Abstract: The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2‐dipalmitoyl‐sn‐glycerol‐3‐phosphoryl[3H]choline and 1‐palmitoyl‐2‐[9, 10(n)‐3H]palmitoyl‐sn‐glycerol‐3‐phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X‐100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of >90% activity at the 4 mM concentration. Nonhydrolyzable guanine nucleotide analogues such as guanosine 5′‐(3‐O‐thio)triphosphate and guanyl‐5′‐yl‐(β,γ‐methylene)diphosphonate but not guanosine 5′‐(2‐thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5′‐(2‐thio)diphosphate was capable of partially blocking guanosine 5′‐(3‐O‐thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was also able to cause a two‐ to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet‐activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide‐binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Neurochemistry|
|State||Published - May 1990|
- Guanine nucleotide
- Guanine nucleotide‐binding protein
- Phospholipase D