DERECHO COMPARADO:

competitive inhibitor for all méthylation reactions in which SAM is the methyl donor (Cantoni,1975 ; Eloranta and Kajander, 1984). The decarboxylated analogue of SAM donates its propylamino group to putrescine to form polyamines (Stipanuk, 1986) . The SAM content of liver influences whether homoysteine will be recycled to form methionine, or undergo trans-sulphurat ion to synthesise cystathionine and cysteine (Finkelstein et al, 1982, 1988) .

1.8.4 The role of PS decarboxylation in SAM-dependent méthylation

The decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) (Fig.10b), is catalysed by the enzyme, phosphatidylserine decarboxylase (EC 4.1.1.65), a membrane-bound enzyme which is predominantly located in the inner mitochondria membrane (Zborowski et al, 1983; Percy et al, 1983.) It has been proposed that PE formed through decarboxylation is preferentially transported from the mitochondria to the ER and plasma membrane (Vance, 1991) . Although the predominant amount of PE is synthesised de novo in many cells via the CDP-ethanolamine

pathway (Kennedy and Weiss, 1956), or through the base- exchange pathway (Fig.10a), in cultured BHK-21 cells PE is most exclusively derived from PS decarboxylation (Voelker et al, 1984). It has also been reported that in cultured neuronal cells, over 30% of the total serine labelled phospholipids were decarboxylated to PE which was a significant substrate for SAM-dependent méthylation (Yavin, 1985) . The latter observation has been challenged by White et al (1986), who rather confirmed that decarboxylation contributed significantly to the synthesis of ethanolamine glycerophospholipids in the brain. Carlini et al (1993) have suggested that decarboxylated-derived PE may follow different utilisation pathways physiologically. In rat hepatocytes newly made PE is preferentially methylated to PC (Samborski et al, 1958) . Vance (1989) proposed that PC and PE derived from PS decarboxylation are rapidly added to very low density lipoproteins (VLDL) , whilst PE and PC made via the CDP-ethanolamine pathway are preferentially excluded from VLDL in cultured hepatocytes (Vance and Vance, 1986) . This further suggests a specific importance of the PS-decarboxylation pathway.

In other studies various investigators have provided evidence that PS decarboxylase and phospholipid-N- methyltransferase can be co-regulated. Concanavalin A was

(a) CDP-ethanolamin 1,2 Diacyl-sn-Glycerol--- CMP 0

^HzC-O-tPi

P

I

Fig. 10 Biosynthesis of phosphatidyethanolamine via (a) the CDP-ethanolamine pathway and (b) the decarboxylation pathway

In (a) ethanolamine phosphotransferase catalyses the transfer of phosphorylethanolamine from CDP- ethanolamine to 1,2 diacyl-sn-glycerol to form PE. In (b) free serine is exchanged with PE to form PS which can be decarboxylated to give PE. (NB: Free ethanolamine can exchange with CPP-choline to PE via DAG pathway)

observed to stimulate both enzymes in mast cells (Hirata et al, 1979), whilst in yeast the activities of both enzymes were shown to be co-downregulated by inositol and choline (Carson et al, 1984; Lamping et al, 1991, and Overmeyer and Waechter, 1991) . It is thus likely that both enzymes could be co-located in cell.

Recent work by Cui et al (1993) indicated that PLMTase resides on a special subcellular fraction associated with mitochondria where the majority of PS decarboxylase activity is also found. They suggested that such c o ­ localization can act as a "bridge" for the synthesis of PC from PS-derived PE. However, no evidence in support of this correlation was offered.

1,9 Research Aims and Objectives

The blood platelet provides an attractive model for this project, first because of its function in blood coagulation and hemostasis and secondly, because of its implicated role in the initiation of thrombosis (Davies et al, 1981) . The first objective was to employ, for the first time, nuclear magnetic resonance (nmr) as a non-invasive, diagnostic technique for the profiling of the lipid content of platelets in both normal and diseased subjects. This could serve as the basis for studying lipid metabolism in platelets and also for monitoring abnormalities in patients with lipid disorders associated with platelet thrombo-

plastic activity or patients with diseases in which platelets have been implicated (Drummond et al, 1987).

It is also well documented that phospholipids play an important role in blood coagulation and membrane function (Chargaff et al, 193 6 ; Papahadjopoulos et al, 1962). The

transmethylation pathway is involved in lipid biosynthesis, particularly the conversion of ethanolamine phospholipids to choline phospholipids; however, neither has it been related to the formation of eicosanoids or to blood clotting, nor has the enzyme(s) involved been purified from p la t e l e t s .

Thus it was also the aim of this project to investigate the metabolism of choline phospholipids via this pathway in platelets, to establish if there exists a cause and effect relationship between phosphatidycholine metabolism in the plasma membrane of platelets and the physiological processes involved in blood clotting. A further aim was to purify and characterise the enzyme(s) involved in the transmethylation.

Finally, since platelets have similar neuroaminergic properties as neurons, we analysed the excitatory sulphur- containing amino acids and polyamine content of platelets and proposed their probable roles in platelet function.

CHAPTER 2