Characterization of the De Novo Biosynthetic Enzyme of Platelet Activating Factor, DDT-Insensitive Cholinephosphotransferase, of Human Mesangial Cells

Abstract

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.

Platelet activating factor (PAF) and activity of its biosynthetic and catabolic enzymes in blood and leukocytes of male patients with newly diagnosed heart failure

“…The relation of PAF-CPT to well established inflammatory parameters (CRP and IL-6) identified in our study, generates novel questions and hypothesis for its implication to pathophysiological processes involving inflammation. Our results are corroborated by the finding that statins which display anti-inflammatory actions can also strongly inhibit PAF-CPT in vitro [21] and that in diseases where chronic inflammation is innermost characteristic such as cancer [38]. Another speculation for the correlation of both PAF-CPT and lyso-PAF AT to CRP is the fact that this protein can exert effects on membrane stabilization in vitro and thus potentially regulate the action of both enzymes which exhibit their activities on membranes [39].…”

“…For the determination of lyso-PAF AT activity, leukocyte homogenates, containing 12 μg of total protein, were incubated with 4 nmol of lyso-PAF and 40 nmol of acetyl-CoA for 10 Both reactions were stopped by adding 1.11 mL of cold chloroform: methanol (2% acetic acid) and 0.3 mL of cold water in order to extract PAF by the acid Bligh-Dyer method [21]. The extracted PAF was further separated by thin-layer chromatography (TLC) on Silica Gel G coated plates with an elution system consisting of chloroform:methanol:acetic acid: water (100:57:16:8, v/v/v/v).…”

“…The extracted PAF was further separated by thin-layer chromatography (TLC) on Silica Gel G coated plates with an elution system consisting of chloroform:methanol:acetic acid: water (100:57:16:8, v/v/v/v). The band corresponding to PAF (between lyso-phosphatidylcholine and phosphatidylcholine) was identified by co-chromatographing lipid standards which were visualized by exposure of the plates to iodine vapors [21]. PAF fractions were scrapped off, extracted by the Bligh-Dyer method and the amount of PAF was determined by the washed rabbit platelet aggregation assay [6].…”

Paf-Metabolic Enzymes and Paf-like Activity inL. InfantumandL. MajorPromastigotes

“…Assay was performed, as previously described (16,22), at 37°C for 20 min in a final volume of 200 ul, containing 0.05-0.25mg/mL protein, 100 mM Tris-HCI (pH 8.0), IS mM dithiothreitol (OTT), 0.5 mM EDTA, 20 mM MgCI 2 , I mg/mL BSA, roo IlM COP-Choline and 100 IlM I-O-alkyl-2-sn-acetyl-glycerol (AAG). The reaction was stopped by adding 2% acetic acid methanol.…”

“…The reaction was stopped by adding 2% acetic acid methanol. The extraction, purification and determination of PAF were performed as previously described (22).…”