Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca.
Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujimura
Y
Y
Department of Blood Transfusion, Nara Medical College, Japan.
Titani
K
K
Usami
Y
Y
Suzuki
M
M
Oyama
R
R
Matsui
T
T
Fukui
H
H
Sugimoto
M
M
Ruggeri
Z M
ZM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001792:Blood Platelets / Q000502:physiology; D002241:Carbohydrates / Q000032:analysis; D003435:Crotalid Venoms / Q000302:isolation & purification* / Q000494:pharmacology; D006388:Hemagglutinins / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012689:Sequence Homology, Nucleic Acid; D012911:Snakes; D013438:Sulfhydryl Compounds / Q000032:analysis; D014841:von Willebrand Factor / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D003435:Crotalid Venoms; D006388:Hemagglutinins; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D014841:von Willebrand Factor; C030961:botrocetin