Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells.
Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006461:Hemolysis; D006801:Humans; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000276:immunology; D007526:Isoelectric Point; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D014174:Transketolase / Q000276:immunology / Q000302:isolation & purification*
D000596:Amino Acids; D000906:Antibodies; D007074:Immunoglobulin G; D008670:Metals; D014174:Transketolase