Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.
Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006358:Hot Temperature; D010446:Peptide Fragments / Q000032:analysis; D010743:Phospholipids / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D010743:Phospholipids; C043697:calcium-binding protein (brain); D011494:Protein Kinases; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium