Studies on thiamine diphosphate kinase (EC 2.7.4.15) from brewer's yeast: purification and some properties.

Radiometric and fluorescent methods for detection of thiamine triphosphate have been used to show the presence of thiamine diphosphate kinase activity in brewer's yeast and to determine optimal conditions for its manifestation. A method of enzyme purification has been developed which involves glycerol-EDTA solution extraction, heat treatment, 2-fold ammonium sulphate fractionation, Sephadex G-200 gel filtration and ion-exchange chromatography on Sephadexes CP-C-50 and QAE-A-25. The protein has been purified 2000-fold in a 19% yield.

Identification of thiamine diphosphate-binding proteins from rat liver supernatant.

Transketolase (EC 2.2.1.

Thiamine-binding protein from rat erythrocytes.

A protein with thiamine-binding activity (14 nmole/mg protein) was isolated from rat red cells by affinity chromatography. Adsorbent with varying degrees of hydrophobicity containing thiamine as ligand were used for the isolation. A 2300-fold purification in a 50% overall yield was attained.

The functional role of histidine and sulfhydryl groups in rat liver thiamine pyrophosphokinase.

The functional role of histidine and sulfhydryl groups of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, E.C.2.