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PubMed2000

Reconciling structure and function in HhaI DNA cytosine-C-5 methyltransferase.

Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme.DNA complex and the lack of catalytic competence of the enzyme.S-adenosyl-L-methionine (AdoMet) complex. The enzyme.AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme.AdoMet.DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme.AdoMet and ternary enzyme. S-adenosyl-L-homocysteine.DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA. We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the 5-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi- and unmethylated substrates alter the fates and lifetimes of discrete enzyme.substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.

Base Sequence↗
PubMed2005

The respiratory substrate rhodoquinol induces Q-cycle bypass reactions in the yeast cytochrome bc(1) complex: mechanistic and physiological implications.

The mitochondrial cytochrome bc(1) complex catalyzes the transfer of electrons from ubiquinol to cyt c while generating a proton motive force for ATP synthesis via the "Q-cycle" mechanism. Under certain conditions electron flow through the Q-cycle is blocked at the level of a reactive intermediate in the quinol oxidase site of the enzyme, resulting in "bypass reactions," some of which lead to superoxide production. Using analogs of the respiratory substrates ubiquinol-3 and rhodoquinol-3, we show that the relative rates of Q-cycle bypass reactions in the Saccharomyces cerevisiae cyt bc(1) complex are highly dependent by a factor of up to 100-fold on the properties of the substrate quinol. Our results suggest that the rate of Q-cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the quinol oxidase site of the enzyme. We conclude that normal operation of the Q-cycle requires a fairly narrow window of redox potentials with respect to the quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.

Adenosine Triphosphate↗
PubMed2000

Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23.

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.

Adenosine Triphosphatases↗
PubMed2003

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation.

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.

Acetic Acid↗
PubMed1983

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: its inhibition by oxidizable substrates and its relationships to purine metabolism.

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.

Adenine↗
PubMed2006

Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein.

Hsp70 alternates between an ATP-bound state in which the affinity for substrate is low and an ADP-bound state in which the affinity for substrate is high, as a result Hsp70 assists the protein folding process through nucleotide-controlled cycles of substrate binding and release. In this work, we describe the cloning and purification of the human 70-kDa heat shock cognate protein, Hsc70, and the use of circular dichroism, intrinsic emission fluorescence, and isothermal titration calorimetry to characterize conformational changes induced by ADP and ATP binding. Binding of either ADP or ATP were not accompanied by a net change in secondary structure suggesting that the conformational rearrangement caused by nucleotide binding is localized. MgADP or MgATP had a greater effect in the stability at stress temperatures than ADP or ATP did. Isothermal titration calorimetry data pointed out that Hsc70 had a lower affinity for ATP (KD=710 nM) than for ADP (KD=260 nM).

Adenosine Diphosphate↗
PubMed2005

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding.

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.

Butyryl-CoA Dehydrogenase↗
PubMed1991

Cortical reflex myoclonus. A study of the relationship between giant somatosensory evoked potentials and motor excitability.

The excitability cycles of the N1-P1-N2 waveforms of the scalp-recorded somatosensory evoked potential (SEP) and of the long-latency, cortical loop reflex electromyographic (EMG) activity were studied in two patients with cortical reflex myoclonus. Long-latency cortical loop reflex EMG activity in the thenar muscles and giant SEPs occurred following median nerve stimulation. The excitability cycle of the EMG paralleled that of the SEP. There was an initial period of attenuation of SEP and EMG amplitude at interstimulus intervals (ISIs) of less than 40 ms followed by a period of amplitude enhancement at an ISI of up to 200 ms followed by a second period of attenuation. The excitability cycle is abnormal and the SEP and EMG amplitude changes parallel each other. It is therefore likely that a common mechanism determines the abnormal excitability cycle. The substrate for this mechanism is unknown and may be diffuse or restricted. Oral 5-hydroxytryptophan (5-HTP) in therapeutic doses altered the SEP excitability cycle. 5-HTP did not attenuate the giant SEPs but did attenuate the long-latency reflex EMG. Therefore, 5-HTP's site of action may be different from the substrate underlying the mechanism that results in the giant SEPs. Additionally, spinal latency reflex EMG activity occurred following treatment with 5-HTP but was absent when the patient discontinued 5-HTP.

5-Hydroxytryptophan↗
PubMed2003

Glutamine cycling in isolated working rat heart.

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.

Adaptation, Physiological↗
PubMed1993

Effects of amino acids on substrate selection, anaplerosis, and left ventricular function in the ischemic reperfused rat heart.

The effect of aspartate and glutamate on myocardial function during reperfusion is controversial. A beneficial effect has been attributed to altered delivery of carbon into the citric acid cycle via substrate oxidation or by stimulation of anaplerosis, but these hypotheses have not been directly tested. 13C isotopomer analysis is well suited to the study of myocardial metabolism, particularly where isotopic and metabolic steady state cannot be established. This technique was used to evaluate the effects of aspartate and glutamate (amino acids, AA) on anaplerosis and substrate selection in the isolated rat heart after 25 min of ischemia followed by 30 or 45 min of reperfusion. Five groups of hearts (n = 8) provided with a mixture of [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose were studied: control, control plus AA, ischemia followed by 30 min of reperfusion, ischemia plus AA followed by 30 min of reperfusion, and ischemia followed by 45 min of reperfusion. The contribution of lactate to acetyl-CoA was decreased in postischemic myocardium (with a significant increase in acetate), and anaplerosis was stimulated. Metabolism of 13C-labeled aspartate or glutamate could not be detected, however, and there was no effect of AA on functional recovery, substrate selection, or anaplerosis. Thus, in contrast to earlier reports, aspartate and glutamate have no effect on either functional recovery from ischemia or on metabolic pathways feeding the citric acid cycle.

Acetyl Coenzyme A↗
PubMed2006

Crystal structures of a multidrug transporter reveal a functionally rotating mechanism.

AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

[Effects of polyunsaturated fatty acids on Krebs cycle in the rat kidney in chronic phosphorus intoxication].

The investigation of Krebs cycle state in kidney homogenates of August rats subjected to oral intoxication with oil solution of yellow phosphorus in a dose of 0.3 mg/kg, has shown that under conditions of balanced nutrition the activity of NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase and accumulation of the substrate fund of the cycle decreased 3.5-fold as compared to the control. The addition of polyunsaturated fatty acids to the ration produced a positive effect on Krebs cycle state: dehydrogenase activity was not significantly changed, accumulation of Krebs cycle substrate was two-fold lower. However, this ration did not completely abolish the toxic action of yellow phosphorus on Krebs cycle.

Animals↗
PubMed1983

Polyamines as modulators of drug oxidation reactions catalyzed by cytochrome P-450 from liver microsomes.

The effect of polyamines on the activity of the mixed-function oxidase (MFO) system from human, rat and rabbit liver microsomes was investigated in detail. It was shown that polyamine (spermine) stimulates NADPH-dependent activity of the MFO system several-fold whatever the substrate (foreign drug or natural), not only with microsomes but also with the reconstituted system consisting of highly purified cytochrome P-450 (LM2 isozyme), cytochrome P-450 NADPH reductase and dilauroylphosphorylcholine. Stimulation (extent and concentration dependence) appeared to be dependent on a number of parameters such as ionic strength, pH, animal species and treatment, nature of the substrate, and was stereospecific (different effect on 6 beta-and 16 alpha-testosterone hydroxylation). Further, the spermine effect was evaluated on some elementary steps of the cytochrome P-450 reaction cycle, like substrate binding, P-450 reduction and second electron transfer. Finally, it was shown that the organic peroxide dependent activity was not stimulated by spermine with microsomes nor with the purified P-450 LM2 isozyme. On the basis of this study, it was concluded that the locus of polyamine action is cytochrome P-450 and that stimulation could result either from increased stability of the oxyferrous intermediate of P-450 or from an increased rate of second electron transfer from reductase to P-450.

Animals↗
PubMed2000

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors.

Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.

Amino Acid Motifs↗
PubMed1991

In vivo evidence for hepatic autoregulation during FFA-stimulated gluconeogenesis in normal humans.

To examine the effect of increased gluconeogenesis [by increasing free fatty acids (FFA)] on hepatic glucose output (HGO) and on the first substrate (glucose) cycle, a primed continuous infusion of [2-3H]- and [6-14C]glucose was infused to isotopic steady state in 12 normal male volunteers after an overnight fast. Blood samples for the determination of glucose specific activity were obtained before and after an infusion of saline (n = 6) or 10% Intralipid and heparin (90 mU.kg-1.min-1, n = 6). Plasma FFA (593.3 +/- 74.5 to 971.1 +/- 127.1 mumol/l, P = 0.007) and glycerol (68.0 +/- 5.9 vs. 222.4 +/- 32.0 mumol/l, P = 0.002) increased during the lipid infusion, and beta-hydroxybutyrate levels rose from 0.24 +/- 0.12 to 0.50 +/- 0.17 mmol/l (P = 0.01). No change in plasma glucose, insulin, or glucagon levels was observed during the study, and levels of the gluconeogenic substrates alanine and lactate were also unchanged. Baseline rates of glucose cycling (rate of appearance of [2-3H]glucose minus rate of appearance of [6-14C]glucose) were similar in the two groups [1.44 +/- 0.33 vs. 1.33 +/- 0.44 mumol.kg-1.min-1, not significant (NS)] and did not change during either saline or lipid infusion, respectively. However, Cori cycle activity (the conversion of [6-14C]- to [1-14C]glucose) increased significantly from 0.59 +/- 0.19 to 1.28 +/- 0.19 mumol.kg-1.min-1 (P = 0.002) after FFA and glycerol levels had been increased, in marked contrast to the saline control (0.51 +/- 0.18 to 0.39 +/- 0.18 mumol.kg-1.min-1, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗