cyclic-gmp and 8-azidoadenosine-3--5--monophosphate

The 8-azidopurine analogs of adenosine and guanine nucleotides have proved to be very useful probes for nucleotide-binding sites. In most systems they have proved to be effective mimics of the natural compounds with 1) both 8-azidoadenosine-3',5'-monophosphate and 8-azidoguanosine-3',5'-monophosphate activating their respective kinases, 2) 8-azidoguanosine-5'-triphosphate effecting tubulin polymerization and activation of adenylate cyclase, and 3) 8-azidoadenosine-5'-triphosphate appearing to be a substrate for a large number of ATPases and several kinases. As photoprobes they have been used to 1) isolate and study active site peptides; 2) determine the membrane sidedness and cellular location of binding sites; 3) detect the availability of various nucleotide-binding sites as cells progress through development, maturation, infectious stages, etc.; 4) study membrane-soluble partitioning of binding sites relative to nucleotide regulation of a biochemical process; 5) detect nucleotide-binding sites exposed by small molecules such as Ca2+ and calmodulin; and 6) detect potential catalytic and regulatory subunits of protein kinases found in preparations that actively phosphorylate endogenous substrates. The difference between the gamma-32P-labeled 8-azidopurine nucleotide triphosphate and the alpha-32P-labeled species has been used to study the in situ hydrolysis of the nucleotides on specific protein receptors and determine the fate of the produced nucleotide diphosphate. Such factors are important in studying the molecular dynamics of such systems as tubulin polymerization, G-actin to F-actin conversions, and GTP activation of adenylate cyclase. A review of techniques used and data obtained with these probes is presented.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Affinity Labels; Animals; Azides; Calcium; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Nucleotides; Phosphorylation; Photochemistry; Receptors, Cyclic AMP

The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.

Topics: Affinity Labels; Animals; Azides; Binding Sites; Cattle; Cyclic AMP; Cyclic GMP; Enzyme Activation; Kinetics; Lung; Photochemistry; Protein Kinases

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.

Topics: Animals; Azides; Cilia; Cyclic AMP; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Paramecium; Phosphorylation; Protein Kinases; Proteins