A study of 3':5'-cyclic mononucleotide-dependent protein kinase from canine prostate glands.
(17/24)
1. An adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1mum-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 10(8)m(-1). 5. Stimulation of the enzyme was also observed with the 3':5'-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing alpha-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent K(m) for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent K(m) for ATP (1.2x10(-5)m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn(2+) for Mg(2+) decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg(2+) was replaced by Ca(2+) and cyclic AMP produced only little stimulation of the prostatic enzyme. (+info)
A unique cyclic nucleotide-dependent protein kinase.
(18/24)
During the course of studying the soluble cyclic nucleotide-dependent protein kinases of a developing insect, three different enzymes were isolated. Two of these were found to be cAMP-dependent enzymes eluting from DEAE-cellulose in a manner identical with protein kinases I and II found in vertebrate muscle. The third enzyme appears to be unique. It has high affinity for either cAMP or cGMP (KA of 43 nM and 25 nM, respectively), the only cyclic nucleotide-dependent kinase described, to have this property. The enzyme has lower affinity for cIMP and cCMP (KA of 160 nM and 340 nM, respectively). Binding to cyclic nucleotide does not alter enzyme size. The KM for ATP is 86 microM, and among several types of histones tried, the slightly lysine-rich subgroup f2a was the best phosphate acceptor. Maximum activity was obtained with 1 mM Mg2+ while Mn2+ was completely ineffective. This new enzyme was purified to homogeneity on a cAMP affinity column as judged by two-dimensional electrophoresis. On the basis of molecular sieving and sodium dodecyl sulfate electrophoresis we have reached the preliminary conclusion that the native enzyme is a dimer of identical subunits with a molecular weight of 180,000. If the mammalian cAMP and cGMP enzymes are indeed homologous proteins, perhaps we have in this new kinase a species that represents a common ancestral protein. (+info)
Cyclic GMP-specific, high affinity, noncatalytic binding sites on light-activated phosphodiesterase.
(19/24)
Two classes of high affinity, cGMP-specific binding sites have been found in association with a peripheral membrane protein in rod outer segments. [3H]cGMP and a photoaffinity label, 8-N3-[32P]cIMP, have been used to study these cGMP binding sites. The cGMP binding sites co-migrated with rod outer segment phosphodiesterase (EC 3.1.4.17) upon Bio-Gel A-0.5m column chromatography, sucrose density gradient centrifugation, and isoelectric focusing (pI 5.35). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 8-N3-[32P]cIMP-labeled protein also migrated in a position identical with that of purified phosphodiesterase. Scatchard analysis, using purified phosphodiesterase, revealed the presence of two classes of cGMP binding sites with apparent KD values of 0.16 and 0.83 microM. A number of observations indicated that these high affinity, cGMP-specific binding sites are distinct from the phosphodiesterase catalytic site. cAMP, which is a substrate for phosphodiesterase, did not bind to the high affinity cGMP specific sites. Limited tryptic proteolysis of phosphodiesterase resulted in a striking activation of the catalytic activity and a 96% loss of cGMP binding. 1-Methyl-3-isobutylxanthine inhibited phosphodiesterase activity and enhanced the specific binding of cGMP. Mg2+ was necessary for phosphodiesterase activity, but not for high affinity cGMP binding. Finally, phosphodiesterase activity and the cGMP-specific high affinity sites showed different stabilities on storage in phosphate buffer. These specific high affinity cGMP binding sites may be involved in the regulation of phosphodiesterase activity. (+info)
Distribution of cyclic-GMP-dependent protein kinase in various rat tissues and cell lines determined by a sensitive and specific radioimmunoassay.
(20/24)
Cyclic-GMP-dependent protein kinase purified from bovine lung was radioiodinated by the Bolton-Hunter procedure yielding a specific radioactivity of 2200 Ci/mmol of enzyme, Using a specific precipitating rabbit antiserum to the cyclic-GMP-dependent protein kinase, a sensitive radioimmunoassay was developed which can detect 200 pg (1.33 fmol) of cyclic GMP-dependent protein kinase. Immunoreactivity like that of cyclic-GMP-dependent protein kinase was detectable in extracts of all rat tissues tested, in extracts of cultured rat brain and heart cells, and in extracts of rat glioma (C6) and neuroblastoma x glioma hybrid cells. In extracts of several tissues and cell lines the presence of cyclic-GMP-dependent protein kinase was also demonstrated by a photoaffinity-labeling procedure using 8-azidoinosine 3',5'-[32P]monophosphate. The results suggest that cyclic-GMP-dependent protein kinase is ubiquitously distributed although its level varies significantly from tissue to tissue and cell type to type. The results also support the hypothesis that cyclic-GMP-dependent protein kinase is involved in mediating some of the intracellular effects of those hormones, neurotransmitters and drugs which regulate the intracellular level of cyclic GMP. (+info)
Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels.
(21/24)
Cyclic nucleotide-gated ion channels of retinal photoreceptors and olfactory neurons are differentially activated by ligands that vary only in their purine ring structure. The nucleotide selectivity of the bovine rod cyclic nucleotide-gated channel (cGMP > cIMP >> cAMP) was significantly altered by neutralization of a single aspartic acid residue in the binding domain (cGMP > or = cAMP > cIMP). Substitution by a nonpolar residue at this position inverted agonist selectivity (cAMP >> cIMP > or = cGMP). These effects resulted from an alteration in the relative ability of the agonists to promote the allosteric conformational change associated with channel activation, not from a modification in their initial binding affinity. We propose a general mechanism for guanine nucleotide discrimination, in common with that observed in high affinity GTP-binding proteins, involving the formation of a pair of hydrogen bonds between the aspartic acid side chain and N1 and N2 of the guanine ring. (+info)
The cyclic GMP-mediated calcium release pathway in sea urchin eggs is not required for the rise in calcium during fertilization.
(22/24)
The mechanisms required for cGMP-induced Ca2+ release in the sea urchin egg were investigated using both egg homogenates and intact eggs. The postulated pathway of cGMP-dependent protein kinase (PKG) activation of ADP-ribosyl cyclase for production of cADPR to activate the ryanodine receptor Ca2+ channel was tested with a variety of activators (cGMP analogs and cIMP) and inhibitors (Rp-8-pCPT-cGMPS, 3-aminopyridine NAD, nicotinamide, and spermine). Our observations are consistent with Ca2+ release by cGMP in the egg being dependent on an isoform of PKG that is distinct from the mammalian enzyme. PKG activity in the sea urchin egg was activated by cIMP, but was insensitive to cGMP analogs, which are potent activators of mammalian isoenzymes. Surprisingly, it appears the activation of the cGMP-dependent Ca2+ release pathway was unnecessary during fertilization. Inhibitors of either PKG or ADP-ribosyl cyclase activities did not prevent the transient rise in intracellular Ca2+ activity in heparin-loaded eggs during fertilization. These results suggest the synthesis of cADPR during fertilization is not necessary for regulating the Ca2+ event. (+info)
Identification of critical determinants for autoinhibition in the pseudosubstrate region of type I alpha cAMP-dependent protein kinase.
(23/24)
The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit. (+info)
Nitric oxide depresses GABAA receptor function via coactivation of cGMP-dependent kinase and phosphodiesterase.
(24/24)
Nitric oxide (NO) is thought to play an essential role in neuronal processing, but the downstream mechanisms of its action remain unclear. We report here that NO analogs reduce GABA-gated currents in cultured retinal amacrine cells via two distinct, but convergent, cGMP-dependent pathways. Either extracellular application of the NO-mimetic S-nitroso-N-acetyl-penicillamine (SNAP) or intracellular perfusion with cGMP depressed GABA currents. This depression was partially blocked by a pseudosubstrate peptide inhibitor of cGMP-dependent protein kinase (PKG), suggesting both PKG-dependent and independent actions of cGMP. cAMP-dependent protein kinase (PKA) is known to enhance retinal GABA responses. 8-Bromoinosine 3',5'-cyclic monophosphate (8Br-cIMP), which activates a type of cGMP-stimulated phosphodiesterase that hydrolyzes cAMP, also significantly reduced GABA currents. 1-Methyl-3-isobutylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, blocked both the action of 8Br-cIMP and the portion of SNAP-induced depression that was not blocked by PKG inhibition. Our results suggest that NO depresses retinal GABAA receptor function by simultaneously upregulating PKG and downregulating PKA. (+info)