Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.
Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.
Egi
Y
Y
Department of Biochemistry, Hiroshima University School of Medicine, Japan.
Koyama
S
S
Shioda
T
T
Yamada
K
K
Kawasaki
T
T
D000263:Adenylate Kinase / Q000302:isolation & purification / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D013836:Thiamin Pyrophosphokinase / Q000302:isolation & purification* / Q000378:metabolism; D013831:Thiamine / Q000097:blood* / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000096:biosynthesis / Q000097:blood; D013838:Thiamine Triphosphate / Q000096:biosynthesis / Q000097:blood
D013838:Thiamine Triphosphate; D000263:Adenylate Kinase; D013836:Thiamin Pyrophosphokinase; D013835:Thiamine Pyrophosphate; D013831:Thiamine