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1.
C L Rutherford R D Taylor L T Frame R L Auck 《Biochemical and biophysical research communications》1982,108(3):1210-1220
A cyclic AMP-dependent protein kinase was found to appear during the time course of development of . No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography. 相似文献
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Cyclic AMP-dependent protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) in Dictyostelium discoideum was shown to be developmentally controlled. No activity was measured in vegetative cells, but activity increased rapidly during differentiation. A simple procedure for the isolation of the catalytic subunit of the kinase from aggregating cells is presented. The cycle AMP-dependent holoenzyme could be reconstituted by adding purified D. discoideum cyclic AMP-binding protein. Molecular weight, kinetic parameters, pH dependence and affinity for cyclic AMP were determined for th enzyme. Most properties are similar to those of cyclic AMP-dependent kinase from mammalian cells. 相似文献
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cAMP-dependent protein kinase from Dictyostelium discoideum 总被引:1,自引:0,他引:1
The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins. 相似文献
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Cells of Dictyostelium discoideum respond to their chemoattractants, cAMP and folate, with a rapid increase of the cellular cGMP content. The molecular mechanisms of cGMP action are not understood. Since in many biological systems cGMP-activated protein kinase is a prominent cGMP acceptor, we searched for such an enzyme in D. discoideum. By means of affinity chromatography on cGMP-Sepharose and other chromatographic procedures (DEAE-Trisacryl, CM-Trisacryl), we separated a novel protein kinase. This preparation did not show any regulation by cGMP and may represent an enzyme modified by proteolysis. In order to establish a rapid and efficient purification step, an antiserum against the kinase preparation was raised and coupled to Sepharose. Chromatography of the supernatant from a cell homogenate on this antibody matrix yielded a protein kinase that was activated 3-fold by cGMP. Half-maximal activation occurred at about 1 nM cGMP. Cyclic AMP at a 20-fold higher concentration also activated the protein kinase. On a Superose 6HR column the cGMP-activated protein kinase eluted in the same volume as enolase (Mr = 82,000). 相似文献
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M Chevalier J de Gunzburg M Veron 《Biochemical and biophysical research communications》1986,136(2):651-656
The purified regulatory (R) and catalytic (C) subunits of cAMP dependent protein kinase (cAK) from the primitive eukaryote Dictyostelium discoideum have been compared with the homologous proteins from bovine heart by SDS-PAGE followed by Western blotting using polyclonal antibodies. No cross-reaction could be demonstrated by this technique although the slime mold subunits share several functional properties with their mammalian counterparts and are able to form functional hybrid holoenzymes. 相似文献
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We have examined protein phosphatase activities that are present during the cellular differentiation of Dictyostelium. Utilizing differential centrifugation, ion exchange, gel filtration, and concanavalin A affinity chromatography we found a number of distinct protein phosphatase activities. Three peaks of soluble Kemptide phosphatase activity and a very broad and heterogeneous soluble histone phosphatase activity were resolved by anion exchange chromatography. Histone phosphatase was associated with the particulate fraction, while Kemptide phosphatase was not. The protein phosphatase activities were able to dephosphorylate sites that had been phosphorylated by the cyclic AMP-dependent protein kinase. Therefore it is possible that their function in vivo may be to oppose the action of the cAMP-dependent protein kinase. In addition several paranitrophenyl phosphate phosphatase activities are shown to be largely separable from the protein phosphatases. An apparent heat-stable inhibitor of histone phosphatase is shown to be artifactual in that instead of interacting with the enzyme it acts by complexing with histone. 相似文献
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A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme. 相似文献
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Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum 总被引:1,自引:0,他引:1
Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme. 相似文献
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Localization and levels of cyclic AMP during development of Dictyostelium discoideum 总被引:4,自引:0,他引:4
Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass. 相似文献
15.
Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum 总被引:10,自引:0,他引:10
P Klein A Theibert D Fontana P N Devreotes 《The Journal of biological chemistry》1985,260(3):1757-1764
We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression. 相似文献
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In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing. 相似文献
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During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum. 相似文献
18.
Kioumars A. Jelveh Rachel Zhande Roger W. Brownsey 《Journal of biological inorganic chemistry》2006,11(3):379-388
Vanadium salts influence the activities of a number of mammalian enzymes in vitro but the mechanisms by which low concentrations
of vanadium ameliorate the effects of diabetes in vivo remain poorly understood. The hypothesis that vanadium compounds act
by inhibiting protein tyrosine phosphatases has attracted most support. The studies described here further evaluate the possibility
that vanadyl sulfate trihydrate (VS) can also inhibit 3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase
(PKA). Using conventional assay conditions, VS inhibited PKA only at high concentrations (IC50>400 μM); however, PKA inhibition was seen at dramatically lower concentrations of VS (IC50<10 μM) when sequestration of vanadyl ions was minimized. Vanadyl appears to be the effective PKA inhibitor because sodium
orthovanadate did not inhibit PKA and inhibition by vanadyl was abolished by potential chelators such as ethylenediaminetetraacetic
acid or glycyl peptides. PKA inhibition by vanadyl appears to be mixed rather than strictly competitive or uncompetitive and
may replicate the inhibitory effects of high concentrations of Mg2+. The effect of vanadyl on PKA provides a possible explanation for the effects of vanadium salts on fat tissue lipolysis and
perhaps on other aspects of energy metabolism that are controlled by cAMP-dependent mechanisms. Considering the high degree
of conservation of the active sites of protein kinases, vanadyl may also influence other members of this large protein family.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
19.
H Ueda N Fukushima H Takagi 《Biochemical and biophysical research communications》1987,142(2):595-602
Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxypeptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5-8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 microM/min/mg protein, while the Km (28.2 microM) was unchanged. 相似文献
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