Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.
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PMID:Changes in calmodulin level and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa. 299 10

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.
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PMID:Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase. 301 Nov 34

The effect of halothane on isoproterenol-stimulated lipolysis was determined in isolated rat epididymal fat cells. The maximal lipolytic response (Emax) activated by isoproterenol was 350 +/- 61 nmol of glycerol/10(5) cells/hr with an EC50 of 5.1 X 10(-9) M. When the adipocytes were simultaneously bubbled with 2.5% halothane, the Emax decreased to 158 +/- 43 nmol of glycerol/10(5) cells/hr and the dose response curve for isoproterenol was shifted to the right (EC50 3.5 X 10(-8) M, p less than 0.05). When lipolysis was maximally stimulated with (-)-isoproterenol (10(-6)M), the inhibitory effect of halothane was found to be both dose dependent (IC50 approximately 2.5%, v/v) and reversible following washout. Neither the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (2 X 10(-3)M), nor forskolin (10(-6) M) was able to normalize lipolysis in the presence of halothane. The activation of cAMP-dependent protein kinase (EC 2.7.1.37) activity by isoproterenol was not different in halothane-exposed cells when compared to unexposed cells. When control adipocytes were exposed to isoproterenol (10(-6) M), there was a 2.5-fold increase in the activity of hormone-sensitive lipase (EC 3.1.1.3) from 0.64 +/- 0.13 to 1.53 +/- 0.32 pkat (pmol/sec) per mg (p less than 0.005, n = 10). However, in the presence of halothane (2.5%, v/v) isoproterenol stimulation of hormone-sensitive lipase was attenuated by 50% to values of 1.06 +/- 0.23 pkat/mg (p less than 0.01, n = 10). Halothane had no direct inhibitory effect on hormone-sensitive lipase since this enzyme's activity was unaffected when homogenates of isoproterenol-stimulated control cells were incubated with halothane. These studies suggest that halothane impairs the activation of hormone-sensitive lipase by cAMP-dependent protein kinase and in this manner inhibits beta-adrenergic-stimulated lipolysis.
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PMID:Mechanism of halothane-induced inhibition of isoproterenol-stimulated lipolysis in isolated rat adipocytes. 335 97

The specific activity of the gamma-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717-720). The HPLC method also allowed the incorporation of 32P into the (alpha + beta)-positions of ATP to be determined. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [gamma-32P]ATP attained a steady-state value after 1-2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the beta-agonist isoprenaline increased this value by 5-10% within 15 min. Under these conditions the steady-state specific activity of [gamma-32P]ATP was 30-40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the gamma-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.
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PMID:Studies on the specific activity of [gamma-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate. 351 72

The changes in intracellular calmodulin levels and cAMP-dependent protein kinase activities have been studied in the testis of normally developing and hypophysectomized rats. It appears that the onset of spermatogenesis which occurs on the first days of the postnatal development, is associated with a major (over fivefold) increase in the calmodulin level and enhancement of the cAMP-dependent protein kinase activity. On the contrary, hypophysectomy of adult animals is associated with a progressive decline in the calmodulin level and a rapid and regular decrease in the cAMP-dependent protein kinase activity. Moreover, measurements of the intracellular calmodulin level and cAMP-dependent kinase activity of isolated testicular germ cells or epididymal spermatozoa have shown that testosterone, administered to hypophysectomized rats as subcutaneous implants, maintains the concentration of these regulatory proteins to normal values.
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PMID:Calmodulin level and cAMP-dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogenesis. 397 73

Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.
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PMID:Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa. 608 45

32P-labeled acetyl-CoA carboxylase was isolated from 32P-labeled rat epididymal fat pads by avidin-Sepharose affinity chromatography after exposure to epinephrine and insulin. Epinephrine led to an inactivation of the isolated enzyme by a reduction of Vmax, while the insulin stimulation observed in crude extracts did not survive enzyme purification. Both insulin and epinephrine caused only small increases in total 32P content of the enzyme. However, mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography revealed that epinephrine and insulin stimulated the phosphorylation of 32P-peptides specific for each hormone. The major 32P-peptide phosphorylated by epinephrine co-migrated with the major 32P-peptide phosphorylated in vitro by the cAMP-dependent protein kinase, while the 32P-peptide phosphorylated in response to insulin co-migrated with that phosphorylated by casein kinase-I and casein kinase-II. The effects of epinephrine on carboxylase activity and phosphorylation can thus be accounted for by the expected epinephrine-induced activation of the cAMP-dependent protein kinase. While the increase in site-specific phosphorylation caused by insulin cannot be directly linked to insulin-induced activation in crude extracts, these data suggest that casein kinase-I and/or casein kinase-II may mediate the insulin-stimulated phosphorylation of acetyl-CoA carboxylase.
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PMID:Stimulation of site-specific phosphorylation of acetyl coenzyme A carboxylase by insulin and epinephrine. 613 73

A putative cAMP-dependent protein kinase substrate associated with the cAMP stimulation of bovine sperm motility has been identified. Optimum conditions for a linear, concentration-dependent incorporation of [32P]ATP into phosphoproteins of an epididymal sperm sonicate by cAMP-dependent protein kinase are described. The motility state of the sperm was reduced by incubation at 37 degrees C and reactivated with theophylline. Endogenous levels of cAMP correlated with the motility state of the sperm. The phosphorylation state of phosphoproteins was frozen by addition of NaF (100 mM final concentration). After sonication and removal of endogenous nucleotides, 32P incorporation into phosphoprotein varied inversely with motility. The inverse relationship results from the procedure monitoring the capacity for incorporation of 32P into dephosphorylated cAMP-dependent protein kinase substrates. Over 70% of the cAMP-dependent label was in the soluble fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one cAMP-dependent phosphoprotein from the soluble fraction which had an inverse correlation with motility. This 55,000-dalton protein did not bind [3H]cholchicine (tubulin) or [3H]cAMP (REGULATORY SUBUNIT OF PROTEIN KINASE). These data indicate the existence of a cytosolic phosphorylated motility protein.
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PMID:A cAMP-dependent phosphorylated motility protein in bovine epididymal sperm. 624 3

The interrelationships among cAMP-dependent protein kinase activity, lipolysis, and cellular concentrations of cAMP were investigated in hamster epididymal adipose tissue. Isoproterenol, norepinephrine, and theophylline increased the protein kinase activity assayed in tissue extracts with no added cAMP, but not in the presence of added cyclic nucleotide. The maximum rate of lipolysis was associated with a nearly three-fold increase in cAMP levels and a protein kinase activity ratio of 0.8 (the ratio of activity assayed without cAMP to that assayed with cAMP). Rates of lipolysis less than maximum were associated with lesser degrees of protein kinase activity and lower levels of cAMP. The relatively pure alpha-adrenergic agent phenylephrine partially suppressed the isoproterenol-stimulated protein kinase activity, lipolysis, and cAMP levels. Conversely, the alpha-adrenergic blocking agent phentolamine increased the activity of protein kinase and cAMP levels in adipose tissues exposed to norepinephrine. These data are consistent with the primary role for cAMP and its dependent protein kinase in control of lipolysis in adipose tissue. Moreover, our data are consistent with the view that the antilipolytic action of alpha-adrenergic agents is mediated by a decrease in activity of protein kinase, caused by a decrease in cellular cAMP concentrations.
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PMID:Activation of adenosine 3',5'-monophosphate-dependent protein kinase and its relationship to cyclic AMP and lipolysis in hamster adipose tissue. 624 85

A protein in rat liver cytosol whose phosphorylation was regulated by hydrocortisone administration in vivo was tentatively identified as the regulatory subunit of a cAMP-dependent protein kinase. Evidence that this protein, whose phosphorylation was regulated by steroid and cyclic AMP, is the regulatory subunit of type-II cAMP-dependent protein kinase included: (a) co-purification of the steroid/cAMP-regulated protein and the regulatory subunit during DEAE-cellulose, Sepharose 4B, and hydroxylapatite column chromatography, (b) co-migration of the two proteins on dodecyl sulfate/polyacrylamide slab gels during the various steps of purification, (c) specific adsorption of the two proteins onto 8(6-aminohexylamino)-cAMP--Sepharose 4B, and (d) a similar pattern of distribution of the two proteins in various subcellular fractions prepared from rat liver homogenate. By each of these criteria, it was found that the steroid/cAMP-regulated protein present in rat liver cytosol behaved identically with the regulatory subunit of type-II cAMP-dependent protein kinase in that tissue. Results qualitatively similar to those obtained in the study of the effect of hydrocortisone on rat liver were also obtained in studies of the effects of other steroid hormones on other target tissues in the rat, including uterus (17 beta-estradiol), ventral prostate and seminal vesicle (testosterone), and epididymal fat pad (hydrocortisone). The tentative identification of the steroid/cAMP-regulated protein as the regulatory subunit of the type-II cAMP-dependent protein kinase in the cytosol of several tissues indicates that autophosphorylation of the regulatory subunit of type-II protein kinase may be regulated by the steroid hormones. The fact that three different classes of steroid hormones appear to affect the phosphorylation of the regulatory subunit of type-II cAMP-dependent protein kinase in their target tissues raises the possibility that this common biochemical action may play an important role in the mechanism of steroid hormone action. It is also possible that this effect of the steroid hormones may provide a molecular basis for some of the known physiological interactions of the steroid hormones with those hormones that act through using cAMP as a second messenger.
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PMID:Steroid hormones may regulate autophosphorylation of adenosine-3',5'-monophosphate-dependent protein kinase in target tissues. 626 19


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