Clark J. Gubler

Studies on the physiological functions of thiamine

Abstract A specific and highly sensitive assay for rat brain thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was developed utilizing the recombination of yeast apotransketolase ( d -sedoheptulose-7-phosphate: d -glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) with thiamine pyrophosphate to form active transketolase. Contaminating thiamine pyrophosphokinase activity was removed from the apotransketolase by chromatography on DEAE-cellulose. With this assay 10 pmoles of thiamine pyrophosphate could be assayed. Thiamine pyrophosphokinase was purified 12-fold from rat brain acetone powders. The pH optimum was 7.8 in glycylglycine buffer, and the Km was found to be 0.02 M for ATP and 0.12 μM for thiamine pyrophosphate. Pyrithiamine gave a Ki of 0.20 μM and oxythiamine 0.15 mM. Oxythiamine was phosphorylated to give an active inhibitor of transketolase.

Molecular weight and coenzyme content of pyruvate decarboxylase from brewer's yeast.

Summary The molecular weight of pyruvate decarboxylase (EC 4.1.1.1), isolated by two different methods from two brewers yeasts, was found to be 175 000 by centrifugation in the analytical ultracentrifuge and in the preparative ultracentrifuge in a sucrose density gradient, using catalase and glucose oxidase as internal reference standards. Determination of the coenzyme content in the gradient fractions by fluorescence assay of thiochrome gave three thiamine pyrophosphates per enzyme molecule. An increase in activity of about 30%, when the holoenzyme was saturated with thiamine pyrophosphate and Mg2+, indicated that the protein molecule probably has a fourth binding site for the coenzyme. Urea treatment gave no cleavage of pyruvate decarboxylase into subunits, but denatured the enzyme almost irreversibly and partially split off the thiamine pyrophosphate.

High-pressure liquid chromatography of α-keto acid 2,4-dinitrophenylhydrazones

Abstract Various α-keto acids were separated as their 2,4-dinitrophenylhydrazine derivatives by ion-pair, reverse-phase, high-pressure liquid chromatography. Excellent baseline resolution was obtained for a seven-component homologous series of α-keto acid dinitrophenylhydrazones at increasing carbon-chain length. Branched-chain keto acids were also separated. Resolution of syn and anti isomers of the α-keto acid derivatives was possible. Pyruvate from biological material was located and identity confirmed by an enzymic peak shift technique. Monitoring at 366 nm permits low-level (nanogram) amounts of keto acids to be detected. Ion pair versus ion exchange is discussed with regard to the mechanism of chromatographic separation.

Separation of Thiamin, Thiamin Antagonists and Their Phosphate Esters by High-Performance Liquid Chromatography

Abstract Methods are presented for the separation of the phosphate esters of thiamin, oxythiamin or pyrithiamin and the separation of thiamin and oxythiamin by high-performance liquid chromatography. Detection by means of a UV monitor and fluorometer with a post-column oxidation system is described which allows for selective detection of thiochrome, pyrichrome and their respective phosphate esters. Anion exchange and ion-pair reverse phase chromatography are the chromatographic modes of separation.

Prostaglandin associated mortality following intravenous injection of catfish epidermal secretions in rabbits

Toxicity of soluble protein extracts from epidermal gel secretions of the catfish, Arius thalassinus, was examined in rabbits. Intravenous injections containing doses as low as 2 mg protein/kg body weight caused mortality in all animals tested. An increase in plasma levels of thromboxane B2 (TXB2) and of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) were observed following injections. Both the mortality and prostaglandin release were prevented by pretreatment of rabbits with either indomethacin or hydrocortisone. A similar indomethacin sensitive induction of prostaglandin release was noted following the in vitro treatment of arterial tissue sections with gel. Lethality appears to result from gel substances stimulating phospholipase activity to yield arachidonic acid, which is then metabolized to give toxic levels of prostaglandins.

A potent thromboxane formation inhibitor in green tea leaves

A ninhydrin positive compound (L2) now identified as 2-amino-5-(N-ethylcarboxyamido)-pentanoic acid, from unprocessed tea leaves was a potent inhibitor of thrombin-stimulated thromboxane formation in rabbit whole blood (Ali and Afzal; Prostaglandins, Leukotrienes and Medicine, 27: 9, 1987). In the present study, processed and unprocessed tea leaf extracts were given to rats to consume for a period of eight weeks. Cholesterol and thromboxane levels were measured in the serum obtained from clotting the blood at 37 degrees C. A significant reduction in thromboxane levels was observed in rats taking unprocessed tea extract. This reduction was equally distributed in adult as well as in juvenile rats. However no appreciable changes in the levels of thromboxane were noticed in the serum of rats taking processed tea extracts. This might be due to the presence of a labile component which is destroyed during the processing of green tea leaves. A decreased level of cholesterol was observed in rats consuming unprocessed tea extract. This decrease could be linked to the decrease in thromboxane levels as observed. Processed tea refers to commercially available tea of different brands while unprocessed tea refers to dried green tea leaves.

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.

Studies on the physiological functions of thiamine. V. Effects of thiamine deprivation and thiamine antagonists on blood pyruvate and lactate levels and activity of lactate dehydrogenase and its isozymes in blood and tissues.

Abstract Rats were made thiamine deficient by thiamine deprivation and treatment with the thiamine antagonists, oxythiamine and pyrithiamine. Thiamine deprivation and oxythiamine treatment caused significant elevations of blood pyruvate and lactate, the changes being greatest after oxythiamine. Neither had any effect on plasma lactate dehydrogenase activity. Pyrithiamine-treated rats showed no elevation in blood lactate nor pyruvate if tested before convulsions started, but showed marked elevations in both, and also in plasma lactate dehydrogenase activity after 24–36 h in convulsions. No significant changes were observed in lactate dehydrogenase activity in brain and kidney in thiamine-deprived and pyrithiamine-treated rats, but an increase was observed in oxythiamine-treated rats. A decrease in lactate dehydrogenase activity was found in the heart in thiamine-deprived rats and in the liver of rats exhibiting all three types of experimental deficiencies. A decrease in the relative abundance of LDH 3 and LDH 4 isozymes and an increase in LDH 1 was observed in the hearts of thiamine-deprived, oxythiamine-treated and pyrithiamine-treated rats.

Changes in the levels of lactic dehydrogenase and transketolase in liver and red cells of rats after treatment with garlic extracts

Summary Rats were injected intraperitoneally with either raw or boiled garlic aqueous extracts. Red cells were assayed for lactate dehydrogenase and transketolase activities while homogenized liver tissue was assayed for total thiamin content. Lactate dehydrogenase activity in liver and lactic dehydrogenase and transketolase activities in the red cells were elevated as a result of treatment with garlic aqueous extracts. The increase was more prominent in the case of raw garlic extract treatment. Neither treatment had any effect on liver thiamin content.

Amino acid composition of cytoplasmic yeast pyruvate decarboxylase

Fyruvate decarboxytase was isolated essentially as described earlier [l] from Saccizaronz,wes carlsbergensis: (a) fresh brewer’s yeast, strain Weihenstephan T34, obtained from Canter-Brauerei, D-78 Freiburg, with a slight modification of the get filtration procedure: (b) dried brewer’s yeast, regular strain, obtained from Anheuser-Busch Inc., St. Louis. MO., USA. The best fractions of 3 different enzyme preparations from source (a) and one from source (b) were exhaustively diatysed against water at 3” for 2---3 days and freeze-dried. Two thirds of the original specific activity of 62, 59, and 74 units/mg [t] for (a) (the highest specific activity reached as yet was 85 units/ mg) and 60 units/mg for (b) were lost during this procedure, the apoenzyme lost all activity by this treatment. 7-10 mg of the resulting hygroscopic white