Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues.
1. The cyclic AMP-dependent protein kinase activity-ratio assay was investigated by comparing histone and a synthetic peptide, malantide [Malencik & Anderson (1983) Anal. Biochem. 132, 32-40], as substrates. 2. In several tissues the activity ratio was higher when assayed with histone as the substrate; this result was obtained in control tissues and also in those incubated with agents known to increase cyclic AMP. The effect of these agents to increase the activity ratio was more clearly demonstrated with malantide. 3. The higher activity ratios observed with histone are due to: (a) measurement of phosphorylation not catalysed by cyclic AMP-dependent protein kinase; (b) activation of cyclic AMP-dependent protein kinase by histone during the assay. 4. When tissues were homogenized in buffers without NACl, lower activity ratios were found, owing to the catalytic subunit being artifactually removed from the supernatant. 5. We conclude that the measured activity ratio more faithfully reflects that in the tissue when NaCl is included in the homogenization buffer and malantide is used in the assay. This was confirmed in experiments where cyclic AMP-dependent protein kinase was added to the tissue before homogenization, and no dissociation of the exogenous enzyme was observed.
Murray
K J
KJ
Department of Cellular Pharmacology, Smith Kline and French Research Limited, Welwyn, Herts, U.K.
England
P J
PJ
Lynham
J A
JA
Mills
D
D
Schmitz-Peiffer
C
C
Reeves
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D000242:Cyclic AMP / Q000502:physiology*; D006168:Guinea Pigs; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology
D006657:Histones; D009842:Oligopeptides; D010455:Peptides; D012965:Sodium Chloride; C064117:malantide; D000242:Cyclic AMP; D011494:Protein Kinases