Characterization of an endogenous cytosolic protein inhibitor (CIP-28) of cytosolic rat testicular neutral cholesteryl ester hydrolase.

摘要

A heal-stable 28 kDa CEH inhibitory protein and copurifying 26.5 kDa protein (25% of total amount of the two proteins) were purified from rat testis cytosol by sequential 40-65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. Polyclonal antibodies raised in rabbits to either the 28 kDa or 26.5 kDa proteins separately or to both proteins immunologically cross-reacted, as well as, immunoprecipitated both proteins. Immunoprecipitation resulted in loss of CEH inhibitory activity.;Both proteins exhibited an identical pI of 4.8. Both were highly hydrophobic, exhibiting non-specific association with apolar surfaces. Amino acid compositions of individual proteins differed from those of other surface active proteins.;Inhibition was non-enzymatic, concentration-dependent and reversible by dilution suggesting a binding phenomenon. Inhibition diminished with increasing (substrate). Moreover, CIP-28 affected hydrolysis of water-insoluble substrates by diverse lipolytic enzymes but not hydrolysis of water-soluble substrates indicating CIP-28 influences lipolysis by association with substrate. Increasing ionic strength enhanced substrate association indicating hydrophobic CIP-substrate interactions. Surface-active, amphipathic molecules disrupted apolar associations but couldn't abolish testicular CEH inhibitory activity possibly suggesting CIP-CEH interactions.;Maximal saturation of inhibition was a function of CIP-28:CEH molar ratios. Inhibition diminished with increasing (CEH) and was measured with CIP-28:CEH molar ratios ;The testicular cytosolic concentration, calculated from the purified measured mass, was ;CIP-28 affected activities of diverse lipolytic enzymes in vitro and may function as a non-specific lipolytic effector. However, CIP-28 appeared to exert the greatest influence on CEH activity in vitro. Ubiquitous distribution of CEH inhibitory activity and cross-immunoreactive 28/26.5 kDa bands may reflect a general role in regulation of the cholesteryl ester cycle and intracellular cholesterol homeostasis.