Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver

Sugden, Peter H.; Holladay, Leslie A.; Reimann, Erwin M.; Corbin, Jackie D.

doi:10.1042/bj1590409

Cited by 298 publications

(109 citation statements)

References 41 publications

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“…On the basis of electrofocussing experiments, several investigators have reported that preparations of the bovine catalytic subunit contain at least three isozymes with identical substrate specificities (9,28,29). However, no primary structure heterogeneity was detected in the present study of the isozyme mixture.…”

Section: Resultscontrasting

confidence: 50%

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

Shoji¹,

Parmelee²,

Wade³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

164

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The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic isolated from protein radiolabeled in vitro by ['4C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in ar-helices and 12% in a-strands.Cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) are widely distributed and play a central role in the regulation of energy metabolism and other physiological functions (1-5). In eukaryotic systems, the actions of cyclic AMP are mediated exclusively through the activation of this protein kinase. Two forms of the enzyme with slightly different regulatory properties have been isolated (6, 7): type I, found predominantly in rabbit skeletal muscle, and type II, found in bovine cardiac muscle. Both types consist of two regulatory (R) and two catalytic (C) subunits. Differences between the two are attributed to differences in the structure of the regulatory subunits; the catalytic subunits are said to be identical (8). In both types, cyclic AMP acts as a positive effector resulting in dissociation of the oligomeric protein and activation of the enzyme according to the following scheme: R2C2 (inactive) + 4 cyclic AMP -± (R-cyclic AMP2)2 + 2 C (active). As a first step toward elucidating the primary structure of the enzyme, Peters et al. (9) developed a large-scale preparation of the catalytic subunit from bovine cardiac muscle and determined its molecular weight, isoelectric point, amino acid composition, and phosphate content and the reactivity of its thiol groups. We recently described the amino acid sequence around the two sites that are phosphorylated (10). In the present communication, the sequence of the 349 amino acid residues in the catalytic subunit is presented and discussed. The experimental details of the sequence determination will be published elsewhere. METHODSThe catalytic subunit of cyclic AMP-dependent protein kinase was prepared from bovine heart as described (9). TPCK-Trypsin, pepsin, and carboxypeptidase A were purchased from Worthington. Myxobacter AL-I protease II (11) 14). Small peptides were analyzed in the sequencer in the presence of Polybrene (15). Some peptides were analyzed with a Sequemat by the method of Laursen (16) after attachment to the solid phase via carboxylte...

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Section: Resultscontrasting

confidence: 50%

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

Shoji¹,

Parmelee²,

Wade³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

164

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“…If the activity of the protein kinase is expressed in terms of its ability to catalyze phosphorylation of histone, 1000-10,000 units/assay was routinely used. It should be emphasized that these protein kinase concentrations are in the physiological range (16). The need to use these levels of protein kinase may explain why previous attempts to phosphorylate rabbit hepatic Fru-P2ase were unsuccessful (22 (1977) in the in vivo phosphorylation of Fru-P2ase (Figs.…”

Section: Discussionmentioning

confidence: 99%

In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Riou¹,

Claus²,

Flockhart³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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Incorporation of 32P from ['y-32PJATP into a homogeneous preparation of rat hepatic fructose-1,6bisphos-phatase (D-fructose-l,6-bisphosphatase I-phosphohydrolase, EC 3.1.3.11) was catalyzed by a homogeneous preparation of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine liver. Approximately 4 mol of phosphate were incorporated per mol ofthe tetrameric enzyme. Tbis phosphorylation was associd with an increase in enzyme activity. In addition, in vivo phosphorylation of the enzyme was observed after injection of radioactive inorganic phosphate into rats and subsequent isolation of the enzyme by conventional purification methods and by immunoprecipitation. All of the labeled phosphate incorporation into the enzyme, both in vitro and in vivo, was precipitated by antibody specific for the enzyme. Furthermore, the 32Pi counts were coincident with the enzyme subunit band when the immunoprecipitates were examined by sodium dodecyl sulfate/disc gel electrophoresis. Acid hydrolysis of the immunoprecipitated enzyme that was phosphorylated in vitro revealed that only seryl residues were labeled. On the basis of the concentration of protein kinase (0.2-1.0 gM) necessary to phosphorylate physiological amounts of fructose-1,6-bisphosphatase (1.0-4.0 jiM), it is suggested that cyclic AMP-ependent protein kinase may catalyze the phosphorylation of fructose-l,6-bisphosphatase in vivo. Neither the mechanism nor the site(s) of action of glucagon on gluconeogenesis has been clearly defined. An attractive hypothesis is that the hormone acts by affecting phosphorylation of an enzyme(s) in the pathway. Recently, it was reported that the activity of and the flux through L-pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase (Fru-P2ase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) are affected by glucagon (1)(2)(3)(4)(5)(6)(7)(8). Both pyruvate kinase and phosphofructokinase are phosphorylated in vitro by protein kinases (9, 10). However, neither enzyme has been shown to be phosphorylated in vivo. The present work provides evidence that rat hepatic Fru-P2ase is phosphorylated in vivo and that phosphorylation of the enzyme in vitro can be catalyzed by the catalytic subunit of a cyclic AMP (cAMP)-dependent protein kinase, with an associated increase in enzyme activity. MATERIALS AND METHODSPurification and Assay of Hepatic Fru-P2ase. Minced rat livers (80 g) were added to 2 volumes (wt/vol) of 0.25 M sucrose/20 mM Na, K phosphate/0.1 mM dithiothreitol/1 mM EDTA, pH 7.4, and homogenized in a Waring blender for 2 min. The homogenate was centrifuged at 30,000 X g for 45 min. The supernatant was then rapidly heated to 65°for 5 min and immediately cooled to 40 by the method of Carlson et al. (11). After centrifugation the enzyme activity was recovered from the supernatant in a 30-50% ammonium sulfate pellet and

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“…Chicken gizzard myosin light chains [3,6], myosin light chain kinase [2,7] and CAMPdependent protein kinase [8] were purified as described. Trypsin (treated with tosylphenylchloromethyl ketone) and chymotrypsin were from Boehringer.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation site sequence of smooth muscle myosin light chain (M_r = 20 000)

et al. 1984

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The ammo terminal sequence of the myosin light cham (Mr = 20 000) Isolated from chicken gizzards was found to be acetyl-Ser-Ser-Lys-Ag-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser(P)-Asn-Val-Phe. This sequence assignment differs from that reported by Msuta et al. [(1981) European J. Biochem. 117, 4171 in the order of the tryptic peptides. The revised amino acid sequence exhibits greater homology with the phosphorylation site sequences of the regulatory light chains from cardiac and skeletal muscle. Moreover it is now apparent why synthetic peptides corresponding to the previously reported sequence were very poor substrates for the myosin light chain kinase.

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Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver

Cited by 298 publications

References 41 publications

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Phosphorylation site sequence of smooth muscle myosin light chain (M_r = 20 000)

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Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver

Cited by 298 publications

References 41 publications

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.

Phosphorylation site sequence of smooth muscle myosin light chain (Mr = 20 000)

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Phosphorylation site sequence of smooth muscle myosin light chain (M_r = 20 000)