Application of a TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate: Acetyl-CoA acetyltransferase in human renal tissue

Prostaglandins Other Lipid Mediat. 2004 Jan;73(1-2):123-40. doi: 10.1016/j.prostaglandins.2004.01.002.

Abstract

The activity of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase, which catalyses the first step of the de novo biosynthesis of PAF, was determined and characterised in cortical and medullary human renal tissues. A novel thin-layer chromatographic system as well as a trichloroacetic acid precipitation method, were utilised in order to determine the enzyme's activity. The acetyltransferase activity was associated with the membranous fractions of the renal tissue, it showed an optimum pH of 8.4 and it had a bell-shaped dependence on BSA concentration. One or more disulphide bonds were necessary for the action of acetyltransferase while the enzyme seemed to be independent from divalent cations. Two assay products were extracted from the incubation mixture namely alkylacetylphosphatidic acid, produced by the acetylating action of the acetyltransferase on alkyllyso-phosphatidic acid and alkylacetyl-glycerol, which is produced by the action of a phosphohydrolase on alkylacetylphosphatidic acid. The presence of NaF in the assay mixture resulted to a decreased degradation of alkylacetylphosphatidic acid, as well as to an increased overall product formation. Cortical and medullary acetyltransferases share similar biochemical properties and there is no statistical difference between the two activities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / analysis
  • Acyltransferases / chemistry*
  • Animals
  • Cattle
  • Cell Membrane / chemistry*
  • Cell Membrane / enzymology
  • Chromatography, Thin Layer / methods*
  • Enzyme Activation / drug effects
  • Glyceryl Ethers / chemistry
  • Humans
  • Hydrogen-Ion Concentration
  • Kidney Cortex / chemistry*
  • Kidney Cortex / enzymology
  • Kidney Medulla / chemistry*
  • Kidney Medulla / enzymology
  • Phosphatidic Acids / chemistry
  • Platelet Activating Factor / biosynthesis
  • Serum Albumin, Bovine / chemistry
  • Sodium Fluoride / pharmacology
  • Trichloroacetic Acid / chemistry*

Substances

  • Glyceryl Ethers
  • Phosphatidic Acids
  • Platelet Activating Factor
  • Serum Albumin, Bovine
  • Trichloroacetic Acid
  • Sodium Fluoride
  • Acyltransferases
  • 1-alkyl-sn-glycero-3-phosphate-acyl CoA acyltransferase