Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour

Abstract

An LH sensitive adenylate cyclase from a tumour Leydig cell has been investigated. The plasma membranes, prepared by a 2 phase (dextran-polyethylene glycol) centrifugation method were found to have the following properties: In the presence of LH plus p(NH)ppG (guanosine 5'-beta, gamma-imido triphosphate) or fluoride ions, maximum adenylate cyclase activity was obtained in the plasma membranes with 4 to 6 mM Mg2+ plus 0.33 to 2 mM ATP. LH alone stimulated adenylate cyclase activity 2-fold when compared with basal activity and the time course of cyclic AMP production was linear up to 45 min. With GTP (10(-5)M) and GTP plus LH, adenylate cyclase activity was increased 3 and 6-fold, respectively, for up to 20 min and thereafter declined. In contrast p(NH)ppG (10(-5)M) and p(NH)ppG plus LH increased adenylate cyclase activity 7 and 14-fold which was maintained for at least 45 min. Fluoride ions increased the enzyme activity linearly over 45 min approx 18-fold. When GTP or p(NH)ppG were added alone there was a lag time of activation of approximately 10 min which was abolished by the addition of LH. GTP but not p(NH)ppG at concentrations greater than 10(-4) inhibited basal and LH stimulated adenylate cyclase when compared with 10(-5)M GTP. The tumour Leydig cell adenylate cyclase is thus essentially similar to other hormone sensitive somatic cells. The present study makes it feasible to prepare plasma membranes by a simple method from large quantities of pure Leydig cells.

Guanine nucleotide mediated desensitazation of adenylate cyclase in cell free preparations from a Leydig cell tumour

“…Activity in the presence of p(NH)ppG + LH was 46% below NaF-stimulated activity. The situation differs from that seen in plasma membranes that were not pre-treated, where stimulation by p(NH)ppG + LH was approximately the same as NaF-stimulated activity (Levi et al 1982), indicating that the pre-treatment may have in some way affected the 'integrity' of the holo-enzyme.…”

“…1980;Maguire et al 1976). This would impair the ability of LH to reduce the lag phase previously observed in p(NH)ppG activation (Levi et al 1982). The effects of GMP and guanosine were entirely reversed during the assay with LH plus p(NH)ppG.…”

“…These were as described by Levi et al (1982). Briefly, Leydig cells were obtained from a Leydig cell tumour by mechanical dispersion and purified by Percoll density gradient centrifugation (Dix & Cooke 1981).…”

“…The plasma membranes (300 yg protein/ml) were pre-incubated in a final volume of 1.0 ml for 30-40 min at 30°C with various additions including ATP and the ATP-generating system (Levi et al 1982) as indicated in the text. The buffer used for the pre-incubation and incubations contained: 40 m M TRIS/HCI pH 7.5; 1 mM Dithiothreitol; 1 mM EDTA; 5 m M MgC12; 0.5 m M methylisobutylxanthine and 0.1 % bovine serum albumin.…”

“…In a previous publication it was demonstrated that the adenylate cyclase in plasma membranes prepared from tumour Leydig cells was stimulated by LH and that this stimulation was modulated by guanine nucleotides and magnesium ions (Levi et al 1982): It was also found that the GTP but not p(NH)ppG (guanosine 5'8, y-imido triphosphate) enhanced adenylate cyclase activity was inhibited at high concentrations (greater than 1 0 -4~ GTP) and that the production rate of cyclic AMP decreased after 10-20 min incubation of the membranes. These results suggested that LH-stimulated adenylate cyclase becomes refractory to further stimulation during incubation in vitro and that this may be mediated by GTP.…”