Phosphatidylinositol assays

Table 3 Phosphatidylinositol assay from in vitro testing. Experimental agents causing an increase in intracellular free inositol phosphate accumulation are considered to be Group I agonists. Agents inhibiting inositol phosphate accumulation induced by ACPDa are considered to be Group I antagonists...

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

Selected entries from Methods in Enzymology [vol, page(s)] Phosphatidylinositol-specific phospholipases C from Bacillus ce-reus and Bacillus thuringiensis, 197, 493 assays for phosphoinosi-tide-specific phospholipase C and purification of isozymes from bovine brain, 197, 502 properties of phospholipase C isozymes, 197, 511 phosphatidylinositol-specific phospholipase C from human platelets, 197, 518 purification of guinea pig uterus phos-phoinositide-specific phospholipase C, 197, 526. 

This enzyme is considered to be important to maintenance of the proper level of calcium ion in the erythrocyte. As such it is also considered to be typical of a membrane-bound enzyme. Upon purification of this enzyme from human erythrocytes, it was found that the purified enzyme exhibited no activity until a mixture of a nonionic detergent and a charged phospholipid (e.g., phospha-tidylserine or phosphatidylinositol), were included in the assay system (Nelson and Hanahan, 1985). Ten- to twelvefold increases in activity could be achieved. If a neutral phospholipid such as phosphatidylcholine were substituted for the phosphatidylserine (or phosphatidylinositol), essentially there was little or no enzymatic activity. 

Fig. 7.6 Analysis of Aventis inhibitor 7600 using the radioactivity-based assay Partially purified HSL from rat epididymal adipose tissue was incubated with lipid droplets prepared by ultrasonic treatment of a mixture of radiolabeled trioleoylglycerol, tripalmitin, phosphatidylcholine and phosphatidylinositol in the presence of increasing concentrations of Aventis inhibitor 7500. After incubation (2 h at 37°C), the radiolabeled oleate released... 

Antibodies raised against transfer proteins may be used to quantitate transfer activities. The decrease in the transfer activity as measured by a standard assay in a pH 5.1 or 105,000g supernatant preincubated with transfer protein-specific antiserum would indicate the contribution of the inhibited protein to the total activity. Helmkamp et al. (1976) used this technique to determine the relative contribution of the phosphatidylcholine-specific and phosphatidylinositol-specific transfer proteins to the total phosphatidylinositol and phosphatidylcholine transfer activities in the bovine brain, heart, and liver. 

Helmkamp (1980a) studied the effect of the fatty acid composition of the acceptor lipid on the stimulation of phosphatidylinositol transfer from rat liver microsomes to phosphatidylcholine vesicles by bovine brain exchange protein. Acceptor vesicles containing egg phosphatidylcholine or dioleoyl phosphatidylcholine gave approximately the same transfer activity, whereas dielaidoyl phosphatidylcholine or dimyristoyl phosphatidylcholine vesicles produced lower transfer rates. Zborowski and Demel (1982) used the same protein and measured the rate of transfer of phosphatidylinositol from a monolayer to phosphatidylcholine vesicles. Vesicles of egg, dioleoyl, dielaidoyl, and dipalmitoyl phosphatidylcholine, even below its phase transition temperature, all gave equivalent transfer rates. However, a reduced rate was found when dimyristoyl and dilin-oleoyl phosphatidylcholine, and other phosphatidylcholines with two polyunsaturated fatty acids, were used. Table IV shows a comparison of the transfer activities measured in the two assays. The transfer rates are expressed as a percent of the transfer rate obtained with egg phosphatidylcholine acceptor vesicles. 

The effect of different phospholipid head groups on the protein-stimulated transfer by phosphatidylcholine- and phosphatidylinositol-specific proteins has been studied. Contradictory results were obtained for the effect of acidic phospholipids on the transfer of phospholipid by the phosphatidylcholine exchange protein from beef liver. DiCorleto et al. (1977) used small unilamellar vesicle-mitochondria and small unilamellar vesicle-multilamellar vesicles to study the effect of varying amounts of acidic phospholipids incorporated into phosphatidylcholine donor vesicles. Up to 20 mol% phosphatidic acid or phosphatidylinositol in the donor was found to stimulate the transfer of phosphatidylcholine in both assay systems. Wirtz et al. (1979) and Hellings et al. (1974) found different results for the phosphatidylcholine exchange protein with unilamellar and multilamellar vesicles. In these assays, the incorporation of acidic phospholipids (phosphatidic acid or phosphatidylinositol) into the donor particles had an inhibitory effect on the rate of phosphatidylcholine transfer. 

Both 39 and 39a inhibited phosphatidylinositol turnover in the A431 cell assay system, with IC50 = 10.0 pg/ml and 1.1 pg/ml, respectively[154], Piericidin B5 A-oxide was active against gram positive and some gram negative bacteria and fungi piericidin B5 was inactive [154]. 

PIP5K produces the versatile phospholipid phosphatidylinositol 4,5-bispho-sphate (PI4,5P(2)). The physiological functions of the various PIP5K isoforms are not yet fully characterized. In an abstract presented at the AACR in 2009, Treinies reported on a novel PIP5K assay developed by CRT Discovery... 

Phosphatidylinositol turnover was assayed by incorporation of labeled inositol into phospholipids. A431 cells were preincubated in HEPES-buffered saline containing [ H]inositol at 37°C for 30 min. Then, test chemical and EGF were added, and the incubation was continued for 60 min. Subsequently, 10% trichloroacetic acid containing 0.01 M sodium pyrophosphate was added, and the acid-insoluble fraction was scraped off from the dish in H2O. The lipid was extracted from it by the addition of CHCI3 and CH3OH (1 1), and [3r]inositol-labeled lipids were counted by liquid scintillation spectrophotometry. 

Three different kinase assays are also presented. Phos-phoinositide 3-kinase, PI3K, converts phosphatidylinositol... 

Fig. 4. Effect of membrane-bound phosphatidylinositol-4-phosphate 5-kinase on the LRP uptake. (A) Effect of kinase inhibitors on the internalization of LRP. Assays were performed in the presence of 1 mM H7, 1 mM A3, or 1 M staurosporine, as indicated. (B) Effect of kinase inhibitors on the production of PI4,5P2 by isolated rat liver PMs.

Very recently, a Japanese team reported an elegant example of molecular recognition of vesicles. It was shown by agglutination assays that vesicles equipped with boronic acid specifically bind to vesicles containing diol lipid such as phosphatidylinositol. It was demonstrated that the interaction of boronic acid vesicles and inositol vesicles is the result of selective coordination of the boronic acid and the carbohydrate diol. 

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