REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

点击翻译按钮获取中文摘要

Hormonal control of adenylate cyclase is mediated by GTP-binding proteins, stimulation via Gs, inhibition via Gi. A similar GTP-binding protein, transducin couples the light receptor rhodopsin to a cGMP phosphodiesterase. Pertussis toxin, an etiologic agent in whooping cough, inactivates Gi and transducin by catalyzing the ADP-ribosylation of a critical cysteine. Pertussis-toxin catalyzed ADP-ribosylation of transducin, and NAD hydrolysis was stimulated by adenine nucleotide and either phospholipid or detergent. NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent CHAPS was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue, adenyl-5'-yl-imidodiphosphate. GTP and guanyl-5'-yl-imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol. The isolated enzymatic (S1) component catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of holotoxin but not the isolated catalytic unit. In animal cells, ADP-ribosylation of proteins is a reversible process, catalyzed by synthetic and degradative enzymes known respectively as ADP-ribosyltransferases and ADP-ribosylarginine hydrolases. The specific substrates for a purified erythrocyte hydrolase were Alpha-ADP-ribosylarginine and Alpha-2'-phospho-ADP-ribosylarginine, products of the transferase reaction. The hydrolase and transferases possess a compatible sterospecificity and substrate specificity consistent with the conclusion that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.