Thiophosphoester analogs of phosphatidic acids: spectrophotometric substrates for phosphomonoesterases

Thiophosphoester analogs of dioctanoyl and didecanoyl phosphatidic acids were synthesized for use as substrates in spectrophotometric assays. These substrates are easily dispersable in aqueous media and release thiodiacylglycerols after phosphomonoesterase catalyzed hydrolysis. The free sulfhydryl of these thiodiacylglycerols reacts with the colorimetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), allowing the reaction to be followed. These analogs were shown to be good substrates for calf intestine alkaline phosphatase (highest activity at alkaline pH) and phosphomonoesterases of partially purified beef brain cytosol (highest activity at physiologic pH). Cationic amphiphilic drugs inhibit the actions of alkaline phosphatase on the dioctanoyl analog, but did not inhibit enzymatic hydrolysis of p-nitrophenyl phosphate. In contrast, the beef brain cytosolic fraction p-nitrophenyl phosphate hydrolysis was mildly inhibited, and the phosphatidic acid analog hydrolysis was increased slightly. Tetramisole inhibited all enzyme activities with p-nitrophenyl phosphate, but was inhibitory only to the alkaline-phosphatase activity with the phosphatidic acid analog.