Molecular interaction of bovine kininogen and its derivatives with papain.
The molecular interaction of bovine kininogen and its derivatives with papain was investigated. High-molecular-weight kininogen (HMWK) or low-molecular-weight kininogen (LMWK) and inactive papain treated with N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64) formed, respectively, a complex, which was dissociable on sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE). The densitometric determination of the bands separated on SDS-PAGE and amino acid analysis of the samples extracted from the electrophoresis gel revealed that the complex between kininogen and papain is formed in a molar ratio of one to one. Moreover, analysis of the inhibition of the caseinolytic activity of papain by these kininogens indicated that HMWK, LMWK, and kinin-free derivatives obtained from both kininogens inhibit active papain with a stoichiometry of 1:1. On the other hand, the papain activity was inhibited by two kinds of cyanogen bromide fragments isolated from the heavy chain of HMWK. These two fragments with Ki values of 38 and 0.64 nM corresponded, respectively, to residue Nos. 47 to 243 and Nos. 244-360 of the HMWK heavy chain. These results suggest that in the intact HMWK and LMWK, one of the two potential reactive sites interacts with papain to form a complex and that the other reactive site becomes active only after separation of the two sites.
Sueyoshi
T
T
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Hara
A
A
Shimada
T
T
Kimura
M
M
Morita
T
T
Kato
H
H
Iwanaga
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000032:analysis; D007704:Kininogens / Q000378:metabolism*; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011288:Prekallikrein / Q000494:pharmacology
D000596:Amino Acids; D004798:Enzymes; D007704:Kininogens; D011288:Prekallikrein; D010206:Papain