Testicular temperature-labile cholesteryl ester hydrolase. Relationship to isoenzymes from other tissues, correlation with spermatogenesis, and inhibition by physiological concentrations of divalent cations.
Temperature-labile cholesterol ester hydrolase (TLCEH) was purified 2,000-fold from rat testis cytosol using sequential ammonium sulfate precipitation, cation exchange chromatography, and isoelectric focusing chromatography. the purified enzyme, which exhibited a single silver-stained band (66 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was inhibited 89% by the elevation of the temperature from 32 to 37 degrees C and 65% by treatment with alkaline phosphatase. Its amino acid composition and amino-terminal sequence differed markedly from those of isoenzymes from other tissues, although 6 of 20 residues were conserved. Polyclonal antibodies raised to TLCEH exhibited no cross-immunoreactivity with cytosolic proteins from other rat tissues and inhibited 70% of testis cytosolic CEH. Western blot analysis demonstrated a high correlation between immunoreactive protein and catalytic activity in the testis during maturation of the rat, with a marked increase at the onset of spermatogenesis. TLCEH was inhibited by physiological levels of Cu2+ (I50 = 0.60 microM) and Zn2+ (I50 = 0.75 microM) and by Cd2+ (I50 = 0.15 microM) but not by 0.5-5 mM Mn2+.
Wee
S
S
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614.
Grogan
W M
WM
D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004795:Enzyme Stability; D006801:Humans; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013091:Spermatogenesis; D002787:Sterol Esterase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013737:Testis / Q000201:enzymology* / Q000254:growth & development
D000906:Antibodies; D007527:Isoenzymes; D002787:Sterol Esterase