|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1995 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1994 : ¥1,300,000 (Direct Cost : ¥1,300,000)
By the support of the Grant-in-Aid for Scientific Research 0680711, we obtained the following results. 1) Arginine-specific ADP-ribosyltransferase from chicken heterophils ADP-ribosylated Arg-28 and Arg-206 of G-actin, and Arg-28 of F-actin in vitro, without affecting the ATPase activity, indicating the modification of Arg-206 prevents the polymerization of actin. 2) Arginine-specific ADP-ribosyltransferase from chicken heterophils ADP-ribosylated Arg-113, Arg-250 and Arg-254 of p33 in vitro. Though 4 mol of ADP-ribose can be stoichiometrically incorporated into one mol of p33, besides these three arginine residues, five candidates (Arg-8, Arg-37, Arg-235, Arg-269, and Arg-285) were detected. Partial reduction of intramolecular disulfide bonds of p33 by sulfhydryl agent, necessary for the ADP-ribosylation, may play a role to change the conformation of the p33 and accessibility of the transferase. 3) Amino terminal sequence of p33-half-2 was SPPFPQQ,indicating that the cleavage site is upstream of Ser-149 in the spacer region of p33.4) cDNA cloning revealed that chicken bone marrow cells have at least two ADP-ribosyltransferases. 5) Enzyme activity or expression of the transferase and amount of the protein or message of p33 found in peripheral polymorphonuclear leukocytes were lower than one tenth of those detected in bone marrow cells.
Though we established the method to detect p33 and p33-halves using anti-p33 antibodies, we have not yet demonstrated the activity of endopeptidase which specifically cleaves p33 and generates p33-halves, in vitro or in reconstituting system. Further studies are required to identify the endopeptidase and elucidate its importance.