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PubMed1991

Metabolic cooperation among cell lines from patients with inborn errors of vitamin B12 metabolism: differential response of cblC and cblD.

Human skin fibroblasts derived from patients with all seven known inborn errors of vitamin B12 metabolism have been studied for functional integrity of methylmalonyl CoA mutase and methionine synthase. Cocultivation of cblC and cblF fibroblasts in the absence of polyethylene glycol resulted in a twofold increase over the expected in both [14C]propionate and [14C]methyltetrahydrofolate incorporation into acid-precipitable material, suggesting that metabolic cooperation between cells occurs. This correction in phenotype seems to be mutant class selective since cblD fibroblasts, which are biochemically similar to cblC cells, do not cooperate metabolically when mixed with cblF cells. These observations lend further support to the division of cblC and cblD diseases into discrete classes.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1991

Metabolic adaptation during erythropoietin-mediated terminal differentiation of mouse erythroid cells.

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly harvested FVA cells have adenosine triphosphate (ATP) levels of 7.23 +/- 2.52 mumol/10(10) cells or 3.76 +/- 1.31 mumol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.

2,3-Diphosphoglycerate↗

PubMed1991

[Myoglobinuria due to enzyme abnormalities in glycolytic pathway--especially lactate dehydrogenase M subunit deficiency].

Glycolysis is an important energy productive system. Enzyme abnormalities the in glycolytic pathway, which cause myoglobinuria, are deficiencies of phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, and lactate dehydrogenase (LDH). Common symptoms of these enzyme abnormalities are muscle cramp, muscle pain, and rhabdomyolysis after strenuous exercise. Acute renal failure owing to myoglobinuria is the most noteworthy symptom. In daily life, symptoms are rarely observed and prognosis is usually good. Correct and fast diagnosis of such latent symptomatic disorders is important to prevent a turn for the worse of these symptoms. LDH M subunit deficiency was first discovered by urinary discoloration and a discrepancy of laboratory data. Since then, only four cases have been reported in the Japanese population. The response to ischemic forearm work is characteristic (an increase of venous lactate concentration after ischemic work is not observed and a marked increase of venous pyruvate is found). The increase of pyruvate concentration is specific in LDH-M subunit deficiency, and is not observed in other abnormalities of the glycolytic pathway. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase (GA3PD) step, possibly due to the impaired reoxidation of NADH produced by GA3PD activity. Then, the excess NADH is reoxidized by alpha-glycerophosphate dehydrogenase and triose phosphates are drained to alpha-glycerophosphate and glycerol. Therefore ATP production is significantly impaired and muscle tissue is damaged. A genetical study revealed a deletion of 20 base-pairs in exon 6 in LDH-M subunit deficiency. This mutation results in a frame-shift translation and premature termination.

Adolescent↗

PubMed1976

Allosteric enzyme-tRNA complexes as regulators of transcription or translation.

The dehydrogenase activity of chorismate mutase-prephenate dehydrogenase, the allosteric enzyme of the tyrosine biosynthetic pathway in Escherichia coli, is inhibited by tRNA. The inhibitory effect of tRNA from E. coli is specific, other RNA species or polynucleotides have no inhibitory effect or only slightly influence the activity of the enzyme. While NAD only slightly influences the inhibitory effect of tRNA from E. coli, prephenic acid at high concentrations suppresses the inhibition. In he presence of a fixed concentration of NAD and low concentration or absence of prephenic acid the inhibitory effect of tRNA is time and temperature dependent. It seems that in the presence of tRNA and low concentration of prephenate the enzyme undergoes a time and temperature dependent conformational change. This process is reversible and can be influenced by the concentration of prephenic acid, the first precursor of the tyrosine biosynthetic pathway. The possible regulatory role of allosteric enzyme-tRNA complexes is discussed.

Chorismate Mutase↗

PubMed1977

Metabolic fate of cyanocobalamin taken up by Spirometra mansonoides spargana.

Analysis of tissue from Spirometra mansonoides spargana has shown that cyanocobalamin (vitamin B12) is metabolized to adenosylcobalamin and hydroxocobalamin. No methylcobalamin was detected. When the tissues were examined for enzymes which are known to utilize coenzyme forms of vitamin B12, only methylmalonyl CoA mutase, which requires adenosylcobalamin was found. The enzyme, tetrahydropteroylglutamate methyltransferase, which requires methylcobalamin as a cofactor, was not detected. A sizable portion of the cyanocobalamin taken up was bound to ammonium sulfate-precipitable material, suggesting that the binding substance is a protein. Vitamin B12 taken up by spargana was found to be released in vivo with a biological half-life of about 7 weeks.

Animals↗

PubMed1990

Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.

2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.

2,3-Diphosphoglycerate↗

PubMed1977

pH-dependent changes of 2,3-bisphosphoglycerate.

A systematic study of the pH dependent changes in the range 6.6--7.4 of 2,3 bisphosphoglycerate (2,3-DPG) was performed in the presence and absence of glucose during transitional and steady states. The results indicate that 2,3-DPGase breaks down 2,3-DPG nealy independent of pH at a rate of 480 mu moles 2,3-DPG/1 cells.h. The 2,3-DPG mutase is practically completely inhibited below pH 6.9. The 2,3-DPG level in the presence of glucose reaches a pH dependent steady state after about 18 h. The share of the 2,3-DPG bypass in the steady state decreases from 24% at pH 7.4 to 12% at pH 7.0. The formation of pyruvate corresponds to the beadkdown of 2,3-DPG after consumption of an unknown reducing substance.

Adenosine Triphosphate↗

PubMed1987

Effect of oxalate and malonate on red cell metabolism.

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

2,3-Diphosphoglycerate↗

PubMed1988

The use of enzymopathic human red cells in the study of malarial parasite glucose metabolism.

The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross-react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co-evolution with their human hosts.

Animals↗

PubMed1986

Perturbation of methionine metabolism in sheep with nitrous-oxide-induced inactivation of cobalamin.

Exposure of sheep to 36% nitrous oxide for 8 days (2-hr per day) led to 90%, 82% and 74% inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase in the liver, heart and brain, respectively, while there was no significant decrease in the activity of methylmalonyl-CoA mutase. There was also no change of betaine-homocysteine methyltransferase activity. The level of plasma methionine in nitrous-oxide-exposed sheep fell to 30% of its initial value. S-Adenosylmethionine level was reduced to 50% of the control value in the liver, and was also significantly decreased in the heart, but not in the brain. Excretion of formiminoglutamic acid and homocystine was also observed in the urine of sheep exposed to nitrous oxide. These results demonstrate that inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase causes a pronounced perturbation of methionine metabolism in sheep, suggesting that dietary methionine plus methionine synthesized from the methyl groups of betaine are not sufficient to meet the methyl needs for biological methylation reactions in this species and, in turn, emphasizing the role of 5-methyltetrahydrofolate-homocysteine methyltransferase in methionine synthesis in the sheep.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1986

Acquired erythroenzymopathy in a monozygotic twin with acute myeloid leukemia.

The specific activity of several red cell enzymes was studied in a pair of monozygotic twins, one of whom presented acute myeloblastic leukemia. When the intrapair variation of these twins was compared with that of a series of nine normal twins, a significant decrease in phosphoglycerate kinase, diphosphoglycerate mutase, pyruvate kinase, lactic dehydrogenase, adenylate kinase and glucose-6-phosphate dehydrogenase activities and an increase in 6-phosphogluconate dehydrogenase activity were demonstrable for the leukemic twin. The heat stability of the leukemic proband's pyruvate kinase at pH 8.0 and 56 degrees C was less than that of the normal twin, suggesting an acquired qualitative disorder.

Adenylyl Cyclases↗

PubMed1986

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency.

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.

Amino Acids↗

PubMed1981

The hematology and biochemistry of canine postnatal anemia.

In conclusion, postnatal changes in the 2,3-DPG concentration of the canine erythrocyte are directly related to changes in erythrocyte 1,3-diphosphoglycerate concentration. There is not apparent differential regulation of 2,3-DPG mutase or phosphatase activities. Glucose consumption and lactate production of the canine erythrocyte do not change during postnatal development, thus, overall rate controlling reactions in the upper portion of the glycolytic pathway do not regulate the changes in 1,3-diphosphoglycerate. The analysis of intermediate and enzyme levels below the 2,3-DPG shunt indicates that pyruvate kinase functions as a controlling sink reaction. Variation in the pyruvate kinase step causes changes in the erythrocyte phosphoenolpyruvate levels. Since the enolase, phosphoglyceromutase and phosphoglycerate kinase reactions are at equilibrium, changes in phosphoenolpyruvate concentrations are paralleled by changes in the concentrations of 1,3-diphosphoglycerate and 2,3-DPG. We propose that the rise in erythrocyte 2,3-DPG, during the first 60 days of postnatal age, results from a decline in the levels of the fetal or M2 isozyme of pyruvate kinase. The subsequent decline in erythrocyte 2,3-DPG to normal adult levels may result from a developmental change in pyruvate kinase isozymes. The changes in erythrocyte glycolysis following birth increase the oxygen transporting efficiency of hemoglobin at the higher oxygen tensions of the neonatal environment. This provides an initial reserve of blood oxygen transport capacity which suppresses erythrocyte production, resulting in the condition defined as postnatal anemia. There is no apparent defect in postnatal erythropoiesis.

Aging↗

PubMed1984

Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro.

Enolase, lactate dehydrogenase, and phosphoglycerate mutase have previously been found to contain phosphotyrosine in fibroblasts transformed by Rous sarcoma virus, which encodes a tyrosine-specific protein kinase. However, these phosphorylations are not stoichiometric, and their significance for any aspect of the transformed phenotype is unknown. We show here that enolase and lactate dehydrogenase are each phosphorylated chiefly at a single tyrosine in Rous sarcoma virus-transformed cells. The purified enzymes can also be phosphorylated at the same tyrosine in vitro when incubated with an immunoprecipitated retroviral transforming protein having associated tyrosine protein kinase activity. The phosphorylated tyrosine in lactate dehydrogenase is amino acid 238. The phosphorylated tyrosine in enolase lies in a sequence homologous to that surrounding histidine 43 in yeast enolase. Although the phosphorylated sequence in lactate dehydrogenase shows some homology to those sequences surrounding phosphotyrosines found in retroviral transforming proteins, the phosphorylated sequence in enolase is quite different.

Amino Acid Sequence↗

PubMed1983

[Biosynthesis of phenylalanine and tyrosine in Flavobacteria].

The enzymes of the terminal steps of phenylalanine and tyrosine biosynthesis, chorismate mutase, prephenate dehydratase, arogenate dehydratase, prephenate dehydrogenase and arogenate dehydrogenase, were studied in 11 different species of the genus Flavobacteria. A comparison of the specific activities, cofactor specificity and regulation of the enzymes, allows a differentiation within the Flavobacteria. All strains studied utilize both arogenate and p-hydroxyphenylpyruvate as an intermediate in L-tyrosine synthesis. Phenylpyruvate was found to be the precursor of phenylalanine in most bacteria. No feedback inhibition of arogenate dehydrogenase by phenylalanine and tyrosine was observed. The diverse strains of the flavobacteria were found to possess different regulatory patterns with respect to the action of phenylalanine and tyrosine on the other enzymes. On the basis of these results a tentative classification of the Flavobacteria within the two groups formed by the different DNA base ratios is proposed.

Chemical Phenomena↗

PubMed1994

Enzymatic activity and morphological differentiation in de novo innervated human muscle cultures.

In the present series of experiments, we studied the effects of developmental neural control on morphological differentiation and on the activity of muscle-specific (creatine kinase, CK; phosphoglycerate mutase, PGAM; phosphorylase, PPL; and phosphofructokinase, PFK) and non-specific (acid maltase, AM; glucose-6-phosphate dehydrogenase, G6PD), human muscle enzymes in de novo innervated muscle cultures. Following innervation of muscle cultures, we noted an increase in the activity of CK, PPL, PFK and AM along with a reduction in G6PD activity. There was also a change of the CK isoenzymes present in the myotubes, i.e. BB and MB are the major isoenzymes in non innervated cultures, but MM becomes predominant following innervation. In the case of PGAM, the only isoenzyme present in the non innervated cultures was BB while the MM isoform appeared only after a prolonged innervation period in most cases--with the exception of AM--these changes in enzyme activity and in the type of isoenzymes present, demonstrate that innervated cultures are more similar to mature muscle. This maturation of enzymatic activity correlates well with the morphological maturation of the myotubes observed following innervation. Such innervated cultures therefore represent a better model with which to study the morphological and biochemical abnormalities associated with neuromuscular diseases.

Creatine Kinase↗

PubMed1996

Effects of low concentrations of dietary cobalt on rumen succinate concentration in sheep.

Sheep fed diets containing less than 70 micrograms Co per kg develop vitamin B12 deficiency. When sheep were fed diets containing 20 micrograms Co per kg or less, mean rumen succinate concentrations increased by more than one hundred-fold within 2 days. This increase was matched by an equimolar decrease in mean rumen propionate concentrations. When diets containing more than 20 micrograms Co per kg were fed to sheep, no such changes occurred. The synthesis of succinyl CoA from propionyl CoA in liver is impaired in ovine cobalt deficiency. We suggested that, paradoxically, accumulation of rumen succinate could reduce the effects of vitamin B12 deficiency on methylmalonyl CoA mutase and consequently result in lower plasma methylmalonic acid (MMA) concentrations than would arise by feeding diets that did not affect rumen succinate concentrations. This hypothesis was tested by feeding diets containing 4, 40 and 1000 micrograms Co per kg to sheep for 23 weeks. However, sheep fed 40 micrograms Co per kg did not have mean plasma MMA concentrations that were higher than those in the sheep fed 4 micrograms Co per kg, indicating that rumen succinate accumulation did not ameliorate the effects of Co deficiency.

Acyl Coenzyme A↗

PubMed1997

Cyanocobalamin [c-lactam] inhibits vitamin B12 and causes cytotoxicity in HL60 cells: methionine protects cells completely.

The [c-lactam] derivative of cobalamin antagonizes vitamin B12 in vivo. Therefore, we investigated its effects in tissue culture to develop a model in which to study vitamin B12-deficient hemopoiesis. HL60 cells were cultured in medium containing either methionine or L-homocysteine thiolactone, and various concentrations of 5-methyltetrahydrofolate or pteroylglutamic acid. In medium with L-homocysteine thiolactone, 5-methyltetrahydrofolate, and dialyzed serum, cyanocobalamin [c-lactam] caused cell death, reversible by additional vitamin B12. Pteroylglutamic acid did not prevent this cytotoxic effect. Methionine completely protected cells against cyanocobalamin [c-lactam] for periods of up to 4 months of culture, irrespective of the folate source. Cyanocobalamin [c-lactam] reversibly impaired the incorporation of 5-[14CH3]-tetrahydrofolate and [1-(14)C] propionic acid by intact cells, consistent with inhibition of methionine synthase and methylmalonyl-CoA mutase. A substantial proportion of 5-[14CH3]-tetrahydrofolate uptake could not be suppressed by methionine and may, therefore, have occurred outside of the methionine synthase pathway. These findings are the first indication that cyanocobalamin [c-lactam] antagonizes vitamin B12 in vitro and causes cell death from methionine deficiency. The model should be valuable for investigating the biochemical pathology of vitamin B12-deficient hemopoiesis. The results suggest that methylfolate is not trapped when methionine synthase is inhibited in HL60 cells, but they do not disprove the methylfolate trap hypothesis as applied to normal blood cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗