Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA.
Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.
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CSIRO Division of Tropical Animal Production, Indooroopilly, Queensland, Australia.
Pearson
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RD
Jarmey
J M
JM
Cadogan
L C
LC
Riding
G A
GA
Tellam
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002918:Chymotrypsin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004175:Diptera / Q000201:enzymology*; D019476:Insect Proteins; D007814:Larva / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D019476:Insect Proteins; D012333:RNA, Messenger; D002918:Chymotrypsin; C092875:Lucilia chymotrypsin b