Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides.

Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolfrom derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated.     

Blood thiamine and thiamine phosphate ester concentrations in alcoholic and non-alcoholic liver diseases.

Thiamine state was investigated in patients with alcoholic liver disease, patients with various non-alcoholic liver diseases, and controls using a direct technique (thiochrome assay) to measure thiamine, thiamine monophospate, and the active coenzyme thiamine pyrophosphate in whole blood after isolating the fractions by ion exchange chromatography. Overall nutrition was similar in all groups as assessed by anthropometry, and no patient had clinical evidence of thiamine deficiency. There was no significant difference among the groups in mean concentration of any form of thiamine. The scatter was much greater in patients with alcoholic liver disease but only 8.7% had biochemical thiamine deficiency (defined as a blood concentration of the active coenzyme greater than 2 SD below the mean control value). An unexpected finding was of abnormally high total thiamine concentrations (greater than 2 SD above the mean control value) in 17.4% of patients with alcoholic liver disease, the highest concentrations being found in two patients with severe alcoholic hepatitis and cirrhosis. The ratio of phosphorylated to unphosphorylated thiamine was calculated as an index of phosphorylation and, although the mean did not differ significantly among the groups, the range was greatest in alcoholic liver disease. The lowest ratios occurred in the two patients with severe alcoholic hepatitis, but neither had evidence of thiamine pyrophosphate deficiency. Contrary to studies using indirect assay techniques, these results suggest that thiamine deficiency is unusual in well nourished patients with alcoholic liver disease. The new finding of unexpectedly high thiamine concentrations in some patients may be due to abnormalities of hepatic storage or release in liver disease, particularly in severe alcoholic hepatitis. There was no convincing evidence of impaired thiamine phosphorylation in any patients with liver disease. Conclusions from studies using indirect assays on the prevalence and mechanisms of thiamine deficiency in liver diseases may not be valid.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Regulation of thiamine transport in Saccharomyces cerevisiae.

Yeast cells were found to be repressed for the uptake of both thiamine and pyrithiamine by growth with exogenous thiamine, and they appeared to regulate the activity of the binding protein for these compounds.     

Membrane-associated thiamine triphosphatase in rat skeletal muscle.

1. Thiamine triphosphatase activity in particulate fraction, but not in soluble, of rat skeletal muscle was stimulated by several anions. 2. The stimulative effect of anions was dependent on pH of reaction medium and was reversible. 3. The activities of ATPase in rat muscle particulate preparation and thiamine triphosphatase in the brain were inhibited by the anions.     

Specific thiamine monophosphate phosphohydrolase in Micrococcus denitrificans.

A phosphohydrolase specific for thiamine monophosphate was isolated from Micrococcus denitrificans, partially purified approximately 100-fold, and separated from acid phosphatase.     

Regulation of thiamine biosynthesis in Saccharomyces cerevisiae.

A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine. The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant. On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S. cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells. These results suggest that thiamine synthesis in S. cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.     

Identification of the two missing bacterial genes involved in thiamine salvage: thiamine pyrophosphokinase and thiamine kinase

The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.