Yeungnam Univ J Med.  1990 Jun;7(1):39-50. 10.12701/yujm.1990.7.1.39.

The Affinity of Calmodulin-Affigel for Inositol Triphosphate Kinase From Bovine Brain

• Lim SW
• Kim JH

• KMID: 2377305
• DOI: http://doi.org/10.12701/yujm.1990.7.1.39

Abstract

The one event on signaling mechanism is the cleavage by adenyl cyclase of ATP into second messenger, cyclic AMP. The other transfer system of inositol metabolism, it is widely recognized that hydrolysis of the minor membrane lipid phosphoinositide bisphosphate (PIPâ‚‚) initiated by occupation of certain receptors and catalyzed by phospholipase C, lead to toe generation of the two intracellular messengers, inositol triphosphate (IP₃) and diacylglycerol (DG). IP₃ is converted to inositol tetrakisphosphate (IPâ‚„) by IP₃ kinase. In the present study, it is that purification of calmodulin is used by phenyl-Sepharose CL-4B chromatography, it's molecular weigh, 17,000 in SDS-polyacrylamide gel electrophoresis. In order to observe the affinity between calmodulin (CaM)-Affigel 15 and IP₃ kinase, and isolated IP₃ kinase, was applied in CaM-Affigel with Ca²âº equilibrium buffer and EGTA equilibrium buffer. We compared with binding and elution effect of IP₃ kinase in several condition of buffer. In affinity of binding, Ca²âº equilibrium buffer was in the most proper condition, and elution, CaM/Ca²âºbuffer (CE 1 10.36, CE2 12.76pM/min/mg of protein) was effected much more than EGTA buffer (E2 1.48, E 2.43pM/min/mg of protein), but CaM/Ca²âºstimulate the activity of IP₃ kinase. And then, several detergents such as sodium deoxycholate, tween 20, cholic acid, polyethylene glycol, chaps were applied. The 0.2% chaps buffer (E2 23.19, E3 8.05pnM/min/mg of protein) was the most effective in elution of IP3 kinase.

MeSH Terms

Adenosine Triphosphate
Adenylyl Cyclases
Brain*
Calmodulin
Cholic Acid
Chromatography
Cyclic AMP
Deoxycholic Acid
Detergents
Egtazic Acid
Electrophoresis
Hydrolysis
Inositol*
Membranes
Metabolism
Occupations
Phosphotransferases*
Polyethylene Glycols
Polysorbates
Second Messenger Systems
Toes
Type C Phospholipases
Adenosine Triphosphate
Adenylyl Cyclases
Calmodulin
Cholic Acid
Cyclic AMP
Deoxycholic Acid
Detergents
Egtazic Acid
Inositol
Phosphotransferases
Polyethylene Glycols
Polysorbates
Type C Phospholipases