Mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli: physical size of enzyme IImtl and its domains IIBA and IIC in the active state.
The size of enzyme IImtl 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 IImtl 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 IImtl 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 IImtl in the associated state are exclusively located in the membrane-bound domain.
Lolkema
J S
JS
Department of Biochemistry, University of Groningen, The Netherlands.
Kuiper
H
H
ten Hoeve-Duurkens
R H
RH
Robillard
G T
GT
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D003902:Detergents; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli