Radiometric assay procedure for thiamine pyrophosphokinase activity

Deus, B.; Blum, Hubert E.; Holzer, Helmut

doi:10.1016/0003-2697(69)90063-3

Cited by 18 publications

(3 citation statements)

References 17 publications

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“…The possible relationship between thiamine transport and phosphorylation in isolated hepatocytes was not investigated in the present study. It is known that phosphorylation of thiamine is catalyzed by thiamine pyrophosphokinase which is primarily associated with a soluble fraction of the subcellular prepara tions (8,30,31), however, several studies have indicated that this process may not play any significant role in the transport of thiamine across cellular mem brane (11,19,21). In the present study, there seemed no significant metabolic alteration of thiamine during the experimental periods (Fig.…”

Section: Discussioncontrasting

confidence: 43%

Active transport of thiamine by freshly isolated rat hepatocytes.

Chen

1978

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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SummaryRat hepatocytes were freshly prepared from adult animals using the collagenase-perfusion technique. The hepatic transport of thiamine was studied in isolated liver cells. The process was found to be saturable with an apparent Kt of 0.31mM and a Vmax of 0.7 umoles/ml intracellular fluid/5 minutes. However, at higher substrate concentrations, the process proceeded in a linear fashion. Both pyrithiamine and oxythiamine were inhibitory on the hepatic uptake of thiamine, the latter showed much weaker activity than the former. The system required the presence of sodium ions and was sensitive to ouabain. Anaerobic condition and metabolic inhibitors, e.g., 2,4-dinitrophenol, cyanide, and iodoacetate suppressed the uptake rate of thiamine. Addition of ethanol in the incubation medium also caused significant reduction of thiamine uptake. Efflux studies indicated that a portion of intracellular thiamine is readily available for exodus. Chromatographic analyses showed that thiamine was only slightly metabolically altered during the transport process. It is suggested that thiamine is transported into isolated hepatic cells by an active, sodium-dependent process.

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Section: Discussioncontrasting

confidence: 43%

Active transport of thiamine by freshly isolated rat hepatocytes.

Chen

1978

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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“…Preparation andpurification of p5S]TPP. This was prepared from [35S]thiamin and ATP by using thiamin pyrophosphokinase (ATP-thiamin pyrophosphotransferase, EC 2.7.6.2) isolated from rat liver by an extensive modification of the methods of Mano (1960) and Deus et al (1969). Unless stated otherwise, operations were carried out at 4°C.…”

Section: Experimental Materialsmentioning

confidence: 99%

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation

Walsh¹,

Cooper²,

Denton³

et al. 1976

Biochemical Journal

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1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...

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“…TPT activity at each purification step was determined by the direct radiometric assay of Deus et al (1969) as modified by Howie (1990) and Molin and Fites (1980), Standard assay final concentrations at pH 7,3 were: 50 mM Tris, 10 mM 2-ME, 10 ^M thiamin (925 GBq mor'), 5 mM MgClj and 270 nM GTP, Pyrophosphorylation of thiamin via TPT requires a divalent cation for the formation of the NTP active complex. Molin and Fites (1980) previously showed that Mg'* was the most effective divalent cation.…”

Section: Standard Tpt Assaymentioning

confidence: 99%

GTP‐specific pyrophosphorylation of thiamin in dark‐grown soybean (Glycine max) seedling axes

Howle

Fites

1991

Physiologia Plantarum

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ATP:thiamin pyrophosphotransferase (TPT: EC 2.7.6.2) was purified 5 900‐fold from 48 h dark‐grown soybean [Glycine max (L.), Merr. cv. Ransom II] seedling axes. TPT activity was monitored during purification by measuring the formation of thiamin pyrophosphate (TPP) from [2‐14C]‐thiamin at optimal pH (7.3). Although other nucleoside triophosphates were active as pyrophosphate donors (apparent Kms from 21 to 138 mM), GTP was the preferred nucleotide with an apparent Km of 0.021 mM. TPT activity was extremely sensitive to TPP formation, suggesting product feedback inhibition of TPT activity in vivo. Sulfhydryl, H+ and Mg2+ concentrations, either independently or in concert, were found to affect TPT activity.

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Radiometric assay procedure for thiamine pyrophosphokinase activity

Cited by 18 publications

References 17 publications

Active transport of thiamine by freshly isolated rat hepatocytes.

Active transport of thiamine by freshly isolated rat hepatocytes.

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation

GTP‐specific pyrophosphorylation of thiamin in dark‐grown soybean (Glycine max) seedling axes

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