Characterization of the de novo biosynthetic enzyme of platelet activating factor, DDT-insensitive cholinephosphotransferase, of human mesangial cells.

Platelet activating factor (PAF), a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT) of human mesangial cell (HMC), the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA), dithiothreitol (DTT), divalent cations (Mg2+ and Ca2+), EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid) and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass "Dicentrarchus labrax," and gilthead sea bream "Sparus aurata"). The results indicated that the above compounds can influence PAF-CPT activity of HMC.

Characterization of acetyl-CoA: lyso-PAF acetyltransferase of human mesangial cells.

Platelet activating factor (PAF) is a potent inflammatory mediator produced by various renal cells and it is implicated in renal pathology. The aim of this study is the characterization of remodeling lyso-PAF acetyltransferase, which is activated under inflammatory conditions, in human mesangial cell. Total membranes of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by trichloroacetic acid precipitation method. The effect of BSA, divalent cations, EDTA, and various chemicals on the activity of lyso-PAF acetyltransferase was also studied. Various detergents were also tested for the solubilization of the enzyme and only glycerol did not affect its activity. Partial purification of solubilized enzyme preparations of human kidney tissue and mesangial cells was performed on anion exchange column chromatography and native-PAGE electrophoresis and two active fractions were detected.