Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80 percent homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii

Difference in the sensitivity of junctional and longitudinal sarcoplasmic reticulum Ca(2+)-ATPase to ADP

The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. We compared Ca2+ accumulation and acetyl phosphate hydrolysis by the Ca(2+)-ATPases present in the longitudinal and junctional membrane of the sarcoplasmic reticulum of rabbit skeletal muscle and found that Ca(2+)-ATPase is more sensitive to ADP inhibition when the enzyme is located on the junctional membrane than when the enzyme is located on the longitudinal membrane (K0.5 = 144 microM for the junctional enzyme vs K0.5 = 415 microM for the longitudinal enzyme). When the enzyme was solubilized in non-ionic detergent (2% v/v Triton X-100) and tested again using 2 mM AcP as substrate, the difference in ADP sensitivity observed with native preparations disappeared. We conclude that the enzyme is regulated differently depending on its localization on the membrane of the sarcoplasmic reticulum