Immunoaffinity purification of rat liver transketolase: evidence for multiple forms of the enzyme.
Rat liver transketolase (TK) has been purified, in a single step, by immunoaffinity chromatography on specific TK antibodies covalently linked to Sepharose 4B. The procedure described also involves the raising and isolation of rabbit TK antibodies to the conventionally purified enzyme [F. Paoletti (1983) Arch. Biochem. Biophys. 222, 489-496]. Affinity chromatography allows a 100-fold purification of TK from the cell cytosol and a recovery of about 70% of the original activity. The TK isolated has a specific activity of 2.7-3.2 at 25 degrees C and migrates as a single band on polyacrylamide gel electrophoresis at pH 9.1. Multiple forms of the enzyme, with distinct pI values in the range 7-8, have been detected in purified preparations by means of analytical isoelectric focusing and staining for TK. No addition of either Mg2+ or thiamine pyrophosphate is required for the activity of the enzyme which, in the native form, exhibits a molecular weight of about 139,000. Two moles of thiamine pyrophosphate can be resolved for each mole of enzyme. Affinity TK preparations are virtually free of glyceraldehyde-3-phosphate dehydrogenase, pentose-phosphate epimerase, and isomerase, although slight contamination by phosphohexose isomerase may occur.
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014174:Transketolase / Q000302:isolation & purification*
D007527:Isoenzymes; D014174:Transketolase; D013835:Thiamine Pyrophosphate