FUNCTIONAL DOMAINS OF PSEUDOMONAS-AERUGINOSA EXOENZYME-S

被引:59
作者
KNIGHT, DA [1 ]
FINCKBARBANCON, V [1 ]
KULICH, SM [1 ]
BARBIERI, JT [1 ]
机构
[1] MED COLL WISCONSIN,DEPT MICROBIOL,MILWAUKEE,WI 53226
关键词
D O I
10.1128/IAI.63.8.3182-3186.1995
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Recombinant exoenzyme S (rHisExoS) of Pseudomonas aeruginosa was expressed in Escherichia coli as a soluble, cytosolic His fusion protein. rHisExoS was purified by Ni2+-affinity chromatography in the presence of protease inhibitors without detectable degradation. rHisExoS possessed a specific activity (within twofold) for the factor-activating exoenzyme S-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) similar to that of native exoenzyme S. Analysis of several deletion peptides showed that Delta N222, which encoded the carboxyl-terminal 222 amino acids of exoenzyme S, possessed factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity. Delta N222 catalyzed the ADP-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relative to rHisExoS, Delta N222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four- to eightfold greater k(cat) for the ADP-ribosylation of SBTI. Like native exoenzyme S, rHisExoS chromatographed as an aggregate with an apparent molecular mass of >300 kDa. In contrast, Delta N222 did not chromatograph as an aggregate, which showed that the amino-terminal 99 amino acids of exoenzyme S were responsible for the aggregation phenotype.
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页码:3182 / 3186
页数:5
相关论文
共 24 条
[1]   MECHANISM OF ACTIVATION OF CHOLERA-TOXIN BY ADP-RIBOSYLATION FACTOR (ARF) - BOTH LOW-AFFINITY AND HIGH-AFFINITY INTERACTIONS OF ARF WITH GUANINE-NUCLEOTIDES PROMOTE TOXIN ACTIVATION [J].
BOBAK, DA ;
BLIZIOTES, MM ;
NODA, M ;
TSAI, SC ;
ADAMIK, R ;
MOSS, J .
BIOCHEMISTRY, 1990, 29 (04) :855-861
[2]   EXOENZYME-S OF PSEUDOMONAS-AERUGINOSA ADP-RIBOSYLATES THE INTERMEDIATE FILAMENT PROTEIN VIMENTIN [J].
COBURN, J ;
DILLON, ST ;
IGLEWSKI, BH ;
GILL, DM .
INFECTION AND IMMUNITY, 1989, 57 (03) :996-998
[3]  
COBURN J, 1989, J BIOL CHEM, V264, P9004
[4]  
COBURN J, 1991, J BIOL CHEM, V266, P6438
[5]   ADP-RIBOSYLATION OF P21RAS AND RELATED PROTEINS BY PSEUDOMONAS-AERUGINOSA EXOENZYME-S [J].
COBURN, J ;
GILL, DM .
INFECTION AND IMMUNITY, 1991, 59 (11) :4259-4262
[6]  
COBURN J, 1992, CURR TOP MICROBIOL, V175, P133
[7]   DIPHTHERIA-TOXIN - MODE OF ACTION AND STRUCTURE [J].
COLLIER, RJ .
BACTERIOLOGICAL REVIEWS, 1975, 39 (01) :54-85
[8]  
CORTINA G, 1991, J BIOL CHEM, V266, P3022
[9]  
CORTINA G, 1991, J BIOL CHEM, V266, P23810
[10]   PSEUDOMONAS-AERUGINOSA EXOTOXIN-A - ALTERATIONS OF BIOLOGICAL AND BIOCHEMICAL-PROPERTIES RESULTING FROM MUTATION OF GLUTAMIC ACID-553 TO ASPARTIC-ACID [J].
DOUGLAS, CM ;
COLLIER, RJ .
BIOCHEMISTRY, 1990, 29 (21) :5043-5049