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)

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70-80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I (Mr 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R2 (Mr 90,000) and a catalytic subunit C (Mr 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg2+ and ATP. The regulatory subunit, R2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R1 (Mr 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase (Mr 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II (Mr 88,000), is the predominatn form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protaimine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same Mr (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.
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PMID:Characterization of the protein kinases in a transplantable islet cell tumor of the Syrian hamster. 17 65

Studies on the gonadotrophin-responsive adenylyl cyclase (AC) system of rabbit and porcine ovarian follicles reveal that hCG or LH-induced desensitization of the AC system can be divided into two phases: an initial, LH-specific phase and a second phase which is not specific for LH. The first phase occurs within the first hour after LH-hCG-receptor interaction, is agonist specific, and is not mediated by protein synthetic events or by cAMP. In view of our previous demonstration of the critical dependence of the LH-induced desensitizing process in cell-free membrane preparations of porcine follicles upon Mg2+ and ATP, we investigated the role of a phosphorylation reaction in the first phase of the AC desensitizing process. Porcine follicular membranes rich in LH-sensitive AC activity were found to contain the molecular requirements necessary for a phosphorylation reaction: namely, cAMP-dependent and cAMP-independent protein kinases as well as phosphoprotein phosphatases. The following lines of indirect evidence indicated that reversal or resensitization of the desenzitized AC system to LH was mediated by a dephosphorylation reaction. Activators of endogenous phosphoprotein phosphatases--Mn2+ and dithiothreitol--promoted a specific resensitization of the follicular AC system to LH. Likewise, a partially purified phosphoprotein phosphatase also resensitized the desensitized, LH unresponsive AC to LH, and boiling of the phosphatase prevented its effect. LH-induced desensitization of the AC system, on the other hand, did not appear to be mediated by a cAMP-dependent protein kinase, as evidenced both by the inability of beef heart protein to promote desensitization of AC and by the inability of an inhibitor of cAMP-dependent protein kinase to prevent LH-induced densensitization. The second phase of desensitization, which occurs after the first hour following hCG-LH-receptor interaction, is characterized by a loss of responsiveness to FSH as well as to LH and can be promoted by dibutryl cAMP (in the absence of LH). These results provide new evidence on the characteristics and molecular mechanism of LH-induced densensitization of the follicular AC system. These results indicate that the level of phosphorylation of membrane-associated components may, in part, regulate the activity of the AC system during this first phase of homologous desensitization.
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PMID:LH-induced desensitization of the adenylyl cyclase system in ovarian follicles. 22 90

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.
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PMID:Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis. 217 26

Partial activation of Mucor rouxii cAMP-dependent protein kinase by cAMP was obtained when kemptide was used as substrate, but complete activation was attained with cAMP plus protamine or histone. Full activation could not be achieved by increasing kemptide or cAMP concentration. Complete activation by cAMP could be obtained by addition of 10 microM polylysine, 10 microM lysine-rich histone or 0.5 mM spermine plus spermidine. The degree of stimulation could be up to 5-fold, depending on the amount of enzyme in the assay. The same concentrations of polycations increased 1.5-2.3-fold the Vmax of kemptide phosphorylation by the free catalytic subunits of both Mucor and bovine heart protein kinases; 10 microM polyarginine inhibited completely the activity of both enzymes.
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PMID:Polyamines and basic proteins stimulate activation by cAMP and catalytic activity of Mucor rouxii cAMP-dependent protein kinase. 217 92

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.
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PMID:Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta. 225 32

Although the major form of soluble cAMP-dependent protein kinase in bovine cerebral cortex can be classified as a type II kinase, the regulatory subunit (RII) can be distinguished from RII found in other tissues such as heart. Heart and brain RII were distinguished qualitatively by autophosphorylation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mobility of dephosphorylated heart RII shifted from an apparent Mr of 55,000 to 57,000 following autophosphorylation. In contrast, when RII purified from brain was autophosphorylated with [gamma-32P]ATP, two radiolabeled bands were visualized, a minor band (less than or equal to 20%) which migrated with an Mr of 57,000 similar to the heart protein and a band with Mr = 55,000 which did not shift its mobility in response to autophosphorylation. Brain RII was further distinguished from heart RII on the basis of cAMP binding. Millipore filtration and equilibrium dialysis indicated that 2 mol of cAMP bound/mol of RII in contrast to 4 mol/mol with heart RII. Immunological differences were also apparent. Radioimmunoassays using monoclonal antibodies to RII showed that the brain protein had less than 4% of the cross-reactivity of heart RII. Both immunoblotting and immunoprecipitation using monoclonal as well as serum antibodies established that the cross-reactivity in phosphorylated brain RII was associated exclusively with the 57,000 component that behaved like heart RII. The lack of cross-reactivity of neural RII with two different monoclonal antibodies targeted the hinge region of RII as an area where structural differences might be anticipated, and comparative sequence analysis of this region definitively established that the major form of RII in brain is a unique gene product from the RII expressed in heart.
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PMID:The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product. 298 23

Both temperature-stable and temperature-labile testicular cholesteryl ester hydrolases are shown to be regulated by an endogenous cAMP-dependent protein kinase activity. The temperature-stable form (Mr = 28,000) was activated 3-fold by the endogenous kinase. This activation was completely blocked by protein kinase inhibitor. Following purification by high performance gel permeation chromatography, the temperature-stable form could also be activated 2-fold by bovine heart protein kinase, type I. The partially purified endogenous protein kinase, type I, which was completely separated from hydrolase activity by ion exchange chromatography, increased hydrolase activity 2-fold in the presence of optimal concentrations of cAMP, ATP, and Mg2+. Cholesteryl ester hydrolase activity could be stabilized indefinitely at -10 degrees C with the addition of 0.1 mM thioglycolate, but not by other thiol reagents. In contrast, the endogenous protein kinase activity was lost from 104,000 X g supernatants after 14 days. However, the property of activation could be restored by addition of bovine heart protein kinase. The temperature-labile hydrolase (Mr = 72,000) could be totally inactivated by a Mg2+-dependent, fluoride-sensitive cytosolic factor and reactivated by cAMP-dependent protein kinase. These observations strongly suggest that the inactivating factor is a phosphoprotein phosphatase.
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PMID:Protein kinase-mediated activation of temperature-labile and temperature-stable cholesteryl ester hydrolases in the rat testis. 308 16

The regulatory subunit of bovine heart isozyme II cAMP-dependent protein kinase contains two different intrachain cAMP binding sites which differ in their rates of cyclic nucleotide dissociation and specificity of cyclic nucleotide analogue binding. The dissociation of cAMP from intrachain Site 1 is slow compared with that from Site 2. Cyclic nucleotide analogues with C-8 alterations show a marked relative preference for Site 1, whereas N6-modified analogues select Site 2. Cyclic IMP, which prefers Site 2, activates the partially purified heart protein kinase holoenzyme as efficiently as, or more efficiently than, cAMP itself. Dissociation studies indicate that the binding of subsaturating or saturating concentrations of cAMP to isolated regulatory subunit occurs at both sites, whereas cAMP in low concentrations binds mainly to Site 1 of the holoenzyme and occupies both sites as the cAMP concentration is raised. Cyclic IMP binding to Site 2 is stimulated by the simultaneous binding of 8-Br-cAMP to Site 1, indicating that at least one function of Site 1 is cooperativity. It is concluded that Site 2 occupancy, either alone or in combination with Site 1 occupancy, is responsible for protein kinase activation. The catalytic subunit of the holoenzyme may prevent cAMP binding to Site 2, but this restraint is relieved as Site 1 becomes occupied.
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PMID:Studies on the function of the two different intrachain cyclic AMP binding sites of heart protein kinase. 630 88

A protein phosphatase from liver which acts preferentially on histone phosphorylated with phospholipid, Ca2+-dependent protein kinase has been purified and the intrinsic specificity determined to reside in the catalytic subunit of the enzyme complex. Comparison with a preparation of pork heart protein phosphatase suggests that this specificity may be a general property of a class of protein phosphatases. Protein kinase C-phosphorylated histone H1 represents an improved substrate for phosphatase detection providing a five to tenfold greater sensitivity than other substrates including cAMP-dependent protein kinase phosphorylated H1.
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PMID:Specificity of a phosphatase for phospholipid, Ca2+-dependent protein kinase-phosphorylated histone H1 resides in the catalytic subunit. 632 Aug 26

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.
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PMID:Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation. 893 82


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