Calmodulin-dependent NAD kinase of human neutrophils.
NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.
Williams
M B
MB
Jones
H P
HP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000097:blood; D009249:NADP / Q000097:blood; D009504:Neutrophils / Q000201:enzymology*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor)
D002147:Calmodulin; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D002118:Calcium