Rapid purification and identification of calcyphosine, a Ca(2+)-binding protein phosphorylated by protein kinase A.
A method is presented for the rapid purification of dog thyroid calcyphosine, a protein previously identified as a major substrate for cyclic AMP-dependent protein kinase in dog thyroid slices stimulated by thyrotropin [Lecocq, Lamy and Dumont (1979) Eur. J. Biochem. 102, 147-152]. The protein was previously identified as a spot on two-dimensional gels and is now purified in its native form by a procedure involving three chromatographic steps. Homogeneous calcyphosine identified by SDS/PAGE, immunoblotting and peptide sequencing can be obtained within 7 h. As for calmodulin, Ca(2+)-dependent conformational changes can be shown by Ca(2+)-dependent hydrophobic interaction chromatography using phenyl-Sepharose. Unlike calmodulin, calcyphosine is a substrate for protein kinase A.
Lecocq
R
R
Interdisciplinary Research Institute (IRIBHN), Brussels, Belgium.
Lamy
F
F
Erneux
C
C
Dumont
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017421:Sequence Analysis; D013961:Thyroid Gland / Q000737:chemistry*
D000838:Anions; C059605:CAPS protein, human; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D017868:Cyclic AMP-Dependent Protein Kinases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase