Phospholipase substrate preparation

Hydrolytic cleavage by phosphatidylinositol phospholipase C (PI-PLC) of the cyclic myo-inositol-l,2-phosphate (cIP) at C-2 is faster than that at C-1. A cyclic monofluorophosphonate has been prepared as a stable analogue of the substrate in... 

As is obvious from the above discussion, the physical state of the phospholipid substrate is very important in any assay involving phospholipases. Besides the ether-methanol-water system, detergents have been used to prepare mixed micelles with the phospholipids. A widely used detergent is the neutral Triton X-100. A more detailed description of this approach can be gained from material presented in a monograph edited by Dennis (1991). 

In order to conduct research studies on this novel lipid mediator, it is necessary to have available a sufficient quantity of purified, well-defined material. This has not been an easy task, but an approach to preparation of sphingosine-l-P in milligram amounts was outlined by Van Veldhoven et al. (1989). In this procedure, a commercial preparation of sphingosylphospho-choline (which contains a free amine group due to removal of a fatty acyl residue from initial substrate, sphingomyelin) is incubated with phospholipase D in an ammonium acetate buffer at pH 8.0 for 1 hr. An insoluble reaction product is collected and subjected to purification by dissolution in water at room temperature followed by cooling to 4°C. A precipitate forms again and is collected and treated in a similar manner as above except that acetone is used as the solvent. 

Some commercial preparations of purified sphingomyehnase from B. cereus may be contaminated with phospholipase C. This is irrelevant when the substrate consists of pure sphingomyelin, but when the LUV contain glycerophospholipids, e.g. phosphatidylcholine, contamination is serious because both enzymes will give off the water-soluble end-product phosphorylcholine. In such cases, the assays include o-phenanthroline, a specific inhibitor of phospholipase C that is innocuous for SMase [92-94]. 

The alcohol once formed is oxidized by an NAD(P)H- and 02-dependent oxygenase to the aldehyde and then to the carboxylic acid. The 4a-carboxylic acid is oxidized by an NAD " -dependent enzyme to the 3-oxo-4a-carboxylic acid. This enzyme has been solubilized and purified from rat liver microsomes. The enzyme has a of 7 juM for the sterol substrate and is maximally active at alkaline pH. The preparation was free of the hydroxylase activities and was uninfluenced by treatment with several phospholipases. It is not known whether the j8-oxo acid formed decarboxylates spontaneously or enzymatically [105]. 

High-performance health beneficial emulsifier (i.e., the DHA-bound lysophos-pholipid) can be prepared from squid meal phospholipid because squid meal phospholipid contains DHA exclusively in position sn-2 (Hosokawa 1996). For this reason, if we eliminate the fatty acid moiety in the sn-1 position by applying position 1,3-specific lipase, or phospholipase Aj, we can obtain the sn-2 DHA-bound lyso-phospholipid. As a substrate for the desired reaction, squid meal phosphatidylcholine (PC) is the most useful so far. 

Saccomani et al. [85] treated a vesicular (K + H )-ATPase preparation from pig gastric mucosa with phospholipase Aj, resulting in a breakdown of 50% of the phospholipids. This treatment also results in partial loss of ATPase activity, but the residual activity is still 25% of the original activity. The K -stimulated phosphatase activity is not affected by this treatment. Schrijen et al. [86] used two phospholipases with shght difference in substrate specificity, alone and in combination. With each of these phospholipases approx. 50% of the phospholipids could be hydrolysed resulting in a 50% loss in enzyme activity. When the two phospholipases were used successively 70% of the phospholipids were hydrolysed and the loss of activity was also 70%. This represents a striking parallelism between residual phospholipid content and ATPase activity. In this case the K -stimulated phosphatase activity... 

Kusunoki, C. Sato, S. Kobayashi, M. Niwa, M. Preparation of phosphatidylethanolamine derivatives as substrates for phospholipase A2 determination. Jpn. Kokai Tokkyo Koho JP 04282391,1992. 

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