Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5- bisphosphate

Vered Chalifa-Caspi, Yona Eli, Mordechai Liscovitch

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of 1-palmitoyl-2-[6- N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP2), and the curve of activation versus PIP2 molar ratio fitted a Michaelis-Menten equation with a K(acl) value between molar ratios of 0.001- 0.002. Maximal activation was observed at a PIP2 molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP2 was shown to elevate by 6-22 fold the specificity constant of PLD towards NBD-PC (K(A), which is proportional to V(max)/K(m)). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V(max) values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K(m) values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K(A)) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP2, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.

Original languageEnglish
Pages (from-to)589-599
Number of pages11
JournalNeurochemical Research
Volume23
Issue number5
DOIs
StatePublished - 28 Apr 1998
Externally publishedYes

Keywords

  • Alcohol
  • Phospholipase D
  • Rat brain
  • Signal transduction
  • Surface dilution kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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