Partial purification and characterization of a novel neutral proteinase from human uterine cervix.
1. Human uterine cervical stroma was found to contain a Ca(2+)-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein-Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4-8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4x10(5) by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10mum) or leupeptin (10mum) was also found to be inhibitory, but chymostatin (40mug/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.
D000328:Adult; D002584:Cervix Uteri / Q000201:enzymology*; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000494:pharmacology; D010958:Plasminogen; D011480:Protease Inhibitors / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D011480:Protease Inhibitors; D012965:Sodium Chloride; D010958:Plasminogen; D010447:Peptide Hydrolases