Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

Alterations of lipid composition in Friend leukemia cell tumors in mice treated with tumor necrosis factor-α

The monocarboxylate (pyruvate) transporter from pea (Pisum sativum) mitochondria was identified by means of a specific monoclonal antibody. The antibody blocked pyruvate-dependent oxaloacetate metabolism without interfering with the metabolism of malate, -ketoglutarate, or glycine. The antibody also blocked the pyruvate/pyruvate exchange reaction of the partially purified transporter reconstituted into phospholipid membranes. Using the specific monoclonal antibody, the transporter was identified on Western blots as a minor 19 kDa protein.     

The role of phosphorylation in the α-adrenergic-mediated inhibition of rat hepatic pyruvate kinase

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40–50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2–3-fold increase in the phosphate content of the enzyme. These changes were blocked by the α-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the α-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by α-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.     

Effects of Dichloroacetate on Brain Tissue Pyruvate Dehydrogenase

The activation of the pyruvate dehydrogenase complex (PDHC) by dichloroacetate (DCA) was studied in brain tissue. Chronic administration of DCA to rats caused no significant change of PDHC activation in brain. DCA brain concentrations were comparable to those of other tissues in which activation is known to occur. No effect of DCA on PDHC could be demonstrated from isolated brain mitochondria, whereas DCA reversed the deactivation of PDHC by ATP, alpha-ketoglutarate plus malate, and succinate in liver mitochondria. This study suggests that the regulation of PDHC activation in neural tissue differs from that in other tissues.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

The mechanism of the hyperglycaemic action of synthetic peptides related to the C-terminal sequence of human growth hormone

Synthetic part sequences of human pituitary growth hormone (hGH 176–191 and hGH 177–191) corresponding to residues 176–191 or 177–191 of the hormone have been tested for their effects on glycogen and pyruvate metabolism in the rat, both in vivo and in vitro. When injected, the peptides caused transient increases in blood glucose and lactate, while decreasing the activity ratio of glycogen synthase in muscle, adipose tissue and liver and of pyruvate dehydrogenase in muscle and adipose tissue, but not in liver. These decreases were associated with the conversion of the enzymes from their active to their inactive forms, since the peptides did not affect the total amount of either the synthase or the dehydrogenase. The time course of the effect on the enzymes was similar to that for the effect on blood metabolites, and responses for synthase were produced over the range 0.07–7 nmols hGH 177–191/kg body weight. Phosphorylase activity was not affected by the peptides, nor was the capacity to dispose of injected L-lactate. Experiments with adipocytes and hepatocytes showed that the peptides also affected glycogen synthase and pyruvate dehydrogenase activities in vitro. The peptides had no effect on the overall rate of gluconeogenesis from lactate by hepatocytes. However, at times corresponding to those at which glycogen synthase was inactivated, the peptides caused increased incorporation of lactate into free glucose and decreased incorporation into glycogen. It was concluded that the peptides acted directly on their target tissues, and that the observed hyperlactataemia was the result of the inactivation of pyruvate dehydrogenase. The addition lactate increased the flux through the gluconeogenic pathway, and appeared as glucose because the peptide also inactivated glycogen synthase. Thus, the hyperglycaemia produced by hGH 177–199 and related peptides is explicable in terms of a modified Cori Cycle.     

Rewiring yeast metabolism to synthesize products beyond ethanol

Saccharomyces cerevisiae, Baker's yeast, is the industrial workhorse for producing ethanol and the subject of substantial metabolic engineering research in both industry and academia. S. cerevisiae has been used to demonstrate production of a wide range of chemical products from glucose. However, in many cases, the demonstrations report titers and yields that fall below thresholds for industrial feasibility. Ethanol synthesis is a central part of S. cerevisiae metabolism, and redirecting flux to other products remains a barrier to industrialize strains for producing other molecules. Removing ethanol producing pathways leads to poor fitness, such as impaired growth on glucose. Here, we review metabolic engineering efforts aimed at restoring growth in non-ethanol producing strains with emphasis on relieving glucose repression associated with the Crabtree effect and rewiring metabolism to provide access to critical cellular building blocks. Substantial progress has been made in the past decade, but many opportunities for improvement remain.     

Long-term day-and-night rotating shift work poses a barrier to the normalization of alanine transaminase

To evaluate the impact of day-and-night rotating shift work (RSW) on liver health, we performed a retrospective analysis of the association between long-term RSW exposure and the normalization of plasma alanine transaminase (ALT) levels over a five-year period. The data from physical examinations, blood tests, abdominal sonographic examinations, personal histories, and occupational records were collected from a cohort of workers in a semiconductor manufacturing company. The sample population was divided into three subgroups for analysis, according to self-reported shift work status over the five-year interval: persistent daytime workers, workers exposed intermittently to RSW (i-RSW), and workers exposed persistently to RSW (p-RSW). Records were analyzed for 1196 male workers with an initial mean age of 32.5 years (SD 6.0 years), of whom 821 (68.7%) were identified as rotating shift workers, including 374 i-RSW (31.3%) and 447 p-RSW workers (37.4%). At the beginning of the follow-up, 275 were found to have elevated ALT (e-ALT): 25.1% daytime workers, 23.0% i-RSW workers, and 21.3% p-RSW workers (p?=?0.098). Of those with e-ALT at the beginning, 101 workers showed normalized serum ALT levels at the end of five-year follow-up: 40 (10.7%) of 375 daytime workers, 32 (8.6%) of 374 i-RSW workers, and 29 (6.5%) of 447 p-RSW workers (p?=?0.016). Compared with the workers having persistent e-ALT at the end of follow-up, the workers normalized serum ALT levels had significantly lesser exposures to RSW during follow-up. By performing multivariate logistic regression analyses, and comparing with the persistent daytime co-workers, after controlling for confounding variables (age, occupational factors, educational levels, lifestyle factors, metabolic syndrome, hepatovirus infection, and fatty liver), analysis indicated that the workers exposed to p-RSW were 46% less likely (OR, 0.54; 95% CI, 0.30–0.95; p?=?0.03) to attain normal ALT levels within a five-year interval. These observations demonstrate that persistent day-and-night RSW pose a vigorous obstacle to the normalization of e-ALT among workers with preexisting abnormal liver function. We suggest that workers and managers approach with caution the consideration of assigning or accepting long-term day-and-night RSW when an employee health screening shows evidence of abnormal liver function.     

Mitochondrial location of pyruvate carboxylase in Aspergillus niger

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.