Isolation and complete amino acid sequence of two 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, 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.
Amarant
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Department of Molecular Biology, Glaxo Research Laboratories, Research Triangle Park, NC.
Burkhart
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W
LeVine
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H
Arocha-Pinango
C L
CL
Parikh
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I
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
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