Amino acid sequence and characterization of a protein inhibitor of protein kinase C.
The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry. The results establish that the protein has a calculated average molecular mass of 13,690 daltons and contains 125 amino acid residues with the following sequence: (sequence: see text) The inhibitor does not show significant homology with any other known protein. Circular dichroism of the freshly prepared apoprotein indicated a secondary structural content of 23% alpha-helix, 31% beta-sheet, and 11% beta-turn. Immobilization on nitrocellulose followed by exposure to a 65Zn2(+)-containing overlay solution showed that, like protein kinase C itself, the inhibitor is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure. Competition with 10-fold molar excess Ca2+ or Mg2+ did not reduce the zinc-binding specificity of this inhibitor.
Pearson
J D
JD
Department of Biopolymer Chemistry, Upjohn Company, Kalamazoo, Michigan 49001.
DeWald
D B
DB
Mathews
W R
WR
Mozier
N M
NM
Zürcher-Neely
H A
HA
Heinrikson
R L
RL
Morris
M A
MA
McCubbin
W D
WD
McDonald
J R
JR
Fraser
E D
ED
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002417:Cattle; D002942:Circular Dichroism; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments; D011487:Protein Conformation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D012697:Serine Endopeptidases
D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C017800:zinc-binding protein; D011493:Protein Kinase C; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008274:Magnesium; D002118:Calcium