title: Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.
abstract: 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, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 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 Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than 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.
authors.affiliation: Department of Molecular Biology, Glaxo Research Laboratories, Research Triangle Park, NC.
authors.forename: T
authors.initials: T
authors.lastname: Amarant
authors.forename: W
authors.initials: W
authors.lastname: Burkhart
authors.forename: H
authors.initials: H
authors.lastname: LeVine
authors.forename: C L
authors.initials: CL
authors.lastname: Arocha-Pinango
authors.forename: I
authors.initials: I
authors.lastname: Parikh
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D002863:Chromogenic Compounds / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000032:analysis; D006458:Hemolymph / Q000201:enzymology; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D012463:Saliva / Q000201:enzymology; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002863:Chromogenic Compounds; D015842:Serine Proteinase Inhibitors; D005337:Fibrin; D005340:Fibrinogen; D012697:Serine Endopeptidases; C071070:achelase I; C071071:achelase II; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator