Choline phosphorylation

Phosphatidylethanolamine synthesis begins with phosphorylation of ethanol-amine to form phosphoethanolamine (Figure 25.19). The next reaction involves transfer of a cytidylyl group from CTP to form CDP-ethanolamine and pyrophosphate. As always, PP, hydrolysis drives this reaction forward. A specific phosphoethanolamine transferase then links phosphoethanolamine to the diacylglycerol backbone. Biosynthesis of phosphatidylcholine is entirely analogous because animals synthesize it directly. All of the choline utilized in this pathway must be acquired from the diet. Yeast, certain bacteria, and animal livers, however, can convert phosphatidylethanolamine to phosphatidylcholine by methylation reactions involving S-adenosylmethionine (see Chapter 26). 

Analogues of phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidic acid with pantoic acid skeleton were prepared by phosphorylation with the respective phosphoric mono-, bis-, and tristriazolide [17]... 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

Because the preceding chromogenic assay rely on choline quantitation, the hydrolysis of substrates with headgroups other than choline cannot be followed. To circumvent this problem, another useful protocol was devised whereby the phosphorylated headgroup produced by the PLCBc hydrolysis is treated with APase, and the inorganic phosphate (Pi) that is thus generated is quantitated by the formation of a blue complex with ammonium molybdate/ascorbic acid 5 nmol of phosphate may be easily detected. This assay, which may also be performed in a 96-well format, has been utilized to determine the kinetic parameters for the hydrolysis of a number of substrates by PLCBc [37,38]. 

The different phosphoglycerides are often named by placing the constituent attached to the phosphate group after phosphatidyl , e.g. phosphatidyl choline (3-in-phosphatidylcholine or l,2-diacyl-sn-glycero-3-phosphoryl-choline). There are many phosphoglycerides because of the possible variation in the fatty acid chains, and when the full chemical structure is known, it should be used (e.g. l-palmitoyl-2-oleoyl-phosphatidylcholine). Nomenclature that entails the use of the DL system should be avoided. 

Figure 12.12 Sphingomyelins. Sphingomyelins are esters of a ceramide and phospho-ryl choline. However, similar compounds are ceramide-1-phosphoryl ethanolamines and phosphono forms of sphingolipids. Ceramides W-acyl-sphingosines) are amides of a long chain di- or trihydroxy base containing 12 to 22 carbon atoms, of which sphingosine (4-sphingenine) is the commonest, and a long chain fatty acid whose acyl chain is shown by R1. This may contain up to 26 carbon atoms.

The majority of PC is synthesized in mammalian cells by the CDP-choline or Kennedy pathway in the endoplasmic reticulum (Eigiue 1). In this pathway, choline taken up from the external medium or released in the cytosol by breakdown of choline containing compoimds, is first converted to phosphocholine by the enzyme choline kinase (CK) (Ishidate, 1997). There are two isoforms of CK cloned which both can convert also ethanolamine to phosphoethanolamine, albeit with a lesser affinity (Aoyama et al 2000). Alternatively phosphocholine can be generated by enzymes that preferentially phosphorylate ethanolamine and are therefore designated ethanolamine kinases (EK). As yet also two different EKs are known (EKI 1 and 2 Lykidis etal., 2001). 

ET), which catalyzes the formation of CDP-ethanolamine, and (iii) an ethanolaminephosphotransferase (EPT), which finally synthesizes PE from DAG and CDP-ethanolamine. As discussed rmder PC synthesis, four enzymes have been cloned that can phosphorylate ethanolamine, two of which preferentially use ethanolamine as a substrate, and two which are more specific for choline. Only one isoform of ET has been cloned, which contains two active sites, but seems to be not as strictly regulated compared to its counterpart CT (Bladergroen et al, 1999a). 

Phosphatidylcholine (lecithin) is the most abundant phospholipid in membranes. Phosphatidylethanolamine (cephalin) has an ethanolamine residue instead of choline, and phosphatidylserine has a serine residue. In phosphatidylinositol, phosphatidate is esterified with the sugarlike cyclic polyalcohol myo-inositol. A doubly phosphorylated derivative of this phospholipid, phosphatidylinositol 4,5-bisphosphate, is a special component of membranes, which, by enzymatic cleavage, can give rise to two second messengers, diacylglycerol (DAG) and inositol l,4,5trisphosphate (InsPsi see p.386). 

This enzyme [EC 2.7.1.32] catalyzes the phosphoryl transfer from ATP with choline to produce ADP and O-phosphocholtne. Ethanolamine and its methyl and ethyl derivatives can also serve as substrates. 

R. Katz-Brull, H. Degani, Kinetics of choline transport and phosphorylation in human breast cancer cells NMR application of the zero trans method. Anticancer Res. 16 (3B) (1996) 1375-1380. 

Synthesis of PE and PC from preexisting choline and ethanolamine These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline-phosphate or ethanolamine-phosphate is transferred from the nucleotide (leaving CMP) to a molecule of diacylglycerol (see Figure 17.5). 

Choline and ethanolamine are activated in much the same way as are sugars. For example, choline can be phosphorylated using ATP (Eq. 17-58, step a) and the phosphocholine formed can be further converted (Eq. 17-58, step b) to cytidine diphosphate choline. Phosphocholine is transferred from the latter onto a suitable acceptor to form the final product (Eq. 17-58, step c). Tire polymerization pattern differs from that for polysaccharide synthesis. When the sugar nucleotides react, the entire nucleoside diphosphate is eliminated (Eq. 17-56), but CDP-choline and CDP-ethanolamine react with elimination of CMP (Eq. 

Fig. 21-5, are also used for formation of both phosphatidylcholine and phosphatidylethanolamine. In both cases, the free base, choline, or ethanolamine180a b is phosphorylated with ATP. Choline phosphate formed in this manner is then converted by reaction with CTP to CDP-choline (Eq. 17-58).181 Phosphatidylcholine is formed from this intermediate1813/b while CDP-ethanolamine is used to form phosphatidylethanolamine (Fig. 21-5). These synthetic reactions occur within cell nuclei as well as on surfaces of cytoplasmic membranes.1810...

The second phase of phospholipid synthesis in eukaryotes. Choline or ethanolamine enters the cell via active transport mechanisms and is immediately phosphorylated by the enzyme, choline (ethanolamine) kinase. The phosphorylated derivatives of choline and ethanolamine... 

Dimyristoyl- and dipalmitoyllecithin were synthesized by acylation of sn-glycero-3-phosphorylcholine with fatty acid anhydrides as described by Cubero Robles and van den Berg (23). The sn-glycero-3-phosphoryl-choline was prepared by deacylation of egg-yolk lecithin according to... 

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