Yoshitsugu Nose

Carrier-mediated transport of thiamine in baker's yeast

Abstract 1. 1. The characteristics of a system of [ 14 C]thiamine uptake were investigated in bakers yeast ( Saccharomyces cerevisaie ). 2. 2. [ 14 C]Thiamine uptake by the system was found to be an energy- and temperature-dependent process which has an optimal pH at 4.5. 3. 3. Most of [ 14 C]thiamine taken up existed intracellularly in free form, which accumulated against a large concentration difference. 4. 4. The apparent K m for [ 14 C]thiamine uptake was 1.8·10 −7 M, and the uptake of labeled thiamine was inhibited by several thiamine analogues, except for oxythiamine. 5. 5. Short-chain fatty acids (C 2 –C 6 ) strongly inbibited [ 14 C]thiamine uptake. 6. 6. Caproate, which has a most pronounced inhibitory effect on [ 14 C]thiamine uptake, brought about the exit of [ 14 C]thiamine taken up. 7. 7. Pyrithiamine inhibited significantly [ 14 C]thiamine exit caused by caproate, whereas oxythiamine did not. 8. 8. It was discussed whether the uptake of thiamine in bakers yeast occurs by a carrier-mediated active process.

Thiamine uptake in Escherichia coli: I. General properties of thiamine uptake system in Escherichia coli

Abstract Properties of a system of 14 C-thiamine uptake have been investigated in Escherichia coli . Uptake of 14 C-thiamine by the system was found to be an energy- and temperature-dependent process which has an optimal pH at 6.5 and follows Michaelis-Menten kinetics; apparent K m was 8.3 × 10 −7 m and V max was 1.1 × 10 −10 moles per min per mg dry weight of the cells. It was characteristic in this system that 14 C-thiamine accumulated as 14 C-thiamine pyrophosphate but not as free 14 C-thiamine. This was confirmed by paper chromatography of the extract of the cells exposed to 14 C-thiamine at 37 °. Intracellular concentration of 14 C-thiamine (pyrophosphate) accumulated when incubated with 1 μ m 14 C-thiamine was approximately 1.75 × 10 −7 moles per ml cellular fluid at equilibrium. This was 30-fold the initial intracellular thiamine concentration and 175-fold the external thiamine concentration. Both pyrithiamine and oxythiamine, anti-thiamine compounds, competed with 14 C-thiamine for the uptake system under conditions in which thiamine kinase (ATP:thiamine pyrophosphotransferase, EC 2.7.6.2.) in the membrane fraction was not inhibited by these analogues. A role of membrane thiamine kinase of E. coli probably participating in a process of the uptake to accumulate 14 C-thiamine as thiamine pyrophosphate in the cell was discussed.

Thiamine transport mutants of Saccharomyces cerevisiae.

Abstract Two mutants, PT-R1 and PT-R2, which are resistant to the inhibitory action of pyrithiamine, were isolated by nitrosoguanidine treatment from Saccharomyces cerevisiae . They were found to be, respectively, partially and almost totally defective in the thiamine-specific transport system. The mechanism of resistance of the mutants to pyrithiamine is discussed.

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae.

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo. In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent Kd of the binding for thiamine was 29 nM which is about six fold lower than the apparent Km (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and O-benzoylthiamine disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R2) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent Kd of 0.17μM at optimal pH 5.0 which is almost the same with the apparent Km for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and O-benzoylthiamine disulfide. In addition, it was found that membrane fraction prepared frtom PT-R2 has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae. These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.

Purification of thiamine-binding protein from Escherichiacoli by affinity chromatography

Abstract An affinity column coupled with thiamine pyrophosphate quantitatively absorbs the thiamine-binding activity from a partially purified preparation of Escherichia coli . The thiamine-binding protein can be eluted from the affinity column in high yield by use of 8 M urea-containing buffer. Approximately 90-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogenous.

Thiamine uptake in Escherichia coli: II. The isolation and properties of a mutant of Escherichia coli defective in thiamine uptake

Abstract A mutant (KG 900) of Escherichia coli, which requires a high concentration of thiamine for growth, has been isolated from a strain KG 33, which is a mutant of E. coli K12, auxotrophic for the thiazole moiety of thiamine and requires a low concentration of either thiamine or thiamine thiazole for growth. The isolation procedure includes mutation by nitrosoguanidine treatment, subsequent growth at a high concentration of thiamine, and penicillin screening during cultivation of the cells at a low thiamine concentration. Thiamine concentration required for a half maximal growth of KG 900 was approximately 150-fold higher than that of the parent KG 33. Thiamine thiazole requirement for growth of KG 900 is only 2-fold that of KG 33. Uptake of 14C-thiamine by cells of KG 900 is negligible when incubated with 0.2, 1.0, and 5.0 μpm14C-thiamine, respectively, for 30 min at 37 ° in the presence of 0.4% glucose, whereas the parent cells take up 14C-thiamine readily from the external medium. The rates of exit of 14C-thiamine taken up by cells of both strains were almost identical. With KG 900 the initial rate and total extent of exchange at 0 ° between a high intracellular thiamine concentration and a low level of extracellular 14C-thiamine were approximately a half as great as those observed with KG 33. Thiamine kinase activity in the sonic membrane fraction, and the activities of four soluble enzymes involved in thiamine synthesis from the pyrimidine and thiazole moieties were higher in preparations from KG 900 than those from the parent strain, KG 33. The results strongly suggest that the defect manifested by strain KG 900 is in the passage of thiamine through the cell membrane. A model for the system of thiamine uptake in E. coli is presented, which assumes that a “carrier” specific for thiamine functions in transporting thiamine across the cell membrane and then thiamine kinase (ATP-thiamine pyrophosphotransferase, EC 2.7.6.2) in the membrane facilitates accumulation of thiamine as thiamine pyrophosphate in the cytoplasm.

Biogenesis of cocarboxylase in Escherichia coli: a novel enzyme catalyzing the formation of thiamine pyrophosphate from thiamine monophosphate.

Abstract An enzyme catalyzing the formation of thiamine pyrophosphate from thiamine monophosphate in the presence of ATP and Mg++ has been found in the soluble fraction of Escherichia coli K12. The enzyme was markedly stimulated by either potassium or ammonium ion. The physiological significance of the enzyme in the biogenesis of cocarboxylase in E. coli is discussed.

Conversion of thiamine to thiamine monophosphate by cell-free extracts ofEscherichia coli

Abstract Enzymatic synthesis of thiamine monophosphate from thiamine in the presence of ATP and Mg2+ by cell-free extracts of Escherichia coli K 12 was demonstrated. The enzyme was markedly stimulated by either K+ or NH4+, and the apparent Km for thiamine was 2.8 · 10−6 M.

Affinity chromatography of thiamin pyrophosphokinase of rat brain

Affinity column chromatography coupled with thiamin monophosphate absorbs thiamin pyrophosphokinase activity in the crude extract of rat brain, and the enzyme can be eluted from the column by 0.01 mM thiamin with approximately 700-fold purification.