Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay

The cathepsin B like proteinase present in ascitic fluid of a patient with neoplasia has been purified and characterized after pepsin activation. From this fluid we have prepared the low molecular weight (LMW) cysteine-proteinase inhibitors. Three major inhibitor forms were found with isoelectric points of 7.4, 5.4, and 5.1, respectively. The interaction of the enzyme with the former inhibitor was studied because this inhibitor was the most abundant. The Ki value was found to be 4.3 × 10−8 M. Two molecules of this proteinase were bound by one molecule of plasma α2 macroglobulin (α2M). The LMW inhibitor was able to bind to the enzyme entrapped in α2M and reduced its endopeptidase activity on benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl-7-coumarylamide. These results may have a physiological significance in the regulation of the enzyme which, among other extracellular hydrolases, probably plays an important role in tumor invasion.

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Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(83)90109-5 ◽

1983 ◽

Vol 757 (2) ◽

pp. 196-201 ◽

Cited By ~ 10

Author(s):

Masayuki Hirado ◽

Michio Niinobe ◽

Setsuro Fujii

Keyword(s):

Molecular Weight ◽

Bovine Serum ◽

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Low Molecular Weight ◽

Cysteine Proteinase Inhibitors

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Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties

Biochimie ◽

10.1016/j.biochi.2013.04.007 ◽

2013 ◽

Vol 95 (8) ◽

pp. 1552-1559 ◽

Cited By ~ 7

Author(s):

Vikash Kumar Yadav ◽

Nirmal Chhikara ◽

Kamaldeep Gill ◽

Sharmistha Dey ◽

Sarman Singh ◽

...

Keyword(s):

Molecular Weight ◽

Seminal Fluid ◽

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Low Molecular Weight ◽

Kinetic Properties ◽

Enzyme Kinetic ◽

Cysteine Proteinase Inhibitors

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Proteinases inTrichomonas vaginalisandTritrichomonas mobilensisare not exclusively of cysteine type

Parasitology ◽

10.1017/s0031182000060418 ◽

1991 ◽

Vol 102 (1) ◽

pp. 113-115 ◽

Cited By ~ 19

Author(s):

P. Bózner ◽

P. Demeš

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Trichomonas Vaginalis ◽

Chelating Agents ◽

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

The Other ◽

Polyacrylamide Gels ◽

Cysteine Proteinase Inhibitors

SUMMARYHigh molecular weight proteinases ofTrichomonas vaginalis(with apparentMrvalues 142 and > 220 kDa) andTritrichomonas mobilensis(Mr67, 86, 104 and 120 kDa), optimally active at pH 8, were analysed in gelatin-containing polyacrylamide gels. All of these proteinases were resistant to serine-, aspartic- as well as cysteine proteinase inhibitors. Both proteolytic bands inT. vaginalisand two proteinases inT. mobilensis(67 and 104 kDa) were inhibited by EDTA and EGTA suggesting that they belong to the metallo-proteinase class. The 67 kDa proteinase ofT. mobilensiswas inhibited also byo-phenanthroline. The other two bands ofT. mobilensis(86, 120 kDa) were not classified to any proteinase group since they appeared to be resistant to the chelating agents tested in this study.

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PURIFICATION AND CHARACTERIZATION OF TWO PROCOAGULANTS FROM WALKER 256 CARCINOSARCOMA TUMORS,

10.1055/s-0038-1643666 ◽

1987 ◽

Author(s):

Stuart Gordon ◽

Bonnie Sloane ◽

Phil Cavanugh ◽

Barbara Cross ◽

Kenneth Honn ◽

...

Keyword(s):

Molecular Weight ◽

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Procoagulant Activity ◽

Coagulation System ◽

Factor X ◽

Cysteine Proteinase Inhibitors ◽

Cancer Procoagulant ◽

Walker 256 ◽

Barbital Buffer

Activation of the coagulation system bytumor cells may play an important role in tumor growth and metastases. Becauseprocoagulant activities have been identified in different tumor cells by different investigators, effective comparison of these activities has been difficult. Therefore, we purified and characterized two different procoagulant proteins from the same Walker 256 tumors. The first procoagulant activity/platelet aggregating activity (PCA/PAA) was purified from a 1% CHAPS detergent extract oftumor homogenate followed by (NH4)2SO4 fractionation, anion exchange and hydrophobic chromatography. The protein had a molecular weight of 58,000, required phospholipid and an intact coagulation pathway from factor X through fibrinogen for activity, but did not require factors VII or IX forits procoagulant activity. The procoagulant activity was not inhibited by 5mMphenyl-methyl sulfonyl fluoride, iodoacetamide or phenanthroline; there was noevidence of proteinase activity. The PAA was due to thrombin generation during coagulation. The second procoagulant,cancer procoagulant (CP), was extracted from tumors in barbital buffer (pH 7.4) without detergent, purified by immunoaffinity (using a polyclonal goat antibody to CP from V2 carcinoma) and mercurial-benzoate affinity chromatography. CP had a molecular weight of 68,000, an isoelectric point of 4.8 and initiated coagulation by directly activating factor X in the coagulation system. CP was inhibited by Hg++ and iodoacetamide, cysteine proteinase inhibitors. The purified CP formed an immunodiffusion precipitin band against the polyclonal anti-CP goat antibody. Thus, thepurified CP had the same physicochemical, enzymatic and immunologic propertiesas CP from rabbit V2 carcinoma. Neither procoagulant had the properties of tissue factor. These results suggest that there aretwo distinct procoagulant activities inWalker 256 and that both may contributeto the coagulation abnormalities that are associated with tumor growthand metastases.

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