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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.
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PMID:N-myristoyl transferase assay using phosphocellulose paper binding. 172 48

The regulation by cAMP of cholesteryl ester hydrolysis and net depletion of cellular cholesteryl ester (cholesteryl ester clearance) in J774 murine macrophages was explored. Using Sandoz 58035 to selectively inhibit acyl CoA:cholesterol acyltransferase, we showed that the absolute rate of cholesteryl ester hydrolysis was stimulated 2-fold in J774 cells by the cAMP analogues 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate and dibutyryl-cAMP. The rate of hydrolysis was also stimulated by prostaglandin E1, by cholera toxin, and by a mixture of forskolin and isobutylmethylxanthine, but was not affected by epinephrine or dibutyryl-cGMP. These data demonstrate that cholesteryl ester hydrolysis in J774 cells can be stimulated by cAMP-dependent protein kinase. Cholesteryl ester clearance from J774 cells was achieved upon incubation with high density lipoproteins (HDL) plus CPT-cAMP but not with HDL alone. HDL-mediated cholesteryl ester clearance was dependent on the concentration of both HDL and CPT-cAMP. The data suggest that the defect responsible for the lack of HDL-mediated cholesteryl ester clearance in J774 cells involves a failure to modulate cAMP levels.
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PMID:cAMP stimulates cholesteryl ester clearance to high density lipoproteins in J7774 macrophages. 184 91

In isolated guinea pig parotid gland lobules the activities of the following enzymes were measured 30 sec after stimulation with either 2 X 10(-5) M isoproterenol or 10(-5) M carbachol: glycerol kinase (EC 2.7.1.30), glycerolphosphate acyltransferase (EC 2.3.1.15), lysophosphatidate acyltransferase (EC 2.3.1.51), phosphatidate phosphohydrolase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol kinase (EC 2.7.1.107), and CDP-diacylglycerol synthetase (EC 2.7.7.41). Lyso-phosphatidate acyltransferase, diacylglycerol kinase, and diacylglycerol acyltransferase exhibited significant increases following stimulation by both types of agonists. Stimulation of the activities of these three enzymes occurred also following in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase II. These effects could be reversed by incubation with various protein phosphatases. When cells were first stimulated with either type of agonist, subsequent incubation with protein kinases was almost ineffective. Activation by the two types of protein kinases was not additive, indicating that they activate by phosphorylating identical sites on the enzyme proteins. The other enzymes examined showed no or only minor changes and their activities could not be affected by in vitro incubation with the two types of protein kinases. The results explain the rapid changes in acyl-group transfer from acyl-CoA to neutral lipids observed previously during the first seconds after stimulation of guinea pig parotid gland lobules with isoproterenol or carbachol (1). An analysis of a potential role of lipocortins for the regulation of phosphoinositide-specific phospholipases C reveals that these proteins do indeed inhibit these enzymes, but that this inhibition results from a calcium-dependent interaction of the lipocortins with the phospholipid substrate. A physiological role of lipocortins for the regulation of phospholipases is doubtful.
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PMID:Mechanisms of short-term (second range) regulation of the activities of enzymes of lipid and phospholipid metabolism in secretory cells. 256 Mar 28

Incubation of Saccharomyces cerevisiae strain JR153 with either [3H]myristate or [3H]palmitate demonstrates the synthesis of proteins that contain covalently bound fatty acids. A unique set of proteins is labeled by each fatty acid. Detailed analysis of a 20-kDa protein labeled with myristic acid demonstrates that myristate is linked to the amino-terminal glycine. We describe an enzymatic activity in yeast that will transfer myristic acid to the amino terminus of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg, whose sequence was derived from a known N-myristoylated acyl protein, the catalytic subunit of cAMP-dependent protein kinase of bovine cardiac muscle. The acylation reaction is dependent on ATP and CoA, is enriched in a crude membrane fraction, and will use myristate but not palmitate as the acyl donor. Specificity of the glycyl peptide substrate is demonstrated by the observation that other glycyl peptides do not competitively inhibit myristoylation of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.
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PMID:Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide. 351 77

Homogeneous rat liver ATP-citrate lyase (EC 4.1.3.8) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. In agreement with other workers, the maximum level of phosphorylation that we observed was approx. 2 mol/mol of tetramer. Phosphorylated and non-phosphorylated forms of ATP-citrate lyase were prepared. Their kinetic properties were examined using an assay system in which the concentrations of Mg.ATP, magnesium.citrate and CoA were varied systematically at a constant concentration of Mg2+. The phosphorylated form had a two-fold higher Km for Mg.ATP than did the non-phosphorylated form, but no other kinetic differences between the two forms were detected. When ATP-citrate lyase was assayed at a concentration of Mg.ATP well below Km, it was found that phosphorylation of the enzyme correlated well with a decrease of approx. 50% in its activity. This is the first demonstration that phosphorylation can affect the activity of ATP-citrate lyase.
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PMID:Effects of phosphorylation on the kinetic properties of rat liver ATP-citrate lyase. 387 37

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.
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PMID:Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue. 614 54

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. 766 53

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT.II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (microliter) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence of myristoylated alanine-rich C-kinase substrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP71) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291:635-639, 1993) potently inhibited highly purified NMT activity (IC50 23.7 nM). A minor NMT activity (NMT.PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP71 inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP71 having an apparent higher affinity for NMT than the NMT.myristoyl.CoA complex. Inhibition by NIP71 at subsaturating concentrations of myristoyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.
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PMID:Mechanisms of action of NIP71 on N-myristoyltransferase activity. 789 74

Post-translational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl and nucleosides. Protein phosphorylation and dephosphorylation is one of the most prevalent and best understood modifications employed in cellular regulation. The bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPEDE) can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for Ca2+ and calmodulin (CaM). The phosphorylation of CaMPDE is blocked by Ca2+ and CaM and reversed by the CaM-dependent phosphatase (calcineurin). The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for CaM. Analysis of the complex regulatory properties of CaMPDE has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaMPDE play a key role in the signal coupling phenomenon. The high molecular weight calmodulin binding protein (HMWCaMBP) was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of HMWCBP was higher in the absence of Ca2+/CaM then in the presence of Ca2+/CaM and reversed by the CaM-dependent phosphatase. Recently, it has become apparent that the binding of myristate to proteins is also widespread in eukaryotic cells and viruses and certainly is of great importance to the correct functioning of an organism. Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the attachment of myristate to the amino-terminal glycine residue of various signal transduction proteins. Cardiac tissue express high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated. The subcellular localization of bovine cardiac muscle NMT indicated a majority of the activity was localized in cytoplasm. Under native conditions the enzyme exhibited an apparent molecular mass of 50 kDa. Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that particulate fraction may contain an inhibitory activity towards NMT. Research in our laboratory has been focusing on the covalent modification of proteins and regulation of various signal transduction proteins. This special review is designed to summarize some aspects of the current work on co- and post-translational modification of proteins in cardiac muscle.
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PMID:Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins. 940 55

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa. The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity. We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction. The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT. Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody. Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA. Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide. Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions. PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT. These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins. We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain. The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin. These observations indicate that calpains may regulate NMT activity.
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PMID:Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain. 963 10


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