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A protein load with an ensuing 20 hr fast was performed to assess the function of biotin dependent carboxylases in 3 girls with Rett syndrome. In plasma a moderate increase of ammonia and propionate was found. The fact that these discrete biochemical alterations were found in all of the patients supports earlier conclusions that further studies on biotin metabolism and carboxylase function should be performed, perhaps preferably in cerebrospinal fluid.
Lipoamide dehydrogenase is a subunit of the alpha-ketoacid dehydrogenases and the glycine decarboxylase complex in mitochondria, and the pyruvate dehydrogenase complex in plastids. We report here the unexpected finding of two plastidic isoforms of lipoamide dehydrogenase from Arabidopsis thaliana that are different from the mitochondrial form of the enzyme. The cDNA clones were confirmed by sequence alignment analysis and their location verified by chloroplast import assay. They are single copy genes that appear to be expressed in parallel in different tissues with highest level in developing siliques. Phylogenetic analysis gives further exemplary evidence for the plastidic evolution derived from cyanobacteria.
We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.
1. Metabolic processes are acutely and chronically regulated in response to changes in the workload of the heart. Acute changes in cardiac work result in activation and inactivation of existing enzymes and in altered fluxes through existing metabolic pathways. Sustained or chronic changes in cardiac work result in both trophic and transcriptional alterations. 2. The metabolic consequences of a sustained increase or decrease in the workload of the heart are surprisingly uniform and consist of a switch from the predominant oxidation of fatty acids to oxidation of glucose. 3. This switch is reflected in the changes of the transcript levels of three key regulators of mitochondrial function: pyruvate dehydrogenase kinase 4 (PDK4), which phosphorylates and inactivates the pyruvate dehydrogenase complex, malonyl-CoA decarboxylase (MCD), which regulates malonyl-CoA levels and, therefore, rates of beta-oxidation of long-chain fatty acids, and uncoupling protein 3 (UCP-3), which uncouples the oxidative phosphorylation of ADP. 4. The transcript levels of all three proteins are downregulated in hypertrophy as well as in atrophy of rat heart. All three transcripts are transcriptionally regulated by the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). 5. Diminished expression of PPARalpha and PPARalpha-regulated genes constitutes an adaptive mechanism in response to altered workload, because reactivation of PPARalpha in hypertrophied heart results in severe contractile dysfunction.
Phosphoenolpyruvate carboxykinase has been partially purified from pineapple (Ananas comosus [L.]) leaves. Specific activities obtained show it to be a major activity in this tissue. Above 15 C, the respective activation energies for decarboxylation and carboxylation are 13 and 12 kcal/mol. Below 15 C, there are discontinuities in Arrhenius plots with an associated large increase in activation energy. The adenine nucleotides are preferred to other nucleotides as substrates. The apparent Km values in the carboxylation direction are: ADP 0.13 mm, HCO(3) (-) 3.4 mm, and phosphoenolpyruvate 5 mm. In the decarboxylation direction, the apparent Km values are: ATP 0.02 mm, ADP 0.05 mm, and oxaloacetate 0.4 mm. The decarboxylation activity had an almost equal velocity with either ADP or ATP. The pH optima are between 6.8 and 7. Inhibition of the carboxylation reaction by ATP, pyruvate, and carbonic anhydrase was demonstrated. Decarboxylase specific activities are over twice carboxylation activities. The data support a model in which phosphoenolpyruvate carboxykinase is of physiological significance only during the light period and then only as a decarboxylase.
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Microbiologically determined plasma biotin levels in 404 epileptics under long-term treatment with anticonvulsants were markedly lower than in 112 controls (p less than 0.0005). Patients with partial epilepsy had lower biotin levels and higher average daily intake of AC than those with generalized epilepsy. Epileptics treated with valproate sodium in monotherapy showed considerably higher biotin levels than epileptics with monotherapy of primidone (PRM), carbamazepine (CBZ), phenytoin (PHT) or phenobarbital (PB). The group of epileptics with high average daily dose of anticonvulsants had lower biotin levels than the group with low dose. In three patients with newly recognized epilepsy biotin levels were normal before starting anticonvulsant medication, increased during the first week and fell under the starting level in the following weeks. Four epileptics treated with PHT, PB, PRM or CBZ had an increased urinary excretion of organic acids, as found in patients with a deficiency of biotin-dependent carboxylases. In 37 epileptics undergoing long-term treatment plasma lactate concentrations were determined; they had a higher mean concentration than that found in controls. Our results suggest, that the lowering of biotin in epileptics is caused by intake of anticonvulsants and has a biochemical effect in these patients. It is discussed, whether this could be a factor in the mode of action of anticonvulsants.
Biotin responsive multiple carboxylase deficiency was suspected in a third trimester conceptus on the basis of enzymatic confirmation in fibroblasts cultured from an earlier sibling who suffered a demise in the immediate neonatal period. Maternal urinary organic acid profile was normal throughout the final 4 wk of pregnancy. Oral administration of biotin, 10 mg/day to the mother resulted in a 100-fold increase in urinary biotin excretion within 7 days. Urinary biotin excretion over the subsequent 2 wk decreased steadily, suggesting either decreased maternal absorption or increased fetal sequestration. After the birth of nonidentical twins, cord blood and urinary organic acid profiles of the infants were normal. However, cord blood biotin concentration was 4 to 7-fold that of normal newborns. Subsequent enzymatic and genetic complementation studies utilizing cultured skin fibroblasts from the infants demonstrated one of them to be affected by the multiple carboxylase defect, although he was clinically and biochemically normal throughout the neonatal period. Thus, prenatal therapy of this inborn enzymatic defect can be safely and effectively accomplished by administration of pharmacologic biotin doses in the last month of pregnancy.
In humans, skeletal muscle is a major site of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) expression, but its function in this tissue is unclear. We investigated the role of hPPAR-alpha in regulating muscle lipid utilization by studying the effects of a highly selective PPAR-alpha agonist, GW7647, on [(14)C]oleate metabolism and gene expression in primary human skeletal muscle cells. Robust induction of PPAR-alpha protein expression occurred during muscle cell differentiation and corresponded with differentiation-dependent increases in oleate oxidation. In mature myotubes, 48-h treatment with 10-1,000 nmol/l GW7647 increased oleate oxidation dose-dependently, up to threefold. Additionally, GW7647 decreased oleate esterification into myotube triacylglycerol (TAG), up to 45%. This effect was not abolished by etomoxir, a potent inhibitor of beta-oxidation, indicating that PPAR-alpha-mediated TAG depletion does not depend on reciprocal changes in fatty acid catabolism. Consistent with its metabolic actions, GW7647 induced mRNA expression of mitochondrial enzymes that promote fatty acid catabolism; carnitine palmityltransferase 1 and malonyl-CoA decarboxylase increased approximately 2-fold, whereas pyruvate dehydrogenase kinase 4 increased 45-fold. Expression of several genes that regulate glycerolipid synthesis was not changed by GW7647 treatment, implicating involvement of other targets to explain the TAG-depleting effect of the compound. These results demonstrate a role for hPPAR-alpha in regulating muscle lipid homeostasis.
Metabolic activity of rat lungs were studied in normal state and in acute hypoxia, caused by an effect of rarefied atmosphere (3 hrs, "height" 10,000 m). Glycolytic splitting of carbohydrates and catabolism of proteins were increased in lungs under hypoxic stress. In hypoxia activities of adlobase, pyruvate kinase, succinate dehydrogenase, 5-hydroxytryptophan decarboxylase were increased, but hexokinase activity was decreased. Activities of lipase, lactate dehydrogenase and NAD-dependent malate dehydrogenase were not altered, whereas the ratio in specific activity of cytoplasmic malate dehydrogenase and lactate dehydrogenase was decreased.
Report on a serious disturbance of amino-acid metabolism in a 56 year old male patient suffering from thiamine-(vitamin B-1) deficiency, as proven by clinical history and examination and by laboratory data. In comparison with a group of 75 normal male persons also evaluated by the same laboratory this patient--while thiamine deficient--had markedly elevated serum concentrations outside the physiologic range of glutamic acid, glutamine, proline, citrulline, ornithine, histidine, lysine, phenylalanine, tyrosine and leucine, whereas his serum concentrations of taurine, serine and isoleucine were comparably definitely lowered. Following six to seven days of daily i.m. injections of 200 mg of thiamine each, this imbalance of amino-acid homeostasis disappeared except for that of taurine and--questionably--that of phenylalanine. In view of the absence of other exogenous or endogenous potential causes for this, it must be presumed that the thiamine-pyrophosphate deficiency caused the imbalance of amino-acid metabolism by consecutively disturbing the function of the alpha-ketoglutarate-, pyruvate- and p-hydroxy-phenylpyruvate- decarboxylase chains respectively. Possible connections between thiamine-deficiency and lowered serum concentrations of serine, taurine and isoleucine are being discussed.
S-Adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines that is the rate limiting step in the formation of spermidine and spermine. The activity of S-adenosylmethionine decarboxylase is known to be regulated negatively by these polyamines and positively by their precursor, putrescine. A specific antiserum to S-adenosylmethionine decarboxylase was raised by immunizing rabbits with the homogeneous enzyme purified from rat prostate and a specific radioimmunoassay for the protein was set up. Using this radioimmunoassay it was found that a number of inhibitors of other steps in the polyamine biosynthetic pathway lead to increases in the amount of S-adenosylmethionine decarboxylase protein. These changes were caused by both a decreased rate of degradation and an increased rate of synthesis of the protein. The increased synthesis was due to two factors; a rise in the amount of translatable mRNA and an enhanced translation efficiency. The mRNA content of the prostate was substantially increased by treatment for 3 days with alpha-difluoromethylornithine (2% in drinking water). The translation of mRNA for S-adenosylmethionine decarboxylase was studied using a polyamine-depleted reticulocyte lysate supplemented with mRNA from rat prostate and the antiserum to precipitate the proteins corresponding to S-adenosylmethionine decarboxylase. These studies indicated that the enzyme was synthesized as an inactive precursor of Mr 37,000 which was converted to the enzyme sub-unit of Mr 32,000. The conversion of the precursor to the active sub-unit in vitro was increased by putrescine. The precursor could also be detected by immunoblotting of extracts from prostates of rats depleted of putrescine by treatment with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The translation of the S-adenosylmethionine decarboxylase mRNA in the reticulocyte lysates was strongly inhibited by the addition of spermidine or spermine demonstrating that polyamines directly inhibit the synthesis of S-adenosylmethionine decarboxylase. cDNA clones corresponding to S-adenosylmethionine decarboxylase were isolated using prostatic mRNA from polysomes enriched in S-adenosylmethionine decarboxylase by immunopurification. The use of these probes showed that rat ventral prostate contains two S-adenosylmethionine decarboxylase mRNA species of approximately 3.4 and 2.1 kb which differ in the 3' non-translated sequence. The sequence of these cDNAs will enable the amino acid sequence of the precursor to be obtained. This will provide evidence on the origin of the pyruvate prosthetic group of S-adenosylmethionine decarboxylase.(ABSTRACT TRUNCATED AT 400 WORDS)
The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.
Clostridium acetobutylicum ATCC 824 using pyruvate as the sole carbon source produced mainly acetate and butyrate as end products of fermentation. Acetate and butyrate kinase activities were higher in cells growing in the presence of pyruvate than glucose, whereas the level of the acetoacetate decarboxylase, an enzyme involved in solvent formation, was lower. Similar activities of glyceraldehyde-3-phosphate dehydrogenase were found in cells grown in pyruvate and glucose mediums. The transfer of C. acetobutylicum from pyruvate to glucose medium suggested that pyruvate represses the "solventogenesis."
Previously we reported that intermittent intraperitoneal administration of ornithine decarboxylase-inducing factor (ODC factor), interleukin 1alpha (IL-1alpha), and tumor-necrosis factor-alpha (TNF-alpha) to normal mice induced biological changes in the hosts which included changes in the pattern of expression of pyruvate kinase (PK) isozymes in the liver and hypertrophy of the spleen. In the study reported here, we investigated the chronic and combined effects of these factors on hepatic enzymes using alzet microosmotic pumps implanted in the subcutis of the backs or abdominal cavities of mice. Continuous administration of ODC factor and recombinant human IL-1alpha (rhIL-1alpha) reduced the activity of L-type PK, which is a glycolysis-related enzyme in the liver, and induced the activity of M2-type PK, a known marker of liver dedifferentiation. Serine dehydratase (SDH) and tyrosine aminotransferase (TAT), enzymes associated with amino acid metabolism, were not significantly influenced at the examined concentration. The simultaneous continuous infusion of ODC factor and rhIL-1alpha or rhTNF-alpha caused alterations in the patterns of expression of PK isozyme activity profiles and reduced overall PK activity. SDH and TAT activities were also significantly induced. Moreover, mice treated with these combined factors displayed many other metabolic changes normally associated with cancer cachexia. These findings suggest that the tumor-derived ODC factor and cytokines such as IL-1alpha and TNF-alpha might function synergistically in the metabolic perturbations observed in Ehrlich ascites tumor bearers.
Soybean (Glycine max [L.] Merr) leaves contain a low level (0.05 micromole per gram fresh weight) of gamma-aminobutyric acid (Gaba) but the concentration of this non-protein amino acid increased to 1 to 2 micromoles per gram fresh weight within 5 minutes after transfer of plants or detached leaves from 33 degrees C to 22 degrees C or lower temperatures. A parallel decrease occurred in the concentration of glutamate. Accumulation of Gaba was also triggered by mechanical damage to the soybean leaves, but in plants subjected to a gradual reduction in temperature (2 degrees C per minute) only a small increase in Gaba occurred. A rapid increase in the concentration of alanine and decrease in glycine occurred upon transfer of the soybean plants to darkness and was not influenced by temperature. When plants were returned to normal growing conditions, all changes in amino acid concentrations were fully reversed in 1 hour.In soybean leaf discs incubated with [(14)C]glutamate, a rapid accumulation of [(14)C]Gaba was detected, and glutamate decarboxylase activity of the soybean leaf considerably exceeded (>30-fold) that of Gaba pyruvate transaminase. Part of the transaminase was localized in the mitochondria, but glutamate decarboxylase was not associated with any organelle or membrane component of the leaf cell. We consider that Gaba accumulation results from some change in intracellular compartmentation of the cell triggered by low temperature shock or mechanical damage. The accumulation of alanine due to a light-dark transition could be accounted for by transamination. [(14)C]Alanine formation was demonstrated when soybean leaf extracts were incubated with glutamate, aspartate, or serine and [(14)C]pyruvate.The changes in amino acid concentrations described for soybean leaves were demonstrated for all the vegetative tissues of the soybean plant and at variable rates in the leaves of a range of plant species. The response in detached tomato (Lycopersicon esculentum Mill.) leaves was of a similar magnitude to soybean. Thus, precautions are necessary to minimize changes in amino acid composition induced by manipulation and extraction of plant material.
Our previous studies showed that polybrominated biphenyl (PBB) induced hepatic microsomal cytochrome P-450 in dairy cattle but did not elevate hepatic cytosolic ornithine decarboxylase or serum isocitrate dehydrogenase. These enzymes would be expected to increase during hepatotoxic injury and regeneration. Thus, PBB appeared to be a hepatotoxin in rats but not in cattle. In order to identify and confirm the response capability of bovine liver to hepatotoxins, we administered thioacetamide, a hepatotoxin known to induce hepatonecrosis, to a dairy calf. A progression of clinical signs of toxicosis was evident until the animal was moribund by 23 hr postdosing. Histolopathologic alterations in the liver included centrilobular necrosis with congestion and subcapsular microhemmorrhage. Marked changes in serum protein profiles were not noted. However, distinct increases in serum Fe and bilirubin occurred with progressing toxicosis, as did sharp declines in glucose and triglycerides. Serum lactic dehydrogenase, alkaline phosphatase, glutamic-oxaloacetic transaminase, isocitrate dehydrogenase and glutamic-pyruvate transaminase were elevated. Elevation of ornithine decarboxylase was dramatic when compared to the level in normal fetal bovine liver. From studies of its kinetic properties, bovine liver ornithine decarboxylase appears to have an apparent Km for ornithine decarboxylase of .45 mM. Liver homogenates from PBB-treated animals did not form inhibitors to ornithine decarboxylase. Compared with the thioacetamide-treated calf, the normal adult bovine, pregnant adult and 6-month fetus had relative activities of .2 .4 and 5.8%, respectively. These studies show that ornithine decarboxylase is low in liver of normal cattle, but is elevated markedly by agents that cause hepatonecrosis.
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