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)

Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.
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PMID:Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity. 167 92

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2-transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.
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PMID:Yeast carbamoyl-phosphate-synthetase--aspartate-transcarbamylase multidomain protein is phosphorylated in vitro by cAMP-dependent protein kinase. 197 85

Phosphorylase phosphatase isolated from rabbit skeletal muscle can be activated in several ways. Trypsin-Mn2+ treatment of the purified Mr = 70,000 complex or addition of Mn2+ alone to the isolated inactive catalytic subunit gives enzyme species that readily dephosphorylate phosphorylase a and the type 2 regulatory subunit of cAMP-dependent protein kinase as well as synthetic phosphopeptides corresponding to the phosphorylation sites of these proteins. In contrast, enzyme activated by phosphorylation of the regulatory subunit using factor FA (glycogen synthase kinase 3) and Mg2+-ATP and thought to be of physiological significance dephosphorylates the protein substrates but not the phosphopeptides. Likewise, the active catalytic subunit isolated following FA treatment could not act on the peptides unless Mn2+ ions (maximal effect at 250 microM) were added. Mg2+ and Ca2+ could not substitute for Mn2+. Such differences in substrate specificity are not seen with p-nitrophenyl phosphate which is dephosphorylated by all forms of the phosphatase. The results suggest that the primary sequence surrounding the phosphorylation site of the substrate is not all that is necessary for recognition by the FA-activated form of the enzyme. They are interpreted in terms of constraints within the enzyme that are relaxed following exposure to Mn2+ or by the additional determinants present in larger protein substrates.
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PMID:Phosphorylase phosphatase. Comparison of active forms using peptide substrates. 282 55

Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.
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PMID:Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis. 349 Aug 79

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.
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PMID:A nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells. 377 74

Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylates serine residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 micrograms of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal insulin-stimulated S6 kinase has a Mr of approximately equal to 50,000-60,000; there is a variable amount of activity that sediments with a Mr of about 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2+ (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.05 mM), and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 micrograms/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate 13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-L1 cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of Mr 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. The data suggest that the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid, and Ca2+-independent proteolytic derivative nor the result of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.
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PMID:Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. 389 33

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.
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PMID:Purification and partial characterization of bovine heart phosphorylase kinase. 630 72