[41] Assay of cyclic AMP-dependent protein kinases

Corbin, Jackie D.; Reimann, Erwin M.

doi:10.1016/0076-6879(74)38044-5

Cited by 487 publications

(156 citation statements)

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“…The immunoprecipitate of 50 ,g of 32P-labeled Fru-P2ase was dissolved in 2 ml of 6 M HCl and hydrolyzed for 20 hr at 1000. After evaporation, the residue was dissolved in 1 ml of 50 mM HCl and the radioactive inorganic phosphate was separated from 32P-labeled amino acid residues by chromatography on Dowex 50-X8 (18). The 32P-labeled amino acid residue was collected, evaporated to dryness, and resuspended in 50 mM HCL.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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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Section: Methodsmentioning

confidence: 99%

“…Rat hepatic Fru-P2ase has been reported to contain no tryptophan (15 (18). The number of moles of 32p incorporated per mol of enzyme was calculated on the basis of a molecular weight of 144,000 for Fru-P2ase (15).…”

Section: Methodsmentioning

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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“…(ii) Protein kinase was determined by the method in [5,6]: 25 ~1 infranatant was added to 75 ~1 12.5 mmol/l potassium phosphate buffer, pH 6.8, containing per liter: 37 mmol NaF, 30 g histone (type II A from calf thymus, Sigma), 4.5 mm01 magnesium acetate, 0.5 mm01 [-y-'"PI-ATP and, when present, 2 I.tmol cyclic AMP (for the determination of total protein kinase activity). Incubation was carried out in triplicates for 5 min at 30°C.…”

Section: Detemzination Of Cyclic Amp and Cyclic Ampdependent Protein mentioning

confidence: 99%

Increased sensitivity to epinephrine of the cyclic AMP‐protein kinase system in adipose tissue of diabetic rats

Zapf¹,

Waldvogel²,

Zumstein³

et al. 1978

FEBS Letters

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“…Protein kinase activity in the total muscle homogenate was measured in a reaction mixture (0.2 ml) consisting of 50 mM KH2P04, 0.5 mM methylxanthine, 5 mM MgC12, 20 mM NaF, 1 mM Tris-ATP (l-5 X 1 O6 cpm [r-'*PI ATP), 100 pg calf thymus histone IIA (pH 7.5) [7,8]. The reaction was initiated by the addition of 100 pg homogenate protein and incubated at 30°C over a time span of O-10 min.…”

Section: L Protein Kinase Determinationmentioning

confidence: 99%