Phospholipid synthesis in mammary tissue. Choline and ethanolamine kinases: kinetic evidence for two discrete active sites.
Choline and ethanolamine kinases are located in the high speed supernatant of lactating bovine mammary gland. Maximum activities of choline and ethanolamine kinases were observed at pH 9.2 and 8.0, respectively, with the rate of ethanolamine phosphorylation being 1/15 that of choline phosphorylation. Activation energies of 29 joules (Q10--1.5) and 31 joules (Q10 = 1.5) were calculated between 3.4 and 31.3 C for choline kinase and ethanolamine kinase, respectively. Above 31.3 C, the Arrhenius plot deviated from linearity for both enzymes, suggesting that denaturation was occurring. An apparent Km of 0.25 mM for choline was obtained for choline kinase activity. The apparent Km of ethanolamine kinase for ethanolamine was unusually high (17 mM), the activity was not linear with increasing protein concentration. Activity was tripled and the Km decreased to 2.5 mM when the enzyme preparation was washed with butanol: benzene mixture, suggesting the presence of an endogenous competitive inhibitor(s), with respect to ethanolamine. Choline kinase was not affected by the solvent wash. Substrate competition studies revealed that choline kinase was slightly inhibited competitively by ethanolamine (apparent Ki = 19-21 mM), whereas choline was a potent competitive inhibitor of ethanolamine kinase (apparent Ki = 0.33-0.50 mM). The results indicated that these two kinase activities were mediated by two distinct active sites, possibly on a single protein. The significance of choline in the regulation of phosphatidylethanolamine synthesis is discussed.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002797:Choline Kinase / Q000378:metabolism*; D004983:Ethanolamines / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D010743:Phospholipids / Q000096:biosynthesis*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011136:Polysorbates / Q000494:pharmacology; D011247:Pregnancy
D004983:Ethanolamines; D010743:Phospholipids; D011136:Polysorbates; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D002797:Choline Kinase; C020125:ethanolamine kinase