Choline kinase and

The enzymatic radioassay method for the analysis of acetylcholine and choline in brain tissue has been reported by Reid et al. [210]. The method describes the determination of nanogram amounts of acetylcholine and choline in as little as 10 mg of brain tissue, involves isolation of acetylcholine by high-voltage paper electrophoresis, alkaline hydrolysis of acetylcholine to choline, and conversion of this into [32P]-phosphoryl choline in the presence of choline kinase and [y32P] ATP. The labeled derivative is isolated by column chromatography on Bio-Rad AG1-X8 resin, using Tris buffer solution as the eluent. Cerenkov radiation from 32P is counted (at 33% efficiency) in a liquid scintillation spectrometer. The amount of phosphorylcholine is proportional to the amount of choline over the range of 0.08-8.25 nmol. 

Phosphatidylcholine is preferentially synthesized in lactating mammary tissue (Kinsella, 1973), possibly regulated by the differential activities of choline kinase and ethanolamine kinase. Choline kinase has a lower Km and a higher Vmax with its substrate than does ethanolamine kinase. Also, choline kinase is inhibited slightly by ethanolamine, whereas choline is a potent competitive inhibitor of ethanolamine kinase. Thus, the intracellular concentration of choline probably regulates the synthesis of these two phos-phoglycerides (Infante and Kinsella, 1976). 

Phosphatidylcholine is degraded by phospholipases that cleave preferentially at specific bonds (Chapter 18). Choline released is phosphorylated by choline kinase and reutilized in phosphatidylcholine synthesis. However, in liver mitochondria, choline is also oxidized to betaine (N-trimethylglycine) ... 

The CT reaction usually limits the rate of PC biosynthesis. The first evidence in support of this conclusion was drawn from the relative pool sizes of the aqueous precursors (in rat liver, choline = 0.23 mM, phosphocholine =1.3 mM, CDP-choline = 0.03 mM). Calculation of these values assumes that 1 g wet tissue is equivalent to 1 ml and that there is no compartmentation of the pools. The second assumption may not be valid as there is evidence for compartmentation of PC precursors (M.W. Spence, 1989). The concentration of phosphocholine is 40-fold higher than that of CDP-choline, consistent with a bottleneck in the pathway at the reaction catalyzed by CT. Pulse-chase experiments illustrate this bottleneck more vividly. After a 0.5 h pulse of hepatocytes with [methyl- H]choline, more than 95% of radioactivity in the precursors of PC was in phosphocholine, with the remainder in choline and CDP-choline. When the radioactivity was chased with unlabeled choline, labeled phosphocholine was quantitatively converted to PC (Fig. 5). The radioactivity in CDP-choline remained low during the chase and CDP-choline was rapidly converted to PC. There was minimal radioactivity in choline which suggests that choline is immediately phosphorylated after it enters the cell. It is important to note that if a cell or tissue is in a steady state, pool sizes and reaction rates do not change. Thus, although the rate of PC synthesis is determined by the CT reaction, the rates of the reactions catalyzed by choline kinase and cholinephosphotransferase are the same as that of the reaction catalyzed by CT, otherwise, the pool sizes of precursors would change. For example, if the choline kinase reaction were faster than the CT reaction, the amount of phosphocholine would increase. Thus, CT sets the pace of the pathway. 

Tanaka, K., N.E. Tolbert, and A.F. Gohlke Choline kinase and phosphorylcholine phosphatase in plants Plant Physiol. 41 (1966) 307-312. 

Additional information <1-8, 10, 11, 14, 17> (<14> choline kinase and ethanolamine kinase activities are mediated by 2 distinct active sites, possibly on a single protein [7] <17> choline kinase and ethanolamine kinase may not have a common active site in a single enzyme protein [8] <1-8, 10, 11, 17> choline kinase and ethanolamine kinase are 2 distinct enzymes [9, 10, 12, 14] <17> ethanolamine kinase II and choline kinase do not use a common active site [11] cf. EC 2.7.1.32) [7-12, 14[... 

Ishidate, K. Furusawa, K.-i. Nakazawa, Y. Complete co-pixrification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme pro-tein(s) in rat tissues. Biochim. Biophys. Acta, 836, 119-124 (1985)... 

Spanner, S. Ansell, G.B. Choline kinase and ethanolamine kinase activity in the cytoplasm of nerve endings from rat forebrain. Biochem. Soc. Trans., 5, 164-165 (1977)... 

The subcellular location of the activities of choline kinase and ethanolamine kinase have not been definitely determined. The study of choline kinase from dodder (Cuscuta reflexa Roxb.) indicated that the activity was primarily mitochondrial (Setty and Krishnan, 1972). On the other hand experiments on spinach leaves showed choline kinase and ethanolamine kinase activities to be cytoplasmic (Macher and Mudd, 1976). Lord et al. (1972), Johnson and Kende (1971), and Morre et al. (1970) all found choline kinase to be cytoplasmically located. 

Figure 18.6 Kinetics of F-choUne. After transport into cells, choline is phosphory-lated by choline kinase, and via intermediate steps, to phosphatidylcholine, a ubiquitous component of cell membrane lipids. Inserted image on the right demonstrates high physiologic hepatic tracer retention (arrow). FocaUy increased activity in the region of the bladder (arrow) is consistent with primary bladder carcinoma, since physiologic urine activity appears later during imaging. The focus of increased activity in the right lower pelvis corresponds to a bone metastasis (arrow).

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