ARGININE-SPECIFIC MONO(ADP-RIBOSYL)TRANSFERASE ACTIVITY ON THE SURFACE OF HUMAN POLYMORPHONUCLEAR NEUTROPHIL LEUKOCYTES

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface o f human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed b y the use of diethylamino(benzylidineamino)guanidine (DEA-BAG) as an A DP-ribose acceptor. Two separate HPLC systems were used to separate AD P-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirm ed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA- BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, whi ch indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs w as not simply a product of a reaction between DEA-BAG and free ADP-rib ose, due possibly to the hydrolysis of NAD(+) by an NAD(+) glycohydrol ase. The assay of mono(ADP-ribosyl)transferase with agmatine as a subs trate was modified for intact PMNs, and the activity was found to be a pprox. 50-fold lower than that in rabbit cardiac membranes. The K-m of the enzyme for NAD(+) was 100.1 +/- 30.4 mu M and the V-max 1.4 +/- 0 .2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol a nchor, since incubation of intact PMNs with phosphoinositol-specific p hospholipase C (PI-PLC) led to a 98 %, decrease in mono(ADP-ribosyl)tr ansferase activity in the cells. Cell surface proteins were labelled a fter exposure of intact PMNs to [P-32]NAD(+). Their molecular masses w ere 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-l inear under these conditions over a period of 4 h. The labelled produc ts were identified as mono(ADP-ribosyl)ated proteins by hydrolysis wit h snake venom phosphodiesterase to yield 5'-AMP.