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1.
Abstract: Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ~80 and ~60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ~7.5 ng/100 μl of suspension), incorporation of 32P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin >7.5 ng/100 μl caused progressive decrease in incorporation of 32P into many synaptosomal proteins; by a concentration of neurocatin of ~45 ng/100 μ/l, the level of 32P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ~7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times >2.0 min, showed progressive decrease in 32P incorporation. Removing extrasynaptosomal Ca2+ with EGTA attenuated the increased 32P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of 32P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane. 相似文献
2.
M.Olivia Pinkett Robert L. Perlman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1974,372(2):379-387
Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of 32P from [32P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The 32P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of 32P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining. 相似文献
3.
Horst Will Tatyana S. Levchenko Dmitri O. Levitsky Vladimir N. Smirnov Albert Wollenberger 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,543(2):175-193
Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA2+ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca2+.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [32P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate. 相似文献
4.
The incorporation of [-32P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca2+ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca2+-independent protein kinase. 相似文献
5.
A nervous system specific protein potentiates the biological effects of the calcium ionophore A23187 总被引:1,自引:0,他引:1
The present study was undertaken to determine whether the nervous system specific protein S-100, whose function is, so far, unknown, could be involved in the regulation of neurotransmitter release from central nerve endings. Our results indicate that the exogenous protein was, by itself, unable to alter the spontaneous and the depolarization-induced release of neurotransmitters from rat brain synaptosomes. However, nanomolar concentrations of S-100 potentiated the effects of the Ca2+ ionophore A23187 on the release of putative transmitter amino acids and catecholamines. The action of the S-100 protein seems to be related to its ability to promote, in combination with the ionophore, a higher influx of Ca2+ into synaptosomes than that elicited by the ionophore alone.We hypothesize that the role of the S-100 present in nerve ending membranes might be that of facilitating the function of an endogenous, voltage-dependent Ca2+ ionophore. 相似文献
6.
Orthovanadate stimulated the incorporation of 32P from [gamma-32P]ATP by Triton X-100-solubilized rat liver plasma membrane into endogenous, trichloroacetic acid-precipitable materials as well as added (Glu4:Tyr1) copolymers. Extraction of incubation mixture with chloroform-methanol-HCl revealed that the increase in 32P incorporation by vanadate was predominantly into endogenous phospholipids. [32P]Phosphatidylinositol 4-phosphate (PtdIns-4-P) was identified by thin-layer chromatography as the major phosphorylated product of vanadate stimulation, which also resulted in elevated 32P, predominantly in P-Tyr in endogenous membrane proteins. Vanadate effects on protein tyrosine and phosphatidylinositol phosphorylation were concomitant and exhibited similar sensitivity. These effects of vanadate were enhanced by the presence of either dithiothreitol or NAD(P)H. Phosphatidylinositol phosphorylation could also be stimulated by a substrate of and inhibited by a synthetic inhibitory copolymer of tyrosine kinase. These results suggest that vanadate, an oxygen radical producer, stimulates a tyrosine kinase-PtdIns kinase coupled system much like those described for a number of growth factors and oncogene encoded products. 相似文献
7.
Erela Gorin Thomas W. Honeyman H. Maurice Goodman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,451(2):491-498
The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [γ-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be accounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day × 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 μg/day × 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 μg of triiodothyronine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process. 相似文献
8.
Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes 总被引:2,自引:0,他引:2
Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [32P]phosphate incorporation into 32P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [32P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level. 相似文献
9.
B A Coughlin H D White D L Purich 《Biochemical and biophysical research communications》1980,92(1):89-94
Brain microtubule protein, prepared by two types of recycling methods, undergoes “flash” phosphorylation in the presence of [γ-32p]ATP through sequential action of protein kinase and phosphoprotein phosphatase present in microtubule protein. SDS electrophoretic analysis indicates that MAP1, tau protein, and tubulin are poorly phosphorylated, and MAP2 is the major site of phosphorylation. To improve [32P]phosphoprotein stability in the presence of the kinase/phosphatase cycle, 3′,5′-cyclicAMP, orthophosphate, or fluoride ion may be added. After separation from tubulin by phosphocellulose chromatography, the MAP fraction exhibits autophosphorylation. Finally, the maximal extent of autophosphorylation is observed with an ATP regenerating system using ADP, [32P]acetyl-P, and bacterial acetate kinase; this results in the incorporation of 3–4 phosphoryl groups per MAP2 subunit. 相似文献
10.
Eugene E. Quist 《Archives of biochemistry and biophysics》1982,219(1):58-64
Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm [γ-32P]ATP and MgCl2 results in incorporation of 32P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol 32P/mg/h, respectively. Incorporation of 32P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with 32P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of 32P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl2 and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways. 相似文献
11.
The S-100 protein family constitutes a subgroup of Ca2+-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a0, S-100a and S-100b, plus a number of structurally related proteins displaying 28–55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca2+-induced Ca2+ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca2+. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities. 相似文献
12.
John P. Hullihan John Eric Wilson Michael Williams 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,499(1):139-147
Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of 32P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?32P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study. 相似文献
13.
Stefano Ferrari Vittorio Moret Noris Siliprandi 《Molecular and cellular biochemistry》1990,97(1):9-16
Summary Incubation of rat liver mitochondria in the presence of either [32P] Pi or
32
y
-P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [32P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca2+, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [32P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed. 相似文献
14.
Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP-dependent protein kinase 总被引:4,自引:0,他引:4
J Zwiller M O Revel P Basset 《Biochemical and biophysical research communications》1981,101(4):1381-1387
Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [γ-32P]ATP, 32P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of 32P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of VM. These results suggest that the activity of guanylate cyclase may be regulated in vivo by phosphorylation. 相似文献
15.
Balwant S. Tuana Brian J. Murphy Christina Schwarzkopf 《Molecular and cellular biochemistry》1987,78(1):47-54
Summary Sarcolemmal vesicles prepared from rabbit heart muscle by differential and discontinuous sucrose density gradient centrifugation, exhibited high marker enzyme activities: Na+ + K+ ATPase 22 µMol Pi × mg–1 × h–1. Adenylate cyclase 500 pmole CAMP × mg–1 × min–1, calcium antagonist receptors 0.7 pmoles × mg–1. Calmodulin in the presence of calcium and -ATP32 stimulated rapidly and specifically the 32P incorporation into two membrane proteins of 54 and 44 kDa. Calmodulin stimulated the phosphorylation of the 44 kDa to a greater extent (17.9 pmol 32P × mg–1 protein) than the 54 kDa protein (1.3 pmoles 32P x mg–1 protein). Removal of endogenous calmodulin from the membrane by EGTA extraction resulted in a 2.5 fold increase in calmodulin dependent 32P incorporation into the two proteins in the presence of exogenous calmodulin. It is suggested that the calmodulin dependent protein kinase activity in heart sarcolemma may mediate the effects of calmodulin in the regulation of Ca2+ transport across the membrane. 相似文献
16.
Protein phosphokinase activity of rat liver nuclear membrane 总被引:3,自引:0,他引:3
The presence of protein phosphokinase activity in a purified nuclear-membrane preparation from adult rat liver was demonstrated by measuring the incorporation of 32P from γ-32P-ATP into endogenous nuclear-membrane proteins as well as into the exogenous protein substrates, dephosphophosvitin (DPV) and lysine-rich histone (LRH). The activity of this enzyme toward DPV was 60 times greater than that toward LRH. cAMP and cGMP did not appear to affect the phosphorylation of endogenous-membrane proteins. 相似文献
17.
James G. Dobson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,675(1):123-131
The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing 32P to partially label intracellular ATP. Subsequent addition of 10?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in 32P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in 32P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of 32P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on 32P incorporation, it prevented the isoproterenol-induced incorporation of 32P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in 32P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of 32P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the 32P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 103 and decreased the incorporation into a 30 · 103 dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility. 相似文献
18.
DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-32P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of 32P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system. 相似文献
19.
E Keyhani 《Biochemical and biophysical research communications》1980,93(3):919-926
The pore complex-lamina fraction obtained from nuclear envelope contains a protein phosphokinase activity capable of phosphorylating endogenous and exogenous protein substrates. Its specific activity in the presence of MgCl2 is approximately twice that of intact nuclear envelope. However, when MgCl2 is replaced by CoCl2 in the reaction mixture, a 7 to 12-fold increase in incorporation of 32P from γ-32P-ATP into protein substrate occurs. This appears not to be due to an effect of the divalent cation on the substrate, or to inhibition of a phosphoprotein phosphatase activity. Substitution of CuCl2, MnCl2, CaCl2, and ZnCl2 for MgCl2 results in a 20 to 30% decrease in incorporation of 32P. Cyclic AMP and cyclic GMP at 1 μM were without apparent effect. Approximately 40% of the total protein phosphokinase activity of the nuclear envelope is associated with the pore complex-lamina fraction. 相似文献
20.
Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype. 相似文献