Biomedical subjects
Publications and source records attributed to M L Sussman.
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Independent contractors, the payment of overtime wages, and negligent hiring practices present legal risks to home care agencies. Conducting an internal review now could save having to defend an agency on a government audit later.
Enzyme IIlac, the membrane-bound component of the lactose phosphotransferase system of Staphylococcus aureus, catalyzes the phosphorylation-transport reaction below: (see article). (The sugar can be lactose or one of its analogs.) The effects of the non-ionic detergents Triton X-100, Brij 35, and Tween 40 on the activity of Enzyme IIlac were studied. Especially striking effects were observed using Triton X-100, a detergent previously used to solubilize and isolate this enzyme. A systematic study of Triton effects over a range of concentrations and temperatures demonstrated three aspects of Triton-membrane interaction. At 0.1% Triton and 25 degrees C Enzyme IIlac is activated, but remains particulate. At 0.5% Triton and 0.5% Triton and 37 degrees C, it is rapidly and irreversibly inactivated. Sugar substrates and inhibitory sugar analogs protect Enzyme IIlac against inactivation; the effect is specific for beta-galactosides. The other substrate of Enzyme IIlac, phospho-Factor IIIlac, does not affect Triton inactivation, and the product analog galactose 6-phosphate slightly enhances the inactivation rate.
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Tosyl galactosides, previously shown to be potent reversible dead-end inhibitors of the membrane-bound Enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus, were used for an investigation of the kinetic mechanism of the sugar phosphorylation/transport reaction catalyzed by this enzyme: phospho-Factor IIIlac&sugar Enzyme IIlac lead to Factor IIIlac&sugar phosphate. Inhibition of Enzyme IIlac was studied in three different systems. Washed membranes, and washed membranes in the presence of 0.1% Triton X-100 were used for phosphorylation experiments, and whole cells were used for transport studies. When washed membranes were used to supply Enzyme IIlac, inhibition of phosphorylation by tosyl galactoside was linear non-competitive against both the sugar and phospho-Factor IIIlac substrates, with an apparent Ki of about 0.5 mM. This Ki decreased with increasing Factor IIIlac concentration. In the presence of 0.1% Triton X-100, the phosphorylation reaction was stimulated; under these conditions the inhibition became strictly competitive against sugar, and completely uncompetitive against phospho-Factor IIIlac. Apparently washed membranes can catalyze phosphorylation both via a reaction sequence in which sugar binds first and via one in which phospho-Factor IIIlac binds first, but in the presence of 0.1% Triton the reaction does not occur by the former sequence. The inability of bound phospho-Factor IIIlac to hinder the binding of tosyl galactosides suggests that the initial binding sites of the two substrates of Enzyme IIlac are separated by at least the distance of the tosyl moiety. Radioactive methyl 6-O-(p-toluenesulfonyl) beta-galactoside was not converted into a phosphorylated product in the reaction mixtures, i.e. it is a true dead-end inhibitor. Inhibition of beta-galactoside transport into whole cells by tosyl galactosides was competitive, with an apparent Ki of 5-10 mM, an order of magnitude higher than the Ki for inhibition of phosphorylation by membrane preparations. This result suggest that a significant level of unphosphorylated phospho-Factor IIIlac is present inside the cells, or that cellular levels of this compound are considerably lower than those used for in vitro sugar phosphorylation assays. Radioactive tosyl galactoside inhibitor was not transported into whole cells.
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The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate:beta-galactoside phosphotransferase system of Staphylococcus aureus was studied. Cells were grown by an improved procedure, which resulted in a 5- to 10-fold increase in cell yield. The four protein components of the system were separated. A membrane fraction containing negligible levels of the soluble components was prepared by alternate cycles of sonic treatment and differential centrifugation. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme IIlac, was rate limiting. Derivaites in which the hydroxyl group of C-6 was missing, or replaced by a fluoro, O-methyl, or carboxyl group had no affinity for Enzyme IIlac, as judged by their inability to inhibit phosphorylation. Surprisingly, derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the O-tosyl compound has an apparent affinity five times that of galactose. The arylsulfonyl substitution in an absolute requirement; neither O-benzyl or O-methanesulfonyl derivatives were inhibitory. The specificity of the inhibition by tosyl derivatives parallels that of unsubstituted substrates; tosyl galactosides of the beta configuration were inhibitory, but those of the alpha configuration were not. The tosyl derivatives also strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme IIlac provides a possible explanation for the unexpected properties of these derivatives.
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