INACTIVATION OF CATHEPSIN-B BY OXIDIZED LDL INVOLVES COMPLEX-FORMATION INDUCED BY BINDING OF PUTATIVE REACTIVE SITES EXPOSED AT LOW PH TO THIOLS ON THE ENZYME

We recently showed that the poor degradation of apo B in oxidized (ox- ) LDL by mouse peritoneal macrophages could be attributed to the inact ivation of cathepsin B by ox-LDL. In this current study, we show that enzyme inactivation involves complex formation of ox-LDL with cathepsi n B rather than the diffusion of reactive components from ox-LDL to th e enzyme. Complex formation between ox-LDL and cathepsin B was far gre ater at pH 4.5 than at pH 7.4 and far greater with ox-LDL than with LD L. Even though complexes were also formed between ox-LDL and other pro teins such as BSA, insulin, and LDL, ox-LDL bound up to 30 times more cathepsin B than BSA, when compared on a molar level and under the sam e conditions. Unlike ox-LDL alone, complexes of ox-LDL and BSA were un able to inactivate cathepsin B, suggesting that BSA was sequestering r eactive sites on ox-LDL. The interaction of ox-LDL with proteins such as cathepsin B appears to represent aldehydic modifications of apo B, since treatment of ox-LDL with the reductant NaBH4, which stabilizes s uch adducts, greatly decreased the binding of ox-LDL to BSA and preven ted ox-LDL from inactivating cathepsin B. It is likely that thiols on cathepsin B or other proteins interact with reactive groups on ox-LDL, since BSA in which thiols were blocked with N-ethylmaleimide (NEM), f ailed to bind to ox-LDL. Moreover, NEM-treated BSA had no effect on th e ability of ox-LDL to inactivate cathepsin B. Similar results were ob tained with LDL modified with 4-hydroxynonenal (HNE). These data sugge st that aldehydic adducts on ox-LDL that are unreactive at neutral pH, possibly HNE bound to apo B, become exposed at acidic pH and then cov alently bind thiols on neighboring proteins such as cathepsin B in lys osomes, inducing crosslinking of proteins and enzyme inactivation. (C) 1997 Elsevier Science Inc.