OCR Text |
Show JESSICA KRAMER, (DALE POULTER), CHEMISTRY DEPARTMENT, UNIVERSITY OF UTAH, SALT LAKE CITY, UTAH 84112 ABSTRACT Phospholipases are a class of enzymes that catalyze the hydrolysis of mem-brane phospholipids. Phospholipase Cs (PLCs) cleave phosphotidylinositol phosphates (PIPs) into a diacyglycerol lipid tail and an inositol phosphate polar head group. These hydrolysis products are crucial secondary messengers for intracellular signaling and have been implicated in several disease states. PLCs are soluble proteins that anchor to the cellular membrane by a phos-phoinositide specific Pleckstrin Homol-ogy (PH) domain. Once anchored to the membrane, the catalytic X and Y domains catalyze many cleavage re--be classified as xi, beta, gamma, and delta. PLCdelta binds PI(4,5)P2 with high affinity. The crystal structure of the PH domain revealed a positively charged surface patch with high affinity interac-tions to the PI(4,5)P2 phosphates. The high specificity of the PH domain of PLCdelta was explored in several experiments. Binding specificity was shown qualitatively in a protein lipid overlay with eight naturally occurring phospholipids. PLCdeltaPH binding was compared to a known promiscuous lipid recognizing protein, LL5alpha. PLCdel-taPH PI(4,5)P2 binding was optimized quantitatively by chemi-luminescence using an Alphascreen assay. Optimal enzyme and substrate concentrations were used in a competition Alphas-creen assay. Five concentrations of eight phosphoinositides were assayed with constant PLCdeltaPH and PI(4,5)P2 conditions. PLCdeltaPH PI(4,5)P2 bind-ing affinity in these varied lipid environ-ments was quantified to see if the spe-cific binding could be competed away. The overlay showed a beautiful ar-ray with PLCdeltaPH binding almost exclusively to PI(4,5)P2 and LL5alpha binding to nonspecifically many PIPs. The Alphascreen showed maximum binding occurring at 0.21 picomoles PLCdeltaPH and 2.5 picomoles PI(4,5)P2 per well in a 25uL Alphascreen assay. The competition assay demonstrated the specific binding of PLC PH to PI(4,5)P2 can be competed away by other vari-ous other PIPs. PI(3,4,5)P3, PI(4)P, and PI(3,4)P2 the most effective inhibitors re-spectively. PI(5)P and PI displayed minor inhibition. PI(3)P and PI(3,5)P2 showed no competition. It is reasonable to con-clude that PIPs with a 4-phosphate were better competitors than PIPs with a 5-phosphate. This suggests that the interaction of the 4-phosphate is more important than the 5-phosphate for the specific bindingofPLCdeltaPHtoPI(4,5)P2. |