SYNTHESIS OF S-35 LABELED MACROMOLECULES BY POLYMORPHONUCLEAR NEUTROPHILS - EVIDENCE FOR THE PRODUCTION OF [S-35] SULFITE WHICH CAN MODIFY BOTH ENDOGENOUS AND EXOGENOUS PROTEINS

被引:7
作者
GARDINER, EE [1 ]
ROBINSON, HC [1 ]
SRIRATANA, A [1 ]
MOK, SS [1 ]
LOWTHER, DA [1 ]
HANDLEY, CJ [1 ]
机构
[1] MONASH UNIV,DEPT BIOCHEM,CLAYTON,VIC 3168,AUSTRALIA
关键词
D O I
10.1042/bj2880577
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The incorporation of [S-35]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of S-35-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80 % of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that S-35-labelled macromolecules isolated from the cell extract migrated with K(av.) of 0.36, while corresponding material isolated from the medium migrated with K(av.) of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise S-35-labelled proteins of various molecular masses. The S-35 label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [S-35]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [S-35]sulphite from [S-35]sulphate. The release of [S-35]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [S-35]sulphite reacted with these proteins to form a stable S-35-labelled conjugate.
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页码:577 / 583
页数:7
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