Activity and distribution of phosphoinositidase C in rat sciatic nerve.
The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by rat sciatic nerve cytosolic phosphoinositidase C [phosphoinositide-specific phospholipase C (PIC)] was studied at neutral pH and at ionic concentrations that approximate intracellular conditions. The principal water-soluble product formed was shown to be inositol trisphosphate by anion exchange chromatography. The maximum hydrolysis rate (2.5 nmol/min/mg protein) was achieved at less than 100 nM Ca2+. Hydrolysis was markedly increased to 15 nmol/min/mg protein by inclusion of K+ in the reaction mixture. In the presence of 200 mM K+, the optimum Ca2+ was increased to approximately 600 nM. Higher Ca2+ concentrations progressively inhibited PIP2 hydrolysis. Mg2+ also inhibited the reaction, but the presence of equimolar amounts of ATP and Mg2+ had no effect. Appreciable degradation of phosphatidylinositol-4-phosphate (PIP) also occurred in the nanomolar Ca2+ range, whereas breakdown of phosphatidylinositol (PI) required millimolar Ca2+. The presence of PIP but not PI inhibited PIP2 hydrolysis. Upon subcellular fractionation of nerve, more than 50% of recovered PIC activity was in the cytosol and about 20% was located in a myelin-enriched fraction. Using PIP2 as substrate, PIC activities in nerves from normal and streptozotocin-induced diabetic animals were not different. However, the myelin-associated enzyme from diabetic animals was more labile to freezing and thawing.
Mathew
J
J
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934.
Date
S
S
Eichberg
J
J
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology; D006868:Hydrolysis; D008297:Male; D010716:Phosphatidylinositols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012584:Sciatic Nerve / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D011188:Potassium