scholarly journals The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1

1991 ◽  
Vol 278 (1) ◽  
pp. 179-187 ◽  
Author(s):  
R V Ward ◽  
R M Hembry ◽  
J J Reynolds ◽  
G Murphy
Keyword(s):  
Molecular Mass ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
U937 Cells ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Gingival Fibroblasts ◽  
Tissue Inhibitor ◽  
Lower Molecular Mass ◽  
Inhibitory Activities

Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.

scholarly journals Effect of diosgenin on biliary cholesterol transport in the rat

1993 ◽  
Vol 291 (3) ◽  
pp. 793-798 ◽  
Author(s):  
A Thewles ◽  
R A Parslow ◽  
R Coleman
Keyword(s):  
Bile Salt ◽  
Molecular Mass ◽  
Bile Salts ◽  
Mass Fraction ◽  
Gel Permeation Chromatography ◽  
High Molecular Mass ◽  
Cholesterol Transport ◽  
Biliary Cholesterol ◽  
Lower Molecular Mass ◽  
Salt Depletion

Biliary cholesterol output in rats was stimulated over 3-fold by feeding diosgenin for 5 days, whereas biliary outputs of phospholipid and bile salts were not changed by diosgenin feeding. Isolating and perfusing the liver without bile salts resulted in a rapid and substantial decrease in biliary bile salt output; bile salt depletion abolished the diosgenin-induced increment in biliary cholesterol output, showing that the diosgenin-elevated biliary cholesterol output was bile-salt-dependent. Diosgenin treatment also produced a significant decrease in biliary alkaline phosphodiesterase I. Fresh bile obtained from control and diosgenin-fed rats was subjected to gel-permeation chromatography in order to separate different-sized biliary cholesterol carriers. Two major peaks of cholesterol were eluted, with cholesterol also being eluted between the peaks. The cholesterol peak eluted at the lower molecular mass (20-30 kDa) was observed in all bile samples. The higher-molecular-mass peak, which was eluted at the void volume, was not observed in all biles; control biles contained very little high-molecular-mass form of cholesterol, whereas biles from the diosgenin group contained up to 47% of cholesterol in the high-molecular-mass fraction. Diosgenin treatment produced a range of elevated biliary cholesterol values which positively correlated with the proportion of cholesterol contained in the high-molecular-mass fraction (r = 0.98). The results show that diosgenin induced a marked bile-salt-dependent increase in biliary cholesterol output and a shift in biliary cholesterol transport to higher-molecular-mass structures.

scholarly journals Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization

1994 ◽  
Vol 304 (3) ◽  
pp. 693-698 ◽  
Author(s):  
L W J Klomp ◽  
L van Rens ◽  
G J Strous
Keyword(s):  
Molecular Mass ◽  
Mechanical Damage ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Apparent Molecular Mass ◽  
Gastric Mucin ◽  
Gastric Mucus ◽  
Apical Side ◽  
Sds Page ◽  
Glycosylation Inhibitor

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.

Cloning and Characterization of Human Tissue Inhibitor of Metalloproteinases-3

1994 ◽  
Vol 13 (7) ◽  
pp. 711-718 ◽  
Author(s):  
CRAIG G. WILDE ◽  
PHIL R. HAWKINS ◽  
ROGER T. COLEMAN ◽  
WENDY B. LEVINE ◽  
ANGELO M. DELEGEANE ◽  
...  
Keyword(s):  
Human Tissue ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Tissue Inhibitor
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