MANNITOL-SPECIFIC ENZYME-II OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - PHYSICAL SIZE OF ENZYME-II(MTL) AND ITS DOMAINS IIBA AND IIC IN THE ACTIVE STATE

被引:24
作者
LOLKEMA, JS
KUIPER, H
TENHOEVEDUURKENS, RH
ROBILLARD, GT
机构
[1] UNIV GRONINGEN,DEPT BIOCHEM,9714 AG GRONINGEN,NETHERLANDS
[2] UNIV GRONINGEN,INST BIOSON,9714 AG GRONINGEN,NETHERLANDS
关键词
D O I
10.1021/bi00057a002
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 size of enzyme II(mtl) solubilized in the active state has been determined by size-exclusion chromatography under conditions that favor the association of the enzyme. The contribution of the detergent bound to the enzyme was determined by solubilizing the enzyme and running the TSK250 column in a number of detergents with decreasing micellar sizes. The size, expressed as the equivalent molecular mass of a globular protein, decreased from 315 kDa in decylPEG, to 275 kDa in octylPEG and octyl glucoside, and then to 245 kDa in cholate. Enzyme II(mtl) is not active in the latter three detergents when at concentrations above their cmc values but still binds mannitol with high affinity without significant loss of sites. This, together with the full reversibility of the inactivation, is taken as evidence that the enzyme does not unfold or dissociate in these detergents. The sizes of the separated domains IIBA and IIC of enzyme II(mtl) were 38 and 175 kDa, respectively. The cytoplasmic domain, IIBA, was monomeric at high concentration, whereas the membrane-bound domain, IIC, was associated at much lower concentration. Apparently, the sites that interact to keep enzyme II(mtl) in the associated state are exclusively located in the membrane-bound domain.
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页码:1396 / 1400
页数:5
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