Guanine nucleotide-binding protein regulation of microsomal phospholipase D activity of canine cerebral cortex.
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 greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-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 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.
Qian
Z
Z
Department of Biochemistry, School of Medicine, University of Minnesota, Duluth 55812.
Reddy
P V
PV
Drewes
L R
LR
D000535:Aluminum / Q000494:pharmacology; D000818:Animals; D002540:Cerebral Cortex / Q000201:enzymology* / Q000648:ultrastructure; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D007477:Ions; D008670:Metals / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D014118:Toxins, Biological / Q000494:pharmacology
D003902:Detergents; D006150:Guanine Nucleotides; D007477:Ions; D008670:Metals; D014118:Toxins, Biological; D000535:Aluminum; D010740:Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D005459:Fluorides