Arginine-specific ADP-ribosyltransferase and its acceptor protein p33 in chicken polymorphonuclear cells: co-localization in the cell granules, partial characterization, and in situ mono(ADP-ribosyl)ation

Abstract

We have reported the purification and characterization of arginine-specific ADP-ribosyltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-glutamate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyltransferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils.(ABSTRACT TRUNCATED AT 250 WORDS)