A comparison of the cyclic nucleotide-dependent protein kinases using chemical cleavage at tryptophan and cysteine.
cGMP-dependent protein kinase (G-kinase) and the regulatory subunit of type I (RI) cAMP-dependent protein kinase (A-kinase) both contain a phosphorylation site located near the NH2 terminus of each enzyme. These sites can be utilized as convenient markers for the determination of the position of an amino acid residue susceptible to either chemical or enzymatic digestion. Using the tryptophan-specific reagent, N-chlorosuccinimide, the approximate location along the polypeptide chain of six reactive tryptophans in G-kinase and three reactive residues in RI were identified. Similarly, cleavage with cyanide was used to locate free and disulfide-bonded cysteines in both proteins. The approximate positions of nine cysteines in G-kinase were determined along with the location of the interchain disulfide bond and an intrachain disulfide bond. RI was found to contain three cyanide-reactive cysteines, two of which are involved in interchain disulfide bonding. A comparison of the positions of the cysteines and tryptophans determined by chemical cleavage in G-kinase and RI, with the positions of cysteine and tryptophan in the known sequence of the type II A-kinase, support the structural relationships between these enzymes. Comparison with subsequently reported primary sequences of all three enzymes indicates the limits of precision of this chemical cleavage procedure.
Monken
C E
CE
Gill
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D004220:Disulfides; D014364:Tryptophan; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D003545:Cysteine