Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease.
The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.
Inomata
M
M
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
Hayashi
M
M
Nakamura
M
M
Saito
Y
Y
Kawashima
S
S
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000097:blood*; D002364:Caseins / Q000494:pharmacology; D002645:Chickens; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D015151:Immunoblotting; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D014746:Vimentin / Q000494:pharmacology
D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D002364:Caseins; D007976:Leupeptins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D014746:Vimentin; D004533:Egtazic Acid; C033668:calpastatin; D002154:Calpain; C032854:leupeptin; D002118:Calcium