Nutrition of Threonine

The threonine content in blood and urine increased and threonine decomposition ability in liver decreased by feeding lower level of lysine, whereas threonine content in blood and urine decreased and the ability of liver increased gradually with increasing lysine content in diet. These phenomena were owing to the increase of threonine dehydratase activity of liver, which was measured from produced α-ketobutyric acid amount, by excess administration of lysine. The phenomena that threonine content in urine decreased and threonine decomposition ability of liver increased with increasing threonine content in diet when adequate amount of lysine was fed, were also ascribed to the increase of the dehydratase activity.

One m mole of threonine was incubated with liver homogenate in presence of PALP*** at pH 8.2 for 20 and 30 min and α-ketobutyric acid produced was introduced to its 2,4-dinitrophenylhydrazone, which was chromatographed on silica-gel thin-layer plate and determined spectrophotometrically at 395 mμ under N,N-dimethylformamide.

Other enzyme systems relating to threonine catabolism were also investigated, including threonine aldolase, threonine dehydrogenase and ornithine transaminase, showing no significant changes in activities by excess administration of lysine and/or threonine.     

L-threonine aldolase is not a genuine enzyme in rat liver.

Activity of L-threonine aldolase in rat liver cytosolic extract was not affected by the omission of alcohol dehydrogenase in a previously established NADPH-linked alcohol dehydrogenase-coupled assay. The liver extract was able to catalyse the dehydrogenation of NADPH with either acetaldehyde (a product of L-threonine aldolase action) or 2-oxobutyrate (a product of L-threonine dehydratase action). When the liver extract was chromatographed on a Sephacryl S-200 column, no threonine aldolase activity was detected in the eluate. However, activity of threonine aldolase re-appeared when the fractions with highest activity of lactate dehydrogenase and threonine dehydratase were mixed. Activity of threonine aldolase could also be abolished by removing threonine dehydratase from the liver extract with a specific antibody. Hence L-threonine aldolase should not be a genuine enzyme in the rat liver, and the apparent enzyme activity may result from a combined effect of threonine dehydratase and lactate dehydrogenase (or an oxo acid-linked NADPH dehydrogenase) in the liver cytosolic extract.     

Dominance and recessiveness at the protein level in mutant x wildtype crosses in Sacchaomyces cerevisiae

Summary The is ₁-locus of the yeast Saccharomyces cerevisiae is the structural gene for threonine dehydratase. is ₁-mutants require isoleucine for growth and do not have active threonine dehydratase.Interallelic complementation is frequent among is ₁-mutants. This is indicative for an aggregate or multimeric structure of yeast threonine dehydratase.Complementing and non-complementing mutants were crossed to wildtype. Properties of threonine dehydratase were assayed in crude extracts of the resulting heterozygotes.Specific activities varied considerably between full wildtype activity and a level about 10% of that. The apparent Michaelis constants were increased in many heterozygotes. This effect was probably due to the aggregation of both mutant and wildtype subunits to form a hybrid threonine dehydratase with reduced substrate affinity in addition to pure wildtype enzyme. This notion is supported by the observation in one heterozygote of two enzyme fractions with increased Michaelis constants in addition to a wildtype-like fraction.The possible formation of hybrid enzymes with normal, reduced or no activity is considered to blur gene dosage relations.A given pair of alleles in a heterozygous cell can generate a new type of enzyme with properties not encountered in the corresponding two homozygous cells. This situation is not accounted for by the classical concepts of dominant-recessive or intermediate behaviour, because the difference between the heterozygotes and the homozygotes is not necessarily only quantitativ but also qualitative.We dedicate this publication to Prof. Dr. C. Auerbach on occasion of her official retirement in admiration for her pioneer work and many contribution to genetics.     

Activities of Threonine Dehydratase and Histidase of Rats Fed on either Threonine or Histidine Imbalanced Diet

Liver threonine dehydratase and histidase activities of rats fed on a threonine and a histidine imbalanced diets respectively, were measured, each of which was the degradation enzyme of the most limiting amino acid for rats on each imbalanced diet. Rats of the imbalanced groups initially lost their body weight, and began to grow again after a few days. Threonine dehydratase activity decreased by changing from stock diet to the experimental diets, and no difference was observed among the control and the imbalanced groups. Histidase activity decreased gradually on the control diet, but, the enzyme activity of the histidine imbalanced group was maintained at the higher level. The inconsistency among the enzyme activities and growth showed that neither the increase of threonine dehydratase activity in threonine imbalance nor that of histidase activity in histidine imbalance would be the main cause of the imbalance.     

Surfactant enhanced l-α-glycerylphosphorylcholine production from phosphatidylcholine using phospholipase A1 in the aqueous phase

Abstract

Enzymatic production of L-α-glycerylphosphorylcholine (L-α-GPC) is difficult due to the limited solubility of phosphatidylcholine (PC) in the aqueous phase. Surfactants can be used to improve the solubility and the dispersibility of non-polar chemicals in the aqueous media. In this study, various surfactants were investigated to improve L-α-GPC enzymatic production using phospholipase A₁ (PLA₁) in the aqueous phase. The results showed that Tween 20 was the most effective surfactant for enhancing L-α-GPC concentration. With 20?g^.L^?1 of Tween 20, the optimal conditions of PC hydrolysis were determined to be enzyme loading of 0.64?g^.L^?1 and substrate concentration of 60?g^.L^?1 at 45?°C for 1?h. In addition, the fed-batch catalytic process of PC was conducted to avoid substrate inhibition and increase product accumulation, resulting in 112.56?g^.L^?1 of L-α-GPC from 360.00?g^.L^?1 PC with yield of 91.36% within 3?h.     

Threonine metabolism in Japanese quail liver

Summary. In general, threonine is metabolized by reaction catalyzed by threonine-3-dehydrogenase (TDH), threonine dehydratase (TH) or threonine aldolase (TA). The activities of these three enzymes were compared in the liver of Japanese quails and rats. The animals were fed a standard or threonine rich-diet, or fasted for 3 days. The specific activity of TDH in the liver from quail fed a standard diet was 11 times higher than that in the liver from rats fed a standard diet. The TDH activities in the livers of the fasting and 5% threonine-rich diet groups of quail were 3 and 2 times higher than those in the livers from quail fed the standard diet, respectively. The TH activity in the liver of rats fed a standard diet was 14 times higher than that in the liver of quail fed a standard diet. The TH activity in the rat liver after fasting was 2.3 times higher than that of the standard diet control. The activity of TA in the livers of rat and quail were so low that its role in threonine metabolism in both animals seemed to be negligible. These results suggest that threonine is a ketogenic amino acid in the quail liver, while it is a glucogenic in the rat liver.     

Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

The influence of B-group vitamins on monooxygenase activity of cytochrome P450 3A4: Pharmacokinetics and electro analysis of the catalytic properties

Simultaneous administration of loading doses of B-group vitamins and diclofenac allow to decrease the daily dose of this drug without reduction of its analgesic effect. In all three schemes of the diclofenac intake (diclofenac alone, diclofenac plus 2 tablets of Gitagamp (a mixture of B-group vitamins), and diclofenac plus 4 tablets of Gitagamp—maximal concentration of blood diclofenal (C_max) was observed 1 h after the treatment. In the case of diclofenac treatment alone, with 2 tablets of Gitagamp, and with 4 tablets of Gitagamp C_max values were 1137.2 ± 82.4, 1326.7 ± 122.5 and 2200.4 ± 111.3 ng/mL, respectively. Thus, loading doses of B-group vitamins caused a statistically significant effect on the C_max value of blood diclofenac concentration; they also reduced manifestations of the pain syndrome. Pharmacodynamics and pharmacokinetics data were confirmed in electrochemical studies of cytochrome P450 3A4 (CYP3A4) activity. This enzyme was immobilized onto screen printed graphite electrodes modified with gold nanoparticles and synthetic membrane-like compound didodecyldimethylammonium bromide (DDAB/Au). Electrochemical analysis revealed the influence of B-group vitamins on metabolism of the non-steroidal anti-inflammatory drug diclofenac catalyzed by cytochrome P450 3A4. Comparative analysis of the effect of 300 μM vitamins of the B-group (B₁, B₂, and B₆) demonstrated that riboflavin was the most effective inhibitor of diclofenac hydroxylation catalyzed by CYP3A4. These data support possibility of regulation of pharmacokinetic parameters and manifestation of pharmacodynamic effects by loading doses of B-group vitamins, which regulate the catalytic activity of drug metabolizing enzymes such as cytochrome P450 3A4.     

The threonine dehydratase structural gene in Aspergillus nidulans

Mutations at the ileA locus of Aspergillus nidulans can lead to loss of threonine dehydratase (l-threonine hydro-lyase (deaminating), EC 4.2.1.16), the first enzyme for isoleucine biosynthesis. A cold-sensitive allele, ileA-13, leads to production of an enzyme having an increased apparent K_m for l-threonine and showing inhibition by l-valine at concentrations which slightly stimulate activity of the wild type enzyme. Hence, ileA codes for a structural component of threonine dehydratase. The ability of very high concentrations of l-threonine to supplement ileA-13 strains at non-permissive temperatures would suggest the auxotrophy conferred by ileA-13 results primarily from the increased apparent K_m of the mutant enzyme for l-threonine.     

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B₆ (B₆) deficiency in rat liver. Male Wistar rats were fed a control, B₆-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B₆-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B₆-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B₆ deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B₆ deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.     

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A₂ and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A₂ resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A₂ is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

The energy-yielding reactions of Peptococcus prévotii,their behaviour on starvation and the role and regulation of threonine dehydratase

The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase.Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit.Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the K _m for threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic.However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity.During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

Lipoamide dehydrogenase in vitamin B2-deficient rat liver

The liver and the kidney of rats fed a B₂-deficient diet showed a decrease in lipoamide dehydrogenase (LADase) activity, 65% and 80% of those of rats fed on a B₂-supplemented diet, respectively, but the heart showed no decrease. The liver of B₂-deficient rats showed also a little decrease in the activity of glutathione reductase, while there were no differences in the activities of GOT and LDH in the liver between the B₂-deficient and B₂-supplemented rats. The activity of LADase in the cytosolic fraction of the liver, which was about one-tenth of that of mitochondrial fraction, was decreased in the B₂-deficient rats in a degree almost equal to the decrease in the activity in the mitochondrial fraction. The results obtained with single radial immunodiffusion test indicated that the decrease in the activity of LADase in B₂-deficient rats resulted from the decrease in the amount of the enzyme protein and that not only the livers of B₂-supplemented rats, but also of B₂-deficient rats contained no apoenzyme of LADase.     

Nutrition of Threonine

By administering 2 mg/day of cortisone acetate to adrenalectomized rats, the hepatic threonine dehydratase activity of these rats increased 5 times as much as that of the control. By administering 5 IU/day of ACTH to hypophysectomized rats, both the hepatic threonine dehydratase activity and the adrenal glucose-6-phosphate dehydrogenase activity increased 3 times and 7 times as much as that of the control group, respectively. The effects of excess feeding of lysine or threonine on the increase of the dehydratase activity by the adminitration of cortisone to the adrenalectomized rats and the administration of ACTH to the hypophysetomized rats were negative. When the intact rats were fed on lysine and/or threonine excess diet, the amount of glucocorticoid secretion as measured by the adrenal glucose-6-phosphate dehydrogenase activity increased and the hepatic threonine dehydratase activity increased accordingly. A linear relationship was found between these two activities and no significant deviation from the relationship due to the difference in diet composition was observed. A mechanism was proposed, based on these results, explaining the fact that the hepatic threonine dehydratase activity increased when rats were fed on lysine or threonine excess diet.     

Biochemical Studies on Fatty Liver

The relation of the fatty liver with some enzyme activities concerning the amino acid metabolism was investigated. Fatty livers were produced by an amino acid imbalanced diet containing 8% of casein supplemented with 0.3% of DL-methionine (threonine deficient), and serine dehydrase ( = threonine dehydrase = cystathionine synthetase), homoserine dehydrase ( = cystine splitting enzyme = cystathionase), and threonine aldolase activities were determined.

Under this condition, the threonine aldolase activity was hardly altered, but the serine dehydrase and the homoserine dehydrase activities were fairly variable. However, the variation of these enzyme activities did not seem to have appreciable relation with the fatty liver, but rather had a connexion with the dietary protein level or calory content.     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.