Nuurosciuncr. 1978. Vol. 3, pp. 83-93. Pergamon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE Press. Printed in Great Bntam zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK GUANYLATE CYCLASE FROM BOVINE ADRENAL MEDULLA: SUBCELLULAR DISTRIBUTION AND STUDIES ON THE EFFECT OF LYSOLECITHIN ON ENZYME ACTIVITY DOMINIQUE AUNIS,’ M. PESCHELOCHE and J. ZWILLER Centre de Neurochimie du CNRS, INSERM, 11, rue Humann, 67085, Strasbourg, France Abstract-The localization of guanylate cyclase in bovine adrenal medulla was explored by preparation of subcellular organelles. Forty per cent of the activity was recovered in the soluble fraction (cytosol), and 46% in the low-speed (8oOg) pellet as particulate. In order to determine the exact localization of the particulate enzyme, continuous sucrose density gradient centrifugation was used. The results indicate that guanylate cyclase activity sedimented together with acetylcholinesterase and adenylate cyclase, two plasma membrane markers. Enrichment of specific activity over that present in the crude chromaffin granule fraction was identical for these three enzymes (from 4.4 to 6.2-fold). Particulate guanylate cyclase appears therefore to be a constituent of plasma membranes. The non-ionic detergent Triton X-100 and the natural phospholipid, lysolecithin, stimulated particu- late guanylate cyclase activity 20-fold, but that of the soluble enzyme only 2- to 3-fold. The kinetic behaviour of particulate guanylate cyclase was not altered by treatment with lysolecithin: the Hill n coefficient for guanosine 5’-triphosphate remained close to 1.35 and the !& value to 320 PM. Among lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce activation of particulate guanylate cyclase. Incubation of membranes with phospholipase A, led to a 5-fold stimulation of particulate guanylate cyclase activity, while the soluble enzyme activity was not affected. These results suggest a possible role for lysolecithin in the regulation of the intracellu- lar levels of cyclic guanosine monophosphate during or following the excitation-secretion coupling process in the medullary cell. RECENT observations have shown that lysophosphatyl choline, a phospholipid with detergent properties which occurs naturally in biological membranes, stimulates the activity of a lot of membrane-bound enzymes, in particular that of guanylate cyclase (GTP pyrophosphate-lyase, EC 4.6.1.2) in fibroblast cell culture (SHIER,BALDWIN, NILSEN-HMILTON, HAMIL- TON & THANASSI, 1976), in rat lung (WHITE & LAD, 1975) and in neuroblastoma cell culture (ZWILLER, CIESELSKI-TRESKA & MANDEL, 1976). The chromaffin granules of the adrenal medulla, which store catecholamines together with adenosine S-triphosphate (ATP), chromogranins and dopamine /3-hydroxylase (3,4-dihydroxyphenylethylamine, acerbate: 0, oxidoreductase (fi-hydroxylating), EC 1.14.17. l), are characterized by an unusually high level of lysolecithin within their surrounding membranes (17% of total membrane lipids, BLASCHKO, FIRE- MARK, SMITH 8c WINKLER, 1967; 12%, DREYFUS, AUNIS, HARTH & MANDEL, 1977). When stimulated, the adrenal medulla releases the soluble content of chromafin granules into the lumen by the process known as exocytosis (KIRSHNER & KIRSHNER, 1971), 1 Chargk de Recherches, INSERM. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED Abbreointions: ATP, GTP, adenosine and guanosine 5’-triphosphate, respectively; cyclic GMP, guanosine 3’:5’-phosphate; cyclic AMP, adenosine 3’: 5’-phosphate; lysolecithin, L-a-lysophosphatidyl choline. and it has been suggested that lysolecithin might be involved in the process of membrane fusion that occurs as the first step during exocytosis (BLASCHKO et al., 1967 ; DOUGLAS, POISNER & TRIFARO, 1966). This interesting hypothesis, combined with our recent studies on the effect of lysolecithin on membrane- bound dopamine /?-hydroxylase (AUNIS, E~WCLIER, PESCHELOCHE & MANDEL, 1977) and Mg’+-dependent adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) (D. AUNIS,unpublished results), prompted us to investigate the effects of lysolecithin on guany- late cyclase activity. Crucial to the questioti of the physiological function of the effects of lysolecithin on enzyme activity is the localization of guanylate cyc- lase in the medullary cell and its relationship to the chromaffin granule membrane. EXPERIMENTAL PROCEDURES Materials L-a-phosphatidyl choline (L-a-lecithin) from egg yolk (Type VII-E); L-a-lysophosphatidyl choline (L-a-lysole- cithin) from egg yolk (Type I), L-a-phosphatidyl serine from bovine brain and L-a-lysophosphatidyl ethanolamine (L-a-lysocephalin) from egg yolk (Type I) were obtained from Sigma (St Louis, U.S.A.) and used without further purification. Phospholipase A, from Crotalus venom (EC 3.1.1.4; 200 U/mg) was purchased from Boehringer (Mann- heim, Germany). Triton X-100 was a Roth product (Karlsruhe, Germany). [a-32P]guanosine triphosphate and 83