2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.
Enolase has been purified from aqueous extracts of Escherichia coli acetone powder by (a) heat treatment, (b) fractionation with acetone, (c) TEAE-cellulose chromatography, (d) Sephadex G-100 chromatography, and (e) crystallization. The purified, crystalline enzyme migrates as a single band in disc gel electrophoresis and is homogeneous by ultracentrifugal analysis. The molecular weight of the enzyme is approximately 90,000, as determined by sedimentation velocity and sedimentation equilibrium experiments. The subunit molecular weight estimated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate is 46,000, suggesting that the enzyme is composed of two subunits of equal size. Functionally there are many similarities between E. coli enolase and other enolases studied. Thus, the dependence on Mg2+ for activity and the inhibition by fluoride in the presence of phosphate are quantitatively very similar for all enolases. Other catalytic parameters (Km, Vmax, and pH optimum) are also similar, but minor quantitative distinction indicates that E. coli enolase is more closely related to yeast enolase than to enolases from vertebrate muscle.
The effect of rhodium(II) acetate, propionate, and methoxyacetate on the activity of 17 enzymes was evaluated. The enzymes were preincubated with the rhodium(II) complexes in order to detect irreversible inhibition. All enzymes that have essential sulfhydryl groups in or near their active site were found to be irreversibly inhibited. Those enzymes without essential sulfhydryl groups were not affected. In each case, the rate of inactivation closely paralleled the observed toxicity and antitumor activity of rhodium(II) carboxylates; that is, rhodium(II) propionate greater than rhodium(II) acetate greater than rhodium(II) methoxyacetate. In addition, those enzymes that have been demonstrated to be most sensitive to established sulfhydryl inhibitors, such as glyceraldehyde-3-phosphate dehydrogenase, were also most sensitive to rhodium(II) carboxylate inactivation. Proton nuclear magnetic resonance measurements made during the titration of rhodium(II) acetate with cysteine showed that breakdown of the carboxylate cage occurred as a result of reaction with this sulfhydryl-containing amino acid.
This spectrophotometric method for the direct determination of potassium in serum or plasma is based on the selective complexing of potassium by a specific macrocyclic polyether, with the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this colorimetric method (y) correlate well with the results obtained by a flame-photometric method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system (y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168). With this system one can use whole-blood specimens in measuring potassium.
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