Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs.
mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.
Kefalas
P
P
Section on Clinical Pharmacology, Imperial College School of Medicine, London, UK.
Saxty
B
B
Yadollahi-Farsani
M
M
MacDermot
J
J
D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002630:Chemotactic Factors / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D018482:Muscle, Skeletal / Q000201:enzymology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism
D002630:Chemotactic Factors; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D036002:ADP Ribose Transferases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase