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Mechanism of the Acute CAMP‐Induced Decrease in P‐450 17α in Cultured Mouse Leydig Cells a
Author(s) -
PERKINS LOUISE M.,
PAYNE ANITA H.
Publication year - 1987
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1987.tb25051.x
Subject(s) - obstetrics and gynaecology , library science , medicine , biology , pregnancy , computer science , genetics
Recent studies from our laboratory have shown that treatment of primary cultures of mouse Leydig cells with a high dose of cAMP results in an oxygen-mediated, steroid product-enhanced decrease in the microsomal 17a-hydroxylase (P-45017,) enzyme activity.'v2 These observations are consistent with the hypothesis proposed by Hornsby3 that these decreases in P-450 enzyme activity occur when steroid products, acting as pseudosubstrates, bind to the P-450 protein and enhance the generation of damaging oxygen free-radicals, which either directly or indirectly inactivate the e n ~ y m e . ~ The present study was designed to investigate the mechanism by which the oxygen-dependent, product-induced decrease in P-45017, activity occurs. The amount of P-45Ol7, was determined in lysates of cultured mouse Leydig cells treated for 48 h with 8-Br-CAMP (CAMP) or steroids by immunoblotting to determine if decreases in P-45017, enzyme activity are due to damage to, and concomitant loss of, the enzyme protein or due to an inactivation of the catalytic activity of the enzyme with no change in the amount of the enzyme protein. In addition, the amount of mitochondria1 cholesterol side-chain cleavage enzyme (P-450,,,) was determined in the same preparations to evaluate if the two enzymes are regulated in the same manner or are regulated differentially. Adult mouse Leydig cells were purified and allowed to attach 3 h prior to the initiation of treatment with cAMP or steroids. Following the 48 h treatment period, P-45017, enzyme activity was determined by the conversion of [3H]progesterone to [3H]products, and cultured cells were lysed with a sodium dodecyl sulfate (SDS)-containing buffer. Aliquots of the lysates were subjected to SDSpolyacrylamide gel electrophoresis and immunoblotting with antibody specific for P-45OI7,. In some cases, immunoblots were treated with antibody to P-450,,, following immunoblotting with anti-P-45017,. Bound antibody was detected with ['2SI]protein A and autoradiography. FIGURE 1 shows that 1.0 mM cAMP or 2 pM testosterone (T) causes a decrease in the amount of P-45017, similar to the decrease in activity of the enzyme. In the presence of aminoglutethimide (AG), which inhibits T synthesis, the