Skin Explants ◽  
Extracellular Activity

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme–inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a `procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.

scholarly journals Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin

1981 ◽  
Vol 199 (3) ◽  
pp. 807-811 ◽  
Author(s):  
G Murphy ◽  
T E Cawston ◽  
W A Galloway ◽  
M J Barnes ◽  
R A D Bunning ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Culture Medium ◽  
Type Iv Collagen ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Bone Culture ◽  
Gel Filtration Chromatography ◽  
Type Iv ◽  
Rabbit Bone ◽  
Type V

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.

scholarly journals The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

1978 ◽  
Vol 172 (2) ◽  
pp. 261-274 ◽  
Author(s):  
G Vaes ◽  
Y Eeckhout ◽  
G Lenaers-Claeys ◽  
C François-Gillet ◽  
J E Druetz
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Neutral Proteinase ◽  
Inhibitor Complex ◽  
The Media ◽  
Parallel Activation ◽  
Cartilage Proteoglycans

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

scholarly journals On the Reaction of Human Plasmin with the Primary Inhibitor of Plasmin from Human Plasma

1977 ◽  
Author(s):  
Ulla Christensen
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Dissociation Constant ◽  
Human Plasma ◽  
Ethyl Ester ◽  
Enzyme Inhibitor ◽  
Specific Time ◽  
Inhibitor Complex ◽  
Reversible Formation ◽  
Rate Of Consumption

The interaction of human plasmin with the recently discovered primary inhibitor of plasmin from human plasma has been investigated. The inhibitor is an α2-glycoprotein with a molecular weight of 60000. Pure preparations of plasmin and the inhibitor were incubated for a specific time, after which residual plasmin was determined from measurements of the rate of consumption of α-N-bensoyl-L-arginine ethyl ester. Experiments were made over ranges of both plasmin and inhibitor concentrations and for a variety of incubation times. The results show that the reaction consists of at least two steps: rapid, reversible formation of an enzyme-inhibitor complex with the dissociation constant, K = 3nM, followed by a slow, irreversible transition with a rate constant, k = 6.5 10-3s-1. Amino acids with anti-fibrinolytic effect inhibit the formation of the first complex, but not its further transition.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

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