ISOLATION AND COMPLETE AMINO-ACID-SEQUENCE OF 2 FIBRINOLYTIC PROTEINASES FROM THE TOXIC SATURNID CATERPILLAR LONOMIA-ACHELOUS

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pI(calc) = 10.55) and Achelase II (214 amino acids, pI(calc) = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22 473 and 22 727) correspond well to M(r) estimates of 24 000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha > beta >> gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg +2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.