EFFECT OF ADP-RIBOSYLATION OF p33 AND ACTIN ON POLYMORPHONUCLEAR LEUKOCYTE DIFFERENTIATION

在科学研究助学金0680711的支持下，我们取得了以下成果。1)ADP-核糖基转移酶体外结合了G-肌动蛋白的Arg-28和Arg-206，F-肌动蛋白的Arg-28，而不影响ATPase的活性，表明Arg-206的修饰阻止了肌动蛋白的聚合。2)鸡嗜异体精氨酸特异性ADP-核糖基转移酶体外作用于p33的Arg-113、Arg-250和Arg-254。虽然4摩尔的ADP-核糖可以化学计量地掺入1摩尔的p33中，但除了这3个精氨酸残基外，还检测到5个候选残基(Arg-8、Arg-37、Arg-235、Arg-269和Arg-285)。ADP-核糖化所必需的分子内二硫键被巯基试剂部分还原，可能会改变p33的构象和转移酶的可及性。3)p33-Half-2的氨基末端序列为SPPFPQQ，表明其切割位点位于p33.4间隔区Ser-149上游。5)外周血多形核白细胞中p33的酶活性或转移酶的表达及蛋白或信息量低于骨髓细胞的十分之一。虽然我们建立了用抗p33抗体检测p33和p33-Half的方法，但我们尚未在体外或重组系统中证明特异性切割p33并产生p33-Half的内肽酶的活性。需要进一步的研究来鉴定内肽酶并阐明其重要性。

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.