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.     

Interaction of rat brain thiamine kinase with thiamine and its derivatives

Participation of the enzyme which provides the phosphorylation of thiamine to thiamindiphosphate (TDP) thiaminkinase (thiaminpyrophosphokinase, KF 2.7.6.2) of rat brain in the realization of thiamine action on the syntheses of acethylcholine (AC) was studied. The thiamine and its structure analogue, which differ the nature of the radicals in the 3-d and 5-e positions of the thiazollium cycle were used: 3-[(4-amino-2methylpyrimidinyl-5)methyl]-4-methylthiazolium chloride, 3-decyloxycarbonylmethyl-4-metyl-5-beta-hydrozyethylthiazolium chloride, 3-decyloxycarbonylmethyl-4-methylthiazolium chloride. All salts in the concentrations lower then Km render active influence on thiaminkinase. The analysis of data shows the presence of the regulation site on the enzyme distinguishing from the active enzyme centre and participating in the interaction with which the hydrophobic fragments of thiamine molecule participating. The comparative studies of thiamine and above mentioned derivatives influence on the inclusion of the labelled carbon with [2-(14)C] pyruvate in acethylcholine confirm an assumption about the key-role of the thiamine interaction with thiaminkinase (meaning its phosphorilation) regarding its action on the acethylcholine syntheses, and probably, on the function of the nervous cells as a whole.     

Action of the thiamine antagonist bacimethrin on thiamine biosynthesis

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.     

Effect of thiamine and its derivatives on acetylcholinesterase activity of the blood and brain of albino mice

Thiamine, thiaminepyrophosphate and 4-methyl-5-beta-oxyethylthiazole are studied for their effect on the acetylcholinesterase activity in the brain, blood plasma and cells. The activity of acetylcholinesterase in blood cells is shown to be inhibited most of all by thiamine and thiazole. Acetylcholinesterase of the brain has been efficiently inhibited only by thiamine pyrophosphate.     

Thiamine, thiamine phosphates and thiamine metabolizing enzymes in synaptosomes of rat brain

Thiamine and thiamine mono-, pyro- and triphosphate were found at detectable levels in synaptosomes isolated from whole rat brain. Synaptosomes prepared from whole brain, cerebellum and medulla were also found to contain uridine and inosine mono- and diphosphatases as well as the thiamine pyrophosphate synthetizing and hydrolyzing enzymes, but no thiamine monophosphatase. By isoelectric focusing on thin layer polyacrylamide gel of Triton X-100 homogenates of synaptosomes, thiamine pyrophosphatase activity could be separated into 10 bands with different isoelectric points. The contents of thiamine compounds and enzymes in synaptosomes were generally lower than those found in neuronal cell bodies.     

Simultaneous liquid chromatographic assessment of thiamine,thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics

An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (P<0.001) and TDP=127.4+/-62.5 nmol/l (P<10(-5)). The diagnostic role of TDP was evaluated and a significant role for thiamine was established in the study of alcohol related problems.     

Relative reactivity of thiamine monophosphate and thiamine diphosphate upon interaction with alkaline phosphatase

Reactivity of thiamin monophosphate (TMP) as calf intestinal alkaline phosphatase substrate in model transformations is lower comparing with thiamin diphosphate (TDP) reactivity. Under these conditions alkaline phosphatase catalyzes TDP, ADP and AMP hydrolysis approximately at same rate. It was shown that TDP competes with p-nitrophenyl phosphate more effectively than TMP for the binding in the active site. At pH 8.5 and 30 degrees C Km values are as follows: (5.2 +/- 1.6) x 10(-3) M for TMP and (3.0 +/- 0.8) x 10(-4) M for TDP. Under the same conditions the Vmax/Km value for TDP hydrolysis is 53 times higher than the one for corresponding reaction of TMP. It was suggested that positively charged thiazolium ion of TMP interacts with the nearest environment at the active center and by this way reduces enzyme activity.     

Phosphorylation of some thiamine analogs by yeast thiamine pyrophosphokinase]

A rapid efficient method of separation of the thiamine pyrophosphokinase reaction products (ATP: thiamine pyrophosphotransferase) on the column packed with DEAE-Sephadex A-25 and their subsequent identification by direct spectrophotometry is suggested. Phosphorylation of some thiamine analogs substituted at the second position of the pyrimidine ring was studied. It was shown that in addition to thiamine, the enzyme transfers the pyrophosphate group to some of its derivatives. The vitamin analogs devoid of quaternary nitrogen in the thiazole cycle, do not form pyrophosphate ethers (thus being unable to act as substrates), whereas 2'-phenoxythiamine, 2'-methoxythiamine and especially 2'-phenylthiamine are phosphorylated at a greater rate than does the "true" substrate, thiamine, under similar conditions.