ABSTRACT
In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H+ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H+ binding. To determine if the lactose permease contains one or more auxiliary H+ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H+ leak, and isolated third site suppressor mutations that blocked the H+ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H+ leak and elevated the apparent Km value for lactose. The M299I and H322Y suppressors could still transport H+ with beta-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H+/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H+ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H3O+).
Subject(s)
Cations, Monovalent/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Hydrogen/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Monosaccharide Transport Proteins , Point Mutation/genetics , Symporters , Thiogalactosides/metabolism , Amino Acid Substitution/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Glutamine/genetics , Glutamine/metabolism , Hydrogen-Ion Concentration , Ion Transport , Kinetics , Lactose/metabolism , Mutagenesis, Site-Directed/genetics , Phenotype , Protons , Suppression, Genetic/geneticsABSTRACT
STUDY OBJECTIVE: To characterize frequency of liver enzyme elevation in patients with type 2 diabetes mellitus receiving troglitazone. DESIGN: Retrospective study. SETTING: Hospital-affiliated medical center. PATIENTS: Two hundred ninety-one patients with type 2 diabetes mellitus. INTERVENTION: Data from patients with an average troglitazone exposure of 412.7 +/- 255.6 days were studied. MEASUREMENTS AND MAIN RESULTS: Enzyme elevations more than 1.5 times the upper limit of normal (ULN) occurred in 17 patients (5.8%) and more than 3-fold elevations in 6 (2.1%). The relationship among enzyme elevation events, demographic factors, duration of troglitazone exposure, frequency of monitoring, and concurrent drugs (limited to glucose and lipid-lowering agents) was assessed by multiple logistic regression. Age was an independent predictor of risk (p=0.009), and concurrent insulin therapy approached statistical significance (p=0.051) for 1.5-fold ULN elevation in liver enzymes. Age and concurrent therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors were the only significant predictors of 3-fold ULN elevations (p=0.03 and p=0.04, respectively). CONCLUSION: Several factors appear to increase the risk of enzyme elevation events in patients treated with troglitazone.
