Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Troponin inhibitory factor, TNI, was prepared by affinity chromatography from different mammalian hearts. (i) Structure. These different TNI have the same M.W. (28000), which is higher than that found in rabbit skeletal muscle (23000). Nevertheless they differ with respect of their charge as shown by alkaline urea polyacrylamide gel electrophoresis using cardiac TNI which has previously been bound to an excess of skeletal troponin Ca2+-binding factor. These changes do not correlate with the PO4 content of TNI. They are associated with structural differences demonstrated by peptide mapping of the unfolded molecule after papain treatment. The structure of cardiac TNI from rat and rabbit differs clearly from that of crow and pig. (ii) Biological activity. These different TNI have the same inhibitory effect on skeletal actomyosin. ATPase, the same content of PO4 and the same ability to be phosphorylated in-vitro by a bovine heart c-AMP-dependent protein kinase.
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PMID:A comparative study of the cardiac troponin inhibitory factor (TNI) from mammalians. 20 99

The effect of the proteases trypsin, thermolysin and papain on the cardiac membrane protein phospholamban was examined before or after phosphorylating the protein with the catalytic subunit of cyclic AMP-dependent protein kinase. The sensitivity of phospholamban to digestion by trypsin and thermolysin was greatly reduced by phosphorylation, suggesting that phospholamban undergoes a conformational change upon phosphorylation. It is suggested that this change in conformation is the mechanism by which phospholamban phosphorylation relieves its inhibition of the sarcoplasmic reticulum Ca2+-ATPase pump.
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PMID:Evidence for a phosphorylation-induced conformational change in phospholamban from the effects of three proteases. 295 52

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.
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PMID:Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. 300 93

The major cAMP-binding proteins isolated from [35S]methionine-labeled S49 mouse lymphoma cells or MDBK bovine kidney cells correspond in isoelectric point and apparent molecular weight to the regulatory subunit (R) of type I cAMP-dependent protein kinase. These proteins were compared directly by two-dimensional gel electrophoresis and by two-dimensional gel electrophoresis of peptides generated either from native R with thermolysin and chymotrypsin or from denatured R with papain. Both the undigested proteins and all their major peptides were identical in charge and apparent molecular weights, indicating a very high degree of structural homology.
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PMID:Homology between regulatory subunits of type 1 cyclic AMP-dependent protein kinases from bovine and murine cells. 609 1

A membranal proteinase from brush-border epithelial cells of the rat small intestine was shown to bring about a restricted and limited degradation of the free catalytic subunit (C) of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) with concomitant inactivation of the kinase. This membranal proteinase exhibits a remarkable specificity. (i) It degrades C in its native conformation, but not after it has been heat-denatured. (ii) The degradation of C (Mr 40,000) does not proceed further, once a distinct clipped product (Mr 34,000) is formed. (iii) The undissociated ("stored") form of the enzyme (R2C2) is not attacked by the membranal proteinase, preserving both its potential catalytic activity and its molecular integrity. Only upon addition of cyclic AMP to release free C does the proteinase attack it. (iv) The membranal proteinase does not degrade the regulatory subunit (R), released by cyclic AMP from R2C2, although R is quite susceptible to degradation by other proteolytic enzymes. None of these features of the membranal proteinase could be reproduced with trypsin, chymotrypsin, clostripain, or papain. The specific, restricted, and limited action of this membranal enzyme raises the possibility that it may have a distinct physiological assignment associated with the bioregulation of cyclic AMP-dependent protein kinase.
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PMID:Degradative inactivation of cyclic AMP-dependent protein kinase by a membranal proteinase is restricted to the free catalytic subunit in its native conformation. 626 95

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.
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PMID:Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin. 629 73

Methods for mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase are described with a view to using such data for fine-structure analysis of mutations and/or modifications affecting the protein's electrostatic charge. Peptides generated from [35S]methionine-labeled regulatory subunit were separated by high-resolution two-dimensional gel electrophoresis. Sites of papain cleavage in denatured regulatory subunit were deduced from the kinetics of the appearance, molecular weights, and relative isoelectric points of the fragments produced. These sites and sites of chymotrypsin digestion in the native protein were confirmed by studying peptide overlaps. Carboxy-terminal peptides were identified both by overlaps with cyclic AMP-binding chymotryptic fragments and by their preferential labeling during polysome runoff mediated by pactamycin, an inhibitor of protein initiation. Since peptides containing modifications or mutations that alter protein charge can be identified by shifts in first-dimension isoelectric focusing gel positions, knowledge of fragment endpoints will permit rapid mapping of sites of such alterations by two-dimensional gel analysis of partial proteolytic digests. Such a mapping procedure is inexpensive, can be applied to partially purified proteins or to proteins eluted from polyacrylamide gels, requires only nanogram amounts of the protein of interest, and does not require sequence data to determine relative positions of peptides. Therefore, it provides an attractive alternative to more classical peptide analysis for studying point mutations in cellular proteins of low abundance.
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PMID:Mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase. 649 30

Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.
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PMID:Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments. 657 36

A preparation procedure has been worked out to obtain a highly purified G type (using GTP as well as ATP) casein kinase from large quantities of bovine lung tissue. It included ion-exchange (DEAE and phosphocellulose) and affinity (casein and ATP-Sepharose) chromatography combined with a flocculation step, and yielded an apparently homogeneous preparation with a 16% yield and a purification factor of more than 1400. The purified lung casein kinase used GTP (Km 16 microM) almost as well as ATP (Km 6.7 microM) and exhibited the major catalytic properties of the casein kinase G previously described in bovine adrenal cortex (Cochet, C., Job, D., Pirollet, F. and Chambaz, E.M. (1981) Biochim. Biophys. Acta 658, 191-201). Mg2+ (30-50 mM) and spermine (2 mM) were potent activators of lung casein kinase G activity, whereas the enzyme was inhibited by heparin and quercetin. The purified enzyme underwent self-phosphorylation in the presence of ATP or GTP, serine being the only target amino acid under these conditions, whereas both serine and threonine were phosphorylated by the enzyme in casein. Lung casein kinase G exhibited an apparent molecular weight between 140 000-160 000 upon gel filtration and appeared formed by the association of two different subunits upon SDS-polyacrylamide gel electrophoresis. The two subunits of Mr 38 000 (alpha) and 27 000 (beta) exhibited a 2:1 ratio upon quantitative scanning, suggesting an alpha 3 beta 2 combination in the oligomeric native enzyme structure. Peptide mapping of the two isolated subunits following 125I-labeling and papain digestion did not disclose any common fragment. The casein kinase catalytic activity was found associated with the alpha (38 kDa) enzyme subunit after recovery from gel electrophoresis in the presence of SDS, whereas the 27 kDa (beta) subunit was the major target of the enzyme self-phosphorylation reaction. alpha and beta subunits appeared strongly associated in the oligomeric enzyme and the possible role of the beta subunit in the casein kinase G activity remains to be examined. The purified casein kinase G, which can be obtained by the present procedure, should facilitate the study of the biological significance of this phosphorylation system in the intact cell.
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PMID:Catalytic and molecular properties of a highly purified G type casein kinase from bovine lung tissue. 657 70

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.
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PMID:Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase. 673 32


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