Assay of a phosphatidylinositol bisphosphate phospholipase C activity in postmortem human brain.
The activity of a phospholipase C which hydrolyses exogenous phosphatidylinositol-4,5-bisphosphate [( 3H]PtdIns(4,5)P2) in membranes prepared from frozen postmortem human brain and rat brain was investigated. Enzyme characteristics were essentially similar in membranes prepared from frozen postmortem brain and fresh or frozen rat brain. The [3H]PtdIns(4,5)P2 solubilization and assay procedure employed resulted in an efficient availability of the substrate for the enzyme. The non-hydrolysable guanosine triphosphate analogue guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) stimulated hydrolysis rapidly with a half maximum activity of approximately 25 microM. This stimulation was not specific for guanine nucleotides as ATP, imidodiphosphate and pyrophosphate also caused enzyme activation. However these activation effects could be distinguished by the polyanion spermine. The non-hydrolysable guanine dinucleotide analogue guanosine 5'-[beta-thio]diphosphate acted as a partial agonist thereby inhibiting the stimulatory effect of Gpp[NH]p. Gpp[NH]p-stimulated enzyme activity showed a maximum response in the presence of 1 mM deoxycholate and displayed a pH optima in the range 7.0-7.5. PtdIns(4,5)P2 hydrolysis was observed in the absence of added calcium, but hydrolytic cleavage was inhibited in the presence of divalent ion chelators. Magnesium inhibited PtdIns(4,5)P2 hydrolysis in a concentration-dependent manner. Elucidation of these aspects of the phosphatidylinositol cycle in normal human postmortem brain will permit comparative studies in CNS disease states.
O'Neill
C
C
Department of Geriatric Medicine, Karolinska Institute, Huddinge University Hospital, Sweden.
Fowler
C J
CJ
Wiehager
B
B
Alafuzoff
I
I
Winblad
B
B
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D011180:Postmortem Changes; D051381:Rats; D011919:Rats, Inbred Strains; D010738:Type C Phospholipases / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D003840:Deoxycholic Acid; D006165:Guanylyl Imidodiphosphate; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium