Basic proteinases from Bothrops moojeni (caissaca) venom--II. Isolation of the metalloproteinase MPB. Comparison of the proteolytic activity on natural substrates by MPB, MSP 1 and MSP 2.
A basic metalloproteinase active on casein was isolated from Bothrops moojeni venom by chromatography on Sephadex G-100, DEAE-Sephacel, SP-Sephadex C-50 and Sepharose 12. The enzyme, named MPB, is not hemorrhagic and presents only traces of blood-clotting activity. On polyacrylamide gel electrophoresis at pH 4.3, MPB presented a single and diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme presented two protein bands corresponding to mol. wts of 65,000 and 55,000, which stained with Schiff's reagent. The proteolytic activity of MPB was inhibited by ethylenediaminetetracetate, 1,10-phenanthroline and dithiothreitol. The proteolytic activity of MPB and the serine proteinases MSP 1 and MSP 2 on natural substrates indicates differences in hydrolytic specificity among these enzymes. All fibrinogen chains were degraded by the three proteinases, but MPB is the most active. On fibrin, the proteinases hydrolyzed only the alpha-chain and alpha-polymer, leaving the beta-chain and gamma-dimer apparently untouched. The native type I collagen was partially hydrolyzed by the three enzymes but no digestion product was detected. On the contrary, calf and guinea-pig skin type I gelatins were readily digested by MSP 1 and MSP 2 producing different hydrolysis patterns. MPB was the least active proteinase on the gelatins. The digestion of fibronectin showed an inversion in the specificity of these proteinases. MPB was the most active on fibronectin, while MSP 1 and MSP 2 promoted a faint, partial hydrolysis on this protein.
Serviço de Bioquímica, Instituto Butantan, São Paulo, Brazil.
S M
SM
Serrano
C A
CA
Sampaio
F R
FR
Mandelbaum
D000818:Animals; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology*; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D045726:Metalloproteases; D010974:Platelet Aggregation / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
D003435:Crotalid Venoms; D005353:Fibronectins; D005780:Gelatin; D005337:Fibrin; D005340:Fibrinogen; D003094:Collagen; D045726:Metalloproteases; C578709:moojenin, Bothrops moojeni; C081402:MSP 1 proteinase, Bothrops moojeni; C081403:MSP 2 proteinase, Bothrops moojeni; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases