Second messengers and phosphoproteins最新文献

Cyclic nucleotide phosphodiesterase (PDE) isozymes isolated by DEAE-Sephacel or Mono-Q High Performance Liquid Chromatography from cardiac left ventricular tissue of normal subjects and patients with end-stage heart failure have been compared. With both separation techniques, four major peaks of PDE activity were evident in the soluble fractions; only one peak of activity was present in particulate fractions. The specific activity of the particulate PDE from myopathics was approximately 30-50% of that of normals while the specific activity of a soluble form of this PDE (peak IIIa) was reduced by 30% in myopathics. No differences in comparison of the other peaks of PDE activity were evident. The particulate PDE isozyme has a low Km for cAMP (0.27-0.29 microM), is inhibited by cGMP (60-80% at 1 microM), is sensitive to inhibition by submicromolar concentrations of CI-930 but not rolipram, and is competitively inhibited by milrinone (Kj = 0.3 microM). The first soluble peak of PDE activity hydrolyzes both cAMP and cGMP and is stimulated by calmodulin while cyclic AMP hydrolysis by peak II PDE is stimulated by cGMP. The other soluble peak III fractions (IIIa and IIIb) hydrolyze cAMP; peak IIIa is inhibited by cGMP or by CI-930 and milrinone, whereas peak IIIb is also inhibited by rolipram when the cardiotonic sensitive PDE is inhibited by CI-930. Thus, cardiotonic-sensitive, cGMP-inhibitable, low Km cAMP PDE is present in both the soluble and particulate fractions of human cardiac left ventricular muscle of hearts from normal and cardiomyopathic subjects while the rolipram-sensitive PDE is present in the soluble fraction. The major differences in PDE activity of myopathic relative to normal left ventricular tissue are a reduced specific activity and Vmax of particulate PDE and one of the soluble peak III PDEs.

Detailed studies of PGE2 binding to isolated rat adipocyte membranes revealed two different classes of binding sites, namely high affinity-low capacity binding sites and low affinity-high capacity binding sites. Addition of albumin or GTP to the incubation medium enhanced the specific binding of PGE2 by decreasing the dissociation constant of the low affinity-high capacity binding sites. Albumin also increased the affinity of PGE2 binding to native canine renal medullary membranes and enhanced the binding of PGE2 to prostaglandin receptors solubilized from these membranes. Pretreatment of the adipocyte membranes with the alkylating agent NEM completely abolished the enhancement of PGE2 binding by GTP, while the enhancement of PGE2 binding by albumin was only partially inhibited. The enhancement of PGE2 binding by GTP was shown to be dependent on the presence of Mg+2, while the albumin effect was independent of Mg+2. These results suggest that the affinity of the prostaglandin receptors is modulated by more than one mechanism.

We have examined the effects of sodium (Na+) salts on rat liver adenylate cyclase. Increasing concentrations of Na+ salts produced biphasic stimulation and inhibition of adenylate cyclase and potentiated enzyme activation by GTP and its hydrolysis resistant analog 5'-guanylyl imidodiphosphate. Salt effects were temperature dependent, of rapid onset, and specific for the Na+ cation though also partly dependent on the accompanying anion. Sodium salt stimulation of adenylate cyclase and enhancement of GTP activation were attenuated by agents (pertussis toxin and N-ethylmaleimide) which inactivate the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase. Cholera toxin, which activates the stimulatory guanine nucleotide-binding regulatory component (Gs) of adenylate cyclase and thereby increases enzyme activity, augmented the inhibitory phase of Na+ salt action. These results suggest that the stimulatory and inhibitory effects of Na+ salts may be due, respectively, to inhibition of Gi and Gs modulation of adenylate cyclase.

Two cyclic-nucleotide independent soluble casein kinase activities (CK I and CK II) from the fungus Mucor rouxii have been isolated, characterized and found to fit in the general classification of type 1 (CK I) and 2 (CK II) casein kinases, according to their enzymatic and structural properties. Both enzymes phosphorylate acidic substrates, require Mg2+ and have a chromatographic behaviour on DEAE-Sepharose and phosphocellulose similar to their mammalian counterparts. CK I has a sedimentation coefficient of 3.5 S, uses ATP as a phosphate donor (Km = 40 microM), phosphorylates casein mainly on serine residues, its activity is strongly inhibited by KCl and polyamines. CK II has a sedimentation coefficient of 7.4 S, uses ATP and GTP as phosphate donors (Km ATP = 10 microM; Km GTP = 40 microM), phosphorylates casein in serine and threonine, its activity is stimulated by KCl and by polyamines and is inhibited by heparin (I50 = 0.5 micrograms/ml). Casein kinase activity associated to particulate fraction (40% of total) has been partially characterized and shown to be similar to the soluble CK I activity.

The sensitivity of the radioimmunoassay for cyclic GMP has been improved to readily detect attomole (200-300) amounts of the nucleotide in tissue extracts. The improved sensitivity has been achieved by using a high specific radioactivity [125I] cyclic GMP 2'O-succinyl tyrosine methyl ester (2200 Ci/mmole) as labeled antigen in conjunction with acetylation of the cyclic GMP in the samples as well as by using a selected cyclic GMP antibody. The high specific [125I] cyclic GMP 2'O-succinyl tyrosine methyl ester was prepared radiochemically pure by radioiodination of cyclic GMP 2'O-succinyl tyrosine methyl ester and purification using HPLC. The new attomole sensitive cyclic GMP radioimmunoassay is simple to perform and fast and has been applied with success to determine attomole quantities of cyclic GMP in cultured smooth muscle cells and AG 1523 fibroblasts.

Extracts of aggregation-competent cells of Dictyostelium discoideum have an S6 protein kinase activity which is inhibited in the presence of the inhibitor of the cAMP-dependent protein kinase. The phosphorylation of S6 is rapid, and decays rapidly. The S6 kinase activity is detectable in the 150,000g supernatant only in the presence of phosphatase inhibitors known for preserving the S6 kinase in other systems, indicating that the activated form of the enzyme is phosphorylated by the cAMP-dependent protein kinase. S6 kinase elutes as a peak from DEAE-Sephacel at 100 mM NaC1, with an activity that is cAMP-dependent.

Platelets permeabilized by means of a high voltage electric field demonstrated time- and ATP-dependent uptake of 45Ca++. Submicromolar concentrations of inositol-1,4,5-trisphosphate (IP3) caused a rapid release of 45Ca++ which was followed by a slower reuptake. Adenosine 3':5'-cyclic monophosphate (cAMP) did not affect 45Ca++ uptake but did reduce IP3-mediated calcium release in a concentration-dependent manner over the range of 1-100 microM. Because cAMP concentrations in this range occur following exposure of platelets to prostacyclin and other agents which interfere with platelet function, it is proposed that cAMP-mediated inhibition of the action of IP3 may play a role in the antithrombotic activity of compounds believed to elevate levels of this cyclic nucleotide.

A fast and sensitive radioimmunoassay for 3':5'cyclic AMP based on a monoclonal antibody has been worked out. Mice were immunized with protein-conjugated 2'-O-succinyl-3':5'-cyclic AMP. The monoclonal antibody detects 0.1 and 1 pmole cAMP with succinyl cAMP (125I)iodotyrosine methyl ester and (3H) cAMP, respectively, as tracers. It shows no cross-reactivity to other adenosine nucleotides up to the millimolar range; cGMP interferes only if present at a 500 fold excess. Plant and animal tissue samples as well as adenylate cyclase activity were analysed directly or after appropriate purification in case of interfering substances. Cyclic AMP levels measured in various tissues by the antibody binding assay correspond to those obtained by HPLC determination using fluorescent etheno-cAMP.

Photolabelling with 32P-8-azido-cAMP identified a major cAMP-binding protein (54 kDa) in isolated rabbit renal apical membranes, whose labelling was competitively inhibited by cAMP. Membrane associated cAMP-binding polypeptides were extensively purified by affinity chromatography on cAMP-Sepharose. The 54 kDa polypeptide represented 70-80% of the total protein eluted with cAMP. This protein was rapidly phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, with a shift in its apparent mobility on SDS-PAGE to Mr 56/58,000. The phosphopeptide maps of autophosphorylated rat skeletal muscle RII and rabbit kidney 56/58 kDa proteins were essentially identical. Western immuno-blot analysis, using antibodies generated against purified rat RI and RII, indicated preferential cross-reactivity of rabbit kidney 54 kDa protein with anti-RII antibodies. The data demonstrates the specific association of the regulatory subunit of type II cAMP dependent protein kinase with rabbit renal brush border membranes.