Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein kinase which phosphorylates and inactivates acetyl-CoA carboxylase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The purification of the kinase involved first partial purification of acetyl-CoA carboxylase through polyethylene glycol precipitation and DEAE-cellulose chromatography. The kinase was then separated from acetyl-CoA carboxylase by Sepharose 2B chromatography. The molecular weight of the kinase subunit was 170,000 as determined by sodium dodecyl sulfate-gel electrophoresis. The incorporation of 1 mol of phosphate/mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The Km values of the kinase for acetyl-CoA carboxylase and ATP are 90 nM and 20 microM, respectively. The kinase was found to be cyclic AMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase, protamine, and histones, but could not act on hydroxymethylglutaryl-CoA reductase or phosphorylase b.
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PMID:Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase. 612 Jan 70

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.
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PMID:Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase). 613 97

Rabbit brain phosphofructokinase was purified to homogeneity by a rapid procedure involving affinity chromatography and gel filtration. The enzyme consists of hybrids of the three phosphofructokinase subunit types C, A, and B. The molecular weights of these subunits are 86,000, 84,000, and 80,000, respectively; they are present in brain phosphofructokinase in a ratio of approximately 5:4:1.5. The enzyme as isolated from rabbit brain contains 0.16-0.18 mol phosphate per mole of subunit; another 0.4-0.5 mol phosphate per mole subunit can be incorporated in vitro in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase. The initial rate of phosphorylation is increased by fructose 2,6-bisphosphate or AMP and decreased by citrate or high concentrations of ammonium sulfate. All three subunit types are phosphorylated in vitro, and the phosphorylation site on each subunit is sensitive to cleavage by trypsin at a terminal region of each subunit. However, these sites show different relative rates of phosphorylation in vitro in the presence of ammonium sulfate. In vitro phosphorylation of brain phosphofructokinase had no affect on specific activity, inhibition by ATP, or activation by fructose 2,6-bisphosphate.
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PMID:Isozyme composition and phosphorylation of brain phosphofructokinase. 623 51

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.
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PMID:Calmodulin triggers the resumption of meiosis in amphibian oocytes. 626 65

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.
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PMID:Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase. 626 16

Relaxation of smooth muscle cells induced by activation of beta-adrenoceptors was investigated in intact and skinned muscles of the guinea-pig mesenteric artery.1. In concentrations over 10(-7) M, isoprenaline reduced the resting tone of intact preparations and also the amplitude of K contractions. When Ca was applied after previous superfusion with Ca-free solution, the amount of Ca accumulated into storage sites was increased by isoprenaline in polarized and depolarized ([K](o) 128 mM) muscles. The amount of Ca stored increased even further when procaine and isoprenaline were applied simultaneously during store loading.2. Isoprenaline increased the concentration of cyclic AMP as determined by radioimmunoassay. Application of isoprenaline at a concentration of 10(-7) M increased cyclic AMP from 2.2+/-0.3 to 2.8+/-0.6 p-mole/mg wet weight and at 10(-6) M increased it to 4.5+/-0.8 p-mole/mg wet weight after 5 min incubation (n = 4).3. Application of cyclic AMP (3 x 10(-6) M) with cyclic AMP-dependent protein kinase (50 mug/ml.) had no effect on the pCa-tension relationship in the skinned muscles. However, an increased concentration of cyclic AMP (> 10(-5) M) suppressed the Ca-induced concentration only in the presence of protein kinase. This protein kinase (50 mug/ml.) alone had no effect on the Ca-induced contraction.4. In skinned fibres, the Ca store could be loaded by applying low concentrations of Ca. If cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was applied during the loading procedure, the amount of Ca accumulated by the store increased if the loading solution contained 10(-6) M-Ca applied for 2 min or less, but if the loading solution was applied for 3 min, or if higher Ca concentrations were used, the presence of cyclic AMP with protein kinase decreased the store size, suggesting that a Ca-induced Ca-release mechanism was also being activated.5. In skinned muscles, accumulation of Ca into the store site in the presence of cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was further accelerated by simultaneous applications of procaine (5 mM), as here the Ca-induced Ca-release mechanism was suppressed.6. These results indicate that activation of beta-adrenoceptors by isoprenaline increases the amount of cyclic AMP in the intact muscles, and leads to an increase in Ca accumulation into the store site. In the skinned muscles, the Ca-induced Ca-release mechanism is activated by cyclic AMP and the Ca receptor for contraction (leiotonin C or calmodulin) is somewhat suppressed. These effects of exogenously applied cyclic AMP require the presence of protein kinase. The relaxation following beta-adrenoceptor activation is more likely to involve Ca extrusion from the cell and accumulation of Ca in internal storage sites than suppression of the binding of calmodulin with the myosin light chain kinase.
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PMID:Mechanisms of relaxation induced by activation of beta-adrenoceptors in smooth muscle cells of the guinea-pig mesenteric artery. 628 50

The cAMP-dependent protein kinases comprise two enzyme forms designated as type I and type II. The type II enzyme can catalyze an autophosphorylation reaction whereby phosphate is transferred from ATP to one seryl residue on each regulatory subunit monomer. Since this reaction can occur in the absence of cAMP-induced enzyme dissociation, it has been used as a probe to identify one site of interaction between the catalytic subunit (C) and the type II regulatory subunit (R11). The type I cAMP-dependent protein kinase does not catalyze an analogous reaction; however, if cGMP-dependent protein kinase is substituted for C, the type I regulatory subunit (R1) becomes phosphorylated. The effects of cyclic nucleotides on this reaction, coupled with the ability of R1 to serve as an inhibitor of cGMP-dependent protein kinase suggest that this phosphorylation also occurs within an important functional domain on R1. A comparison of the autophosphorylation site on R11 with the cGMP-dependent protein kinase catalyzed phosphorylation site on R1 indicates that each modification takes place within a similar proteolytically sensitive region. On each subunit, this sensitive "hinge" region lies distal to the functional domain responsible for regulatory subunit dimerization and proximal to that responsible for cAMP binding. Phosphorylation of the "hinge" region decreases the affinity of each regulatory subunit for C, although the magnitude of this change appears greater for R1 than for R11. Phosphorylation of R1 also reduces the stoichiometry of cAMP binding from two to one mole of cAMP bound per mole of R1 monomer. These results suggest that the "hinge" regions of both R1 and R11 form part of the interaction site between the regulatory subunit and C; and, in the case of R1, it also forms a portion of one of two cAMP-binding sites. The amino acid sequence surrounding the phosphorylated serine of each regulatory subunit has been determined: R11: D-R-R-V-S(P)-V R1: R-R-R-R-G-A-I-S(P)-A It is thought that the number and position of the basic amino acid residues proximal to the modified serine may be responsible, in part, for determining the susceptibility of each site to phosphorylation by cAMP or cGMP-dependent protein kinase. Both R1 and R11 exist as phosphoproteins in vivo. Dephosphorylation of purified "native" phospho-R1 is without effect on the ability of R1 to interact with either C or cAMP. The site phosphorylated in vivo is therefore distinct from that modified in vitro by cGMP-dependent protein kinase. In addition to the autophosphorylation site, R11 possesses a second, less enzymatically reactive, phosphorylation site that is modified in vivo. Dephosphorylation of this site is also without apparent effect on the functional properties of R11. The kinases responsible for catalyzing the phosphorylation of R1 and the cryptic site on R11 and the role that these modifications play in modulating kinase activity are currently unknown but are under active investigation.
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PMID:Phosphorylation of cAMP-dependent protein kinase subunits. 628 16

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.
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PMID:Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex. 628 91

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.
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PMID:Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus. 629 Aug 71

Two isoforms (I and II) of soluble cAMP-dependent protein kinase with basal activity of 2.1 and 10.87 nmole of 32P/min/mg of protein, respectively, were detected in rabbit myometrium at functional rest. cAMP (5 microM) activates 1.5-fold both isoforms of the enzyme. The apparent Km values for ATP of isoforms I and II is 0.9 X 10(-5) M and 2.1 X 10(-5) M, respectively; Km for histone H1 are 0.15 and 0.29 mg/ml, respectively. The pH optimum for both isoforms lies at 7.3-7.6; the pI values are 5.0 and 5.5, respectively. Na-DS electrophoresis in polyacrylamide gel demonstrated that the molecular weight of the regulatory subunit (R) of isoform I is 47000, that of the catalytic subunit (C) is 31000. No difference in the electrophoretic mobility of C for forms I and II were found. The molecular weight of R II is 54000. Isoform II reveals the ability for autophosphorylation. The plot for the dependence of the reaction rate versus enzyme concentration is linear; up to 1.5 mole of 32P per mole of the holoenzyme is incorporated. The myometrium of pregnant rabbits contains one isoform of cAMP-dependent protein kinase which is identical to isoform II in terms of its elution profile on DEAE-cellulose, molecular weight of R, pI and the ability for autophosphorylation. The optimal conditions for the pregnant rabbit myometrium enzyme activity are as follows: pH 7.0-9.0, cAMP--10(-8) M, basal activity--3.68 nmole of 32P/min/mg of protein, cAMP activation--2.4-fold. The values of apparent Km for ATP and histone H1 are 5.6 X 10(-5) M and 0.42 mg/ml, respectively. During autophosphorylation 0.4 mole of 32P per mole of the holoenzyme is incorporated.
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PMID:[Soluble cAMP-dependent protein kinases from the rabbit myometrium]. 630 99


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