Re-evaluation of the specificity of adenylyl (beta,gamma-methylene)diphosphonate as a substrate for adenylate cyclase.
The conversion of the ATP-analogue adenylyl(beta,gamma-methylene)diphosphonate (AMPPCP) to cyclic AMP by adenylate cyclase of rat liver membranes was demonstrated using a radioimmunoassay for cyclic AMP. The conversion was only insignificantly lower than with adenylylimidodiphosphate (AMPPNP), another ATP-analogue which is usually used in the histochemical adenylate cyclase assay. The unspecific phosphate production was lower with AMPPCP as compared to AMPPNP. Therefore AMPPCP is considered to be a more suitable substrate for the histochemical assay. Unspecific phosphate deposition in the histochemical assay was due to ATP:pyrophosphatase activity and could be significantly inhibited by 1 mM NAD. However, a residual phosphate deposition due to cleavage of NAD could not be suppressed. Adenylate cyclase activity could be markedly activated by 5 x 10(-5) M forskolin, an activator of the catalytic subunit of the enzyme, and inhibited by 1 mM 2'5'-dideoxyadenosine, a specific inhibitor of adenylate cyclase. Adenylate cyclase was localized predominantly in the sinusoidal part of the plasma membrane, while ATP-pyrophosphatase seemed to be restricted to the canalicular part. It is concluded that at least three parallel assays are necessary for routine histochemical demonstration of adenylate cyclase, namely (1) basal activity (2) activation by forskolin and (3) inhibition by 2'5'-dideoxyadenosine, to demonstrate a specific enzyme reaction.
Mayer
D
D
Abteilung Cytopathologie, Deutsches Krebsforschungszentrum, Heidelberg, FRG.
Hacker
H J
HJ
Bannasch
P
P
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000032:analysis*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D005629:Frozen Sections; D006651:Histocytochemistry; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013773:Tetramisole / Q000494:pharmacology
D008565:Membrane Proteins; D010710:Phosphates; D000266:Adenylyl Imidodiphosphate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D013773:Tetramisole; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D015056:1-Methyl-3-isobutylxanthine