Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides.
Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.
Department of Pharmacology and Toxicology, Molecular Graphics and Modeling Laboratory, the University of Kansas, Lawrence, KS 66045-7582, USA.
Andreas
A
Gille
Gerald H
GH
Lushington
Tung-Chung
TC
Mou
Michael B
MB
Doughty
Roger A
RA
Johnson
Roland
R
Seifert
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry*; D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006162:Guanylate Cyclase; D020128:Inhibitory Concentration 50; D007313:Insecta; D007700:Kinetics; D008223:Lymphoma / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D009711:Nucleotides / Q000737:chemistry* / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D000071756:Soluble Guanylyl Cyclase; D013873:Thionucleotides / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D009711:Nucleotides; D020033:Protein Isoforms; D011687:Purines; D011743:Pyrimidines; D018160:Receptors, Cytoplasmic and Nuclear; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D008274:Magnesium