Kinetic studies of human and rat neutrophil lysoPAF acetyltransferase using lysoPAF and dansyllysoPAF as substrates.
Enzyme kinetic studies of lysoPAF acetyltransferase from microsomal preparations of human and rat neutrophils were carried out using lysoPAF or dansyllysoPAF as substrate. With the human enzyme, incomplete conversion of the substrate into the product was observed at 37 degrees C with both substrates. The acetyltransferase was inactivated at 37 degrees C in the absence of substrate with a half-life of 7.5 min. However, the initial rate of product formation under the assay conditions was linear up to 10 min. Both enzymes were optimally active at 40 microM concentration with either substrate, but enzyme activity was inhibited at higher substrate levels. At a constant substrate concentration (40 microM), the Km (microM) and Vmax (nmol product/min/mg protein) values for the human acetyltransferase, with respect to acetyl-CoA were 132 and 23.1, respectively, with lysoPAF as substrate, and 105 and 26.7, respectively, when dansyllysoPAF was used. The Km and Vmax values for the rat enzyme were 105 and 6.5, respectively, with lysoPAF as substrate, and 120 and 5.4, respectively, when dansyllysoPAF was used. Under our standard conditions, lysoPAF required 1 mg of BSA per mL in the assay, whereas full activity of both enzymes was seen with dansyllysoPAF even in the absence of BSA. The results show that dansyllysoPAF can replace lysoPAF in the assay without any significant changes in kinetic parameters.
Schindler
P W
PW
Pharma Forschung, Hoechst AG, Frankfurt, Germany.
Ninio
E
E
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000097:blood*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; C058864:dansyl-platelet activating factor; D000001:Calcimycin; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C046035:1-alkylglycerophosphocholine acetyltransferase