Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.     

Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]

The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC 2.7.6.2), pyruvate dehydrogenase (EC 1.2.4.1) and transketolase (EC 2.2.1.1) with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.     

Interrelationship between the thiamine content, thiamine diphosphate-dependent enzyme activity and reduced glutathione level in the rat liver

Changes in the amount of thiamine, reduced glutathione, thiamine diphosphate-dependent dehydrogenase activity has been traced after thiamine injection to thiamine-deficient rats and oxythiamine to normal rats. The obtained data show that a drop in reduced glutathione level was a primary reason of the alpha-keto-acid dehydrogenase activity reduction under conditions of the thiamine deficiency. The existence of immediate connection between thiamine and glutathione metabolism is supposed.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

The stimulation by treatment in vivo with tri-iodothyronine of amino acid incorporation into protein by isolated rat-liver mitochondria

1. Normal and thyroidectomized rats were treated with near-physiological doses of tri-iodothyronine. Liver mitochondria were isolated and incubated with radioactive amino acids. In normal rats tri-iodothyronine caused only a slight stimulation of incorporation into mitochondrial protein, but in thyroidectomized animals the incorporation was doubled. 2. There was a lag period of about 36 hr. after injection and the maximum effect was observed after 2 days. 3. Direct addition of tri-iodothyronine to the incubation medium had no effect on mitochondrial incorporation. 4. The incorporation was not due to bacterial, nuclear, lysosomal or microsomal contamination and the labelled particles had sedimentation properties identical with those of mitochondria, as followed by suitable enzyme markers. 5. Thyroid hormone treatment did not cause any marked alterations in the pattern of labelling of submitochondrial fractions and in all cases the most radioactive protein was in an insoluble lipid-rich fraction. The amino acid compositions of the total mitochondrial protein and the more radioactive lipoprotein were also unaltered. 6. Increases in the content of RNA and various cytochromes per mg. of mitochondrial protein were observed after treatment with tri-iodothyronine. These occurred slightly later than the stimulation of amino acid incorporation. 7. No uncoupling of oxidative phosphorylation was observed and the ATP production per mg. of mitochondrial protein increased. 8. It was concluded that tri-iodothyronine stimulated amino acid incorporation into mitochondrial protein and that the result is consistent with the view that treatment with thyroid hormone results in an enhanced selective synthesis of mitochondrial respiratory units.     

Effect of naloxone and D-Met2-Pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met²-Pro⁵-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of ³H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of ³H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met²-Pro⁵-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.     

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue

Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 hours. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [³⁵S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro.     

Transfer of proteins synthesized in the cytoplasm into mitochondria. Stimulation of mitochondrial protein synthesis by microsomal fraction]

The in vitro transport into mitochondria of proteins synthesized in the cytoplasm was studied. The system, in which the microsomes synthesize protein in the presence of mitochondria directly during the experiment proved to be the most efficient one. The microsomal fraction significantly stimulated the incorporation of 14C-valine into the isolated mitochondria proteins. The effects of EDTA treatment of the mitochondrial fraction, the dependence of protein synthesis stimulation on the ratio of mitochondria and microsomal proteins and the kinetic pattern of the reaction suggest that the stimulation of the labelled precursor incorporation into mitochondrial proteins is not probably due to the labelled microsomes adsorption on the mitochondria.     

Effects of cycloheximide,d-threo-chloramphenicol,erythromycin and actinomycin D on De-novo synthesis of cytoplasmic and mitochondrial proteins in the cotyledons of germinating pea seeds

Summary Inhibitors of, and radioactive substrates for, protein synthesis were introduced into germinating pea (Pisum sativum L.) seeds, and protein synthesis was allowed to proceed in vivo. Subsequent analyses of subcellular fractions showed the following: Cycloheximide strongly inhibited the incorporation of [¹⁴C]leucine into both mitochondrial and cytoplasmic proteins. d-Threo-chloramphenicol and erythromycin did not affect cytoplasmic protein synthesis, but partially inhibited mitochondrial protein synthesis. These results suggest that most of the new mitochondrial proteins were originally synthesized in the cytoplasm. Actinomycin D did not appreciably affect the initial incorporation of [¹⁴C]leucine into either mitochondrial or cytoplasmic proteins, suggesting that information (mRNA) concerning the initially synthesized proteins may be present in the quiescent seeds. The lack of appreciable incorporation of [³H]thymidine into mitochondrial DNA supported our previons report that mitochondria may not be synthesized de novo in pea cotyledons.     

A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.     

High adrenaline sensitivity of the mitochondrial processes in the mucosa of the small intestine in rats

Higher adrenalin sensitivity of mitochondrial processes in the small intestinal mucosa compared to that in liver mitochondria, was revealed under specially devised conditions of work with isolated mitochondria retaining their natural properties. Fifteen minutes after intraperitoneal injection of adrenalin into rats in a dose of 5 micrograms/100 bw an increase in Ca2+ capacity was seen only in intestinal mucosa mitochondria. The adrenalin-induced activation of oxidative phosphorylation was more remarkable in intestinal than in liver mitochondria at the initial stages of adrenalin action. The effect of adrenalin was completely reversed by the beta-blocker propranolol only in liver mitochondria. After 3 hours the adrenalin-induced activation of phosphorylation in the mitochondria ceases, whereas in the small intestinal mucosa it still persists.     

Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea

The reaction of the carcinogen N-methyl-N-nitrosourea with mitochondrial DNA from various rat tissues was examined in vivo and in vitro. After incubation of isolated mitochondria or cell nuclei with N[(14)C]-methyl-N-nitrosourea in vitro and subsequent isolation and purification of the DNA the specific radioactivity of the mitochondrial DNA was 3-7 times that of the nuclear DNA. The incorporation of (14)C into embryonic mitochondrial DNA in vitro was about twice that into the liver mitochondrial DNA. Identical incorporation rates, however, were found for the reaction of isolated mitochondrial DNA or nuclear DNA respectively with N[(14)C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3-58.5mg of N[(14)C]-methyl-N-nitrosourea/kg body wt. to adult rats the labelling of the mitochondrial DNA was on average 5 times that of the nuclear DNA. A smaller specific labelling was observed for the ribosomal RNA, transfer RNA, and mitochondrial RNA as well as for the mitochondrial protein as compared with the mitochondrial DNA. After hydrolysis of the alkylated nucleic acids with hydrochloric acid, fractionation was carried out on Dowex 50 cation-exchange columns. In most experiments 70-80% of the input (14)C radioactivity was eluted in the 7-methylguanine fraction. The preferential alkylation of the mitochondrial DNA by N-methyl-N-nitrosourea in situ is discussed in connexion with the cytoplasmic-mutation hypothesis of carcinogenesis.     

Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats

Hepatic mitochondria isolated from rats 40 h after dosage with 1.1 ml/kg CCl4 are uncoupled and display structural damage. Mitochondrial function returns during hepatic recovery. Because the products of mitochondrial protein synthesis are essential to mitochondrial structure and function, the effects of CCl4 on the rate of mitochondrial protein synthesis, and on the products, was studied using mitochondria from CCl4-exposed rats during the early, maximum development and resolution stages of CCl4-induced mitochondrial damage. Rates of mitochondrial protein synthesis (incorporation of [35S]methionine) were elevated 300% over that of mitochondria from non-exposed rats 17 h after exposure; depressed by 50% at 40 h and above control at 113 h. When the radiolabeled products of incorporation were separated and examined by autoradiography, a novel, low-molecular-weight band, of approx. 9700, was apparent 40 h after CCl4 exposure. A band of similar molecular weight appeared when control mitochondria were incubated without an exogenous supply of ATP. Mitochondria from exposed rats which displayed rates of protein synthesis greater than control consistently had a relative increase in a band that corresponded in size to that of cytochrome oxidase subunit I. It was concluded that the loss of mitochondrial function induced by CCl4 could not be attributed to inhibition of mitochondrial protein synthesis, and that the mitochondria may not always synthesize protein in constant proportions.     

Effect of temperature on protein synthesis and leucine transport by yeast mitochondria

Protein synthesis in yeast mitochondria shows biphasic Arrhenius plots both in vivo and in vitro, with a twofold increase in the activation energy below the transition temperature suggesting a functional association between mitochondrial protein synthesis and the inner membrane. Analysis by gel electrophoresis of mitochondrial translation products labeled in vivo showed that the same proteins are synthesized and then inserted into the membrane above and below the transition temperature of the membrane. The rate of leucine uptake into mitochondria was decreased at least fivefold in the presence of chloramphenicol, suggesting that leucine is used mainly for protein synthesis. In the absence of chloramphenicol, the rate of leucine uptake was always slightly higher but comparable to the incorporation rate of leucine into protein at all temperatures studied, suggesting that the transport of leucine into mitochondria is not rate-limiting for protein synthesis. The ionophore valinomycin or the uncoupler carbonyl phenylhydrazone (CCCP) inhibited 75-80% of the leucine uptake in the presence of chloramphenicol. In addition, the omission of respiratory chain substrates and the ATP-regenerating system led to a 93% inhibition of uptake, suggesting that leucine uptake may occur by an active transport mechanism.     

Effects of cortisol on lipid and lipoprotein synthesis in rat hepatocytes]

Under in vivo conditions cortisol induces moderate hyperlipidemia followed by an increase in the phospholipid and triglyceride concentrations in the blood and a decrease of cholesterol; similar changes were observed in the liver. At all time intervals studied cortisol inhibits the phospholipid and cholesterol syntheses and decreases the specific radioactivities of the lipids in the mitochondrial fraction. The hormone has an inhibiting effect on the fatty acid synthesis at early postinjection stages. The phospholipid synthesis is increased after adrenalectomy and is then inhibited after injection of the hormone. A single injection of ACTH or cortisol causes suppression of phospholipid and cholesterol syntheses and a decrease in their specific radioactivities in the mitochondria. A similar effect is observed under stress conditions. In addition, the hormone inhibits the synthesis of lipoprotein apoproteins of very low and high densities. After 5 hours following the hormone injection the lipoprotein apoprotein synthesis in the liver is activated; the activation of apoprotein synthesis is also observed after adrenalectomy. However, the injection of the hormone to adrenalectomized rats decreases the apoprotein synthesis. It was shown that in blood serum cortisol affects the conversions of very low density lipoproteins into low density lipoproteins, thus providing for hyperlipidemia.     

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.