Determination of the Binding Constant of a Protein Kinase C Substrate, NG(28–43), to Sodium Dodecyl Sulfate via the Diffusion Coefficient Measured by Pulsed Field Gradient Nuclear Magnetic Resonance

1998 ◽  
Vol 264 (2) ◽  
pp. 211-215 ◽  
Author(s):  
Wei-Jyun Chien ◽  
Shu-Fang Cheng ◽  
Ding-Kwo Chang
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Diffusion Coefficient ◽  
Protein Kinase C ◽  
Magnetic Resonance ◽  
Protein Kinase ◽  
Sodium Dodecyl Sulfate ◽  
Binding Constant ◽  
Sodium Dodecyl ◽  
Kinase C

scholarly journals Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C

1985 ◽  
Vol 232 (2) ◽  
pp. 559-567 ◽  
Author(s):  
J R McDonald ◽  
M P Walsh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Large Scale ◽  
Binding Proteins ◽  
Potent Inhibitor ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Heat Stable ◽  
Kinase C

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.

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