Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.
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PMID:Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation. 630 4

3-Oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase ("NADH-5 alpha-reductase", EC 1.3.1.?) is rapidly inactivated in the presence of 17 beta-hydroxy-4-androsten-3-one (testosterone). This activation is prevented by increasing the phosphate concentration. When the enzyme assay is carried out in Tris-HCl, only a small activity (1.7 nmol X min-1 X mg-1) is observed which may be further decreased by addition of phosphatases. Addition of the phosphatase inhibitor dextran sulphate or ATP, Mg++ and c-AMP results in a significant increase of activity (228% and 273%, respectively) compared with the Tris-HCl control. Glycerol 2-phosphate and glycerol 3-phosphate have a stabilizing effect on 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase by decreasing the Km towards the substrate testosterone from 1.2 X 10(-5) mol/l to 3.3 X 10(-6) mol/l. V remains unchanged. Half maximal velocity (testosterone reduction) is achieved with 20 mumol/l glycerol 2-phosphate and glycerol 3-phosphate. Addition of c-AMP dependent protein kinase (EC 2.7.1.37) to a microsomal preparation pretreated with alkaline phosphatase (EC 3.1.3.1) results in a significant increase of 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase activity compared with the control.
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PMID:Rat liver 3-oxo-5 alpha-steroid delta 4-dehydrogenase. Modulation of enzyme activity by changes in phosphorylation state. 652 91

A new and rapid method of protein kinase assay is presented which is suitable for low-molecular-weight substrates, irrespective of their electrophoretic or chromatographic mobility. It depends on the phosphorylation of the substrates with [gamma-32P]ATP, hydrolysis of the pyrophosphate bonds by boiling in 1 N HCl, extraction of 32P with isobutanol-benzene, and measurement of the radioactivity of 32P-labeled phosphoesters in the water phase. The method is shown to be suitable for both tyrosine- and serine-phosphorylating protein kinases.
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PMID:A rapid assay for protein kinases phosphorylating small polypeptides and other substrates. 666 May 12

Both metabolic acidosis and phosphorus (Pi) deprivation have been shown to alter not only renal Pi metabolism independent of parathyroid hormone (PTH), but also the phosphaturic response to PTH. In the present studies, we examined the interaction between metabolic acidosis and Pi deprivation on renal handling of Pi in an animal model in which metabolic acidosis was superimposed on 3 days of dietary Pi deprivation. The effect of metabolic acidosis was evaluated after acute (for 3 h) and chronic (for 3 days) administration of HCl. In Pi-deprived and thyroparathyroidectomized rats, chronic acidosis increased both plasma Pi and the basal fractional excretion of Pi (FEPi). Furthermore, chronic acidosis partially restored the phosphaturic response to PTH, which was totally absent in nonacidotic Pi-deprived rats. These effects metabolic acidosis were not demonstrable in acutely acidotic animals. Determination of PTH-dependent cAMP generation and cAMP-dependent protein kinase activation revealed that neither of these parameters was altered by chronic metabolic acidosis in vivo. These results show that in Pi-deprived rats chronic metabolic acidosis induced by HCl administration further modifies the renal handling of Pi associated with Pi deprivation. Further, the renal interaction between acidosis and Pi deprivation is at a step (or steps) after cAMP generation and protein kinase activation.
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PMID:Effect of metabolic acidosis on renal response to parathyroid hormone in phosphorus-deprived rats. 678 36

The native form of ATP citrate lyase (2 mol of phosphate/tetramer) and the dephospho-ATP citrate lyase (phosphate-free) purified to homogeneity from rat liver, are phosphorylated by ATP and by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle. A total of 2 mol of phosphate/tetramer were incorporated into native enzyme, while with the dephospho form, 4 mol of phosphate were incorporated. The phosphopeptides resulting from trypsin treatment which were isolated from phosphorylated forms of both native enzyme and the dephospho enzyme were similar. The ATP citrate lyase, phosphorylated to an extent of 4 mol of phosphate/tetramer, has the same Vmax as the native enzyme (2 mol of phosphate/tetramer). Native ATP citrate lyase, trypsin-treated to remove the phosphopeptide, could not be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle, suggesting a common trypsin-sensitive specific phosphorylation site. The phosphorylation rate varied with pH in potassium phosphate, imidazole/HCl, and Tris/HCl buffers. Divalent cations were essential for the activity of the protein kinase. The apparent Km value for ATP was found to be 50 microM.
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PMID:Phosphorylation of dephospho-ATP citrate lyase by the catalytic subunit of cAMP-dependent protein kinase. 705 76

Separation of phosphorylated sarcoplasmic reticulum (SR) fragments by polyacrylamide gel disc electrophoresis in the presence of Na-DS revealed that the radioactivity is distributed in protein zones with molecular weights of 95,000 and 6000-8000. The phosphorylation of the protein with m. w. of 95,000 is Ca2+-dependent. The tryptic hydrolysis of the phosphorylated SR fragments from fast skeletal muscles results in a loss of radioactivity by 60-70%; phospholipase C from Clostridium welchii reduces the labelled phosphate content by 40-50%. The cAMP-dependent protein kinase inhibitor decreases the phosphorylation of both substrates. The substrate of phosphorylation with m. w. of 6000-8000 is not stained with Amidoschwartz 10B or Coumassie brilliant blue. Extraction by an acidified chlorophorm--methanol mixture results in a proteolipid with specific radioactivity exceeding that of the original preparation of phosphorylated SR membranes 3-4-fold. Thin-layer chromatography on Silufol plates and Silicagel KSK showed that the proteolipid is not chromatographically homogeneous after 2-fold precipitation by diethyl ether and is localized in a band with Rf varying from 0.6 to 0.8. The fluorescence spectrum of the proteolipid in a chlorophorm--methanol--HCl solution is represented by an assymmetrical structure-free band with a maximum at 350 nm. A possible role of phosphorylase b and proteolipid in manifestation of the functional activity of the SR fragments is discussed.
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PMID:[Characterization of endogenous phosphorylation substrates of sarcoplasmic reticulum fragments from fast skeletal muscles of the rabbit]. 711 8

A cyclic AMP-binding protein of human erythrocyte membranes was solubilized with 0.3% Triton X-100 in 10 mM Tris-HCl (pH 7.4) at 4 degrees C, and purified by DEAE-cellulose column chromatography and affinity chromatography on a cyclic AMP derivative-fixed Sepharose 4B column. The purified cyclic AMP-binding protein showed a single band (molecular weight: 49,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the band was specifically labeled with a photoaffinity analogue of cyclic AMP, 8-N3-cyclic [2-3H]AMP. This protein bound 1.6 mol of cyclic AMP per molecule with an association constant of 3.8 x 10(9) M-1 and the optimum pH for binding was 7.4. The protein inhibited the activity of a purified protein kinase from human erythrocyte membranes [Suzuki, K., Terao, T., & Osawa, T. (1981) J. Biochem. 89, 1--11], while cyclic AMP restored the enzymic activity. The amino acid composition of this protein was different from those of cytoplasmic cyclic AMP-binding proteins. These observations indicate that the cyclic AMP-binding protein purified in this work is the regulatory subunit of a membrane bound cyclic AMP-dependent protein kinase.
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PMID:Purification and some properties of a cyclic AMP-binding protein from human erythrocyte membranes. 714 23

Rat liver nuclear protein kinase NII, which is independent of cyclic nucleotides, phosphorylated both the acidic protein casein and the basic histone preparation, soluble in 0.25 M HCl, which was extracted from the cell nuclei. When the isolated nuclei were incubated with [32P]ATP in the presence of protein kinase NII, more than 95% of the 32P-labelled proteins was recovered in the 0.25 M HCl extract. In order to analyze the substrate proteins of protein kinase NII, a histone preparation free from the endogenous protein kinase activities was used as substrate. The results demonstrated that histones were not phosphorylated, except for a faint 32P incorporation into the H3 fraction. Instead, multiple non-histone proteins were highly phosphorylated, which were present in a minor quantity in the histone preparation. The molecular weights of the main phosphorylated proteins were 72000, 68000, 56000, 47000, 46000, 43000, 38000 and 32000. Isoelectric focusing demonstrated the basic nature of the phosphorylated proteins, and as much as 72% of 32P radioactivity was distributed in the pH region higher than 7.0. Furthermore, many proteins phosphorylated in the isolated nuclei with added protein kinase NII were also found to be basic non-histone proteins. These results indicate that protein kinase NII preferentially phosphorylates in vitro a set of nuclear basic non-histone proteins.
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PMID:Preferential phosphorylation of basic non-histone proteins by nuclear protein kinase NII from rat liver. 740 86

1. A study has been made of the behaviour of the radiochloride efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. In the majority of the fibres studied, the fractional rate constant for 36Cl efflux is a constant and unaffected by the injection of distilled water (approximately 0 . 3 microliter. in volume). 3. Acidification of the HCO3(-)-containing medium causes stimulation of the Cl efflux, the threshold value being pH 7 . 0. The magnitude of the response is a logarithmic function of the external H+ and HCO3 concentration over a wide concentration range. 4. (i) Total replacement of the external Cl and NO3 fails to alter the course of the Cl efflux. However, the magnitude of the response to acidification is reduced to a marked degree. (ii) Replacement of the external Na by Li reduces not only the Cl efflux but also the size of the response to acidification. 5. Injection of HCl, HCO3 or KCl fails to alter the Cl efflux. Injection, however, of 4 M-KCl or NaCl causes a fall in the efflux. 6. 10( 4)M-ouabain is ineffective. It also fails to alter the response of the Cl efflux to acidification. 7. (i) Injection of cyclic AMP stimulates the Cl efflux in a dose-dependent manner, but only transitorily. (ii) Preinjection of pure protein kinase inhibitor causes a marked reduction in the magnitude of the response to cyclic AMP. 8. Preinjection of pure protein kinase inhibitor fails to affect the response to external acidification. 9. (i) Pretreatment externally with ethacrynic acid reduces the response to external acidification. (ii) External application of 4-acetoamineo-4'-isothiocyano-2,2'-stilbene disulphonate (SITS) reduces the resting Cl efflux. It also abolishes completely the response to acidification. (iii) The effect of 4,4'-diisothiocyano-2,2'-stilbene disulphonate (DIDS) resembles that of SITS. (iv) Injection of H2DIDS fails to reduce the resting efflux but tends to reduce the magnitude of the response to acidification. 10. (i) 5 X 10(-4) M-benzolamide is without effect on the basal Cl efflux. (ii) Benzolamide in high concentration reduces the magnitude of the response to acidification. This occurs within a rather narrow concentration range. 11. (i) A sudden reduction in environmental temperature from 24 to 0 degrees C causes a marked fall in the Cl efflux. (ii) Acidification of the artificial sea water at 0 degrees C stimulates the efflux. 12. The present experiments have led to evidence which is consistent with the view that the Cl efflux is modulated by at least two distinct mechanisms: one is responsive to acidification when HCO3 as buffer is present and involves participation of a benzolamide-sensitive component presumably lying in the fibre membrane. The other is responsive to injection of cyclic AMP, and, and probably involves cyclic AMP-protein kinase.
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PMID:Chloride efflux in single barnacle muscle fibres. 741 34

Previous studies have shown that protein kinase stimulators can induce the release of the metaphase II arrest in mouse ova. The present report is about the role of protein kinase in parthenogenic activation of pig oocytes, which was studied using a broad-spectrum protein kinase inhibitor. Metaphase II oocytes were obtained via in vitro maturation. Two sources of H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl] were tested--Sigma (H7s) and Calbiochem (H7c). Both were found to be equally effective in promoting release of the metaphase II block. Activation, release of the metaphase II arrest, and progression to the first interphase could be induced at the highest percentage with an exposure to 2.0 mM H7s for 80 min (68.1%). In another experiment, H7c resulted in 69.5% activation, while iso-H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine, HCl] at a similar concentration and exposure duration resulted in 25.7% activation. H7s and H7c were more effective than iso-H7 in inducing the appearance of a 22-kDa protein that is associated with normal fertilization in the pig. In contrast, although pronuclei could form and the protein profiles were indicative of activation, cortical granule exocytosis did not occur, and oocytes failed to develop to the blastocyst stage after H7 treatment. In contrast to the H7-treated oocytes, electrostimulation resulted in pronuclear formation, the appearance of the 22-kDa protein, release of cortical granules, and development to the blastocyst stage. These data demonstrate that broad-spectrum inhibitors of protein kinase are unable to induce all the events associated with normal fertilization.
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PMID:Parthenogenic activation of pig oocytes by protein kinase inhibition. 749 78


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