P. Kefalas et al., Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs, EUR J BIOCH, 259(3), 1999, pp. 866-871
mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed w
ith cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransf
erase (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 fragm
ent, corresponding to the amino acid sequence of the processed human skelet
al muscle enzyme, was generated by reverse transcription-PCR amplification
of RNA from human PMNs, and was found to be identical to the ART1 cDNA deri
ved from human skeletal muscle. ART1 was expressed as a fusion protein with
glutathione S-transferase (GST) in insect cells, and antibodies were raise
d against the fusion protein in a rabbit. Following removal of GST immunore
activity by immunoprecipitation, these antibodies were used to measure the
abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs t
o formyl-Met-Leu-Phr (FMLP) was followed by a rapid increase in the abundan
ce of cell surface ART1 (T-1/2 = 1.9 min), and the concentration of FMLP fo
r half-maximum response was 28.6 nM. Similar responses were observed after
exposure of the cells to platelet-activating factor or interleukin-8, and w
e conclude that some of the effects of these chemotaxins are mediated by tr
anslocation of an intracellular pool of ART1 to its site of catalytic activ
ity on the outer aspect of the plasma membrane.