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R B Gregory

Publications and source records attributed to R B Gregory.

At least 19 recordsLinked to original sources
PubMed2006

Influence of hydration on the dynamics of lysozyme.

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T approximately 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h approximately 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h approximately 0.2. Activation of the slow process is responsible for the dynamical transition at T approximately 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.

Animals↗
PubMed2005

Onsets of anharmonicity in protein dynamics.

Two onsets of anharmonicity are observed in the dynamics of the protein lysozyme. One at T approximately 100 K appears in all samples regardless of hydration level and is consistent with methyl group rotation. The second, the well-known dynamical transition at T approximately 200-230 K, is only observed at a hydration level h greater than approximately 0.2 and is ascribed to the activation of an additional relaxation process. Its variation with hydration correlates well with variations of catalytic activity suggesting that the relaxation process is directly related to the activation of modes required for protein function.

Cold Temperature↗
PubMed2003

Evidence that store-operated Ca2+ channels are more effective than intracellular messenger-activated non-selective cation channels in refilling rat hepatocyte intracellular Ca2+ stores.

Liver cells possess store-operated Ca2+ channels (SOCs) with a high selectivity for Ca2+ compared with Na+, and several types of intracellular messenger-activated non-selective cation channels with a lower selectivity for Ca2+ (NSCCs). The main role of SOCs is thought to be in refilling depleted endoplasmic reticulum Ca2+ stores [Cell Calcium 7 (1986) 1]. NSCCs may be involved in refilling intracellular stores but are also thought to have other roles in regulating the cytoplasmic-free Ca2+ and Na+ concentrations. The ability of SOCs to refill the endoplasmic reticulum Ca2+ stores in hepatocytes has not previously been compared with that of NSCCs. The aim of the present studies was to compare the ability of SOCs and maitotoxin-activated NSCCs to refill the endoplasmic reticulum in rat hepatocytes. The experiments were performed using fura-2FF and fura-2 to monitor the free Ca2+ concentrations in the endoplasmic reticulum and cytoplasmic space, respectively, a Ca2+ add-back protocol, and 2-aminoethyl diphenylborate (2-APB) to inhibit Ca2+ inflow through SOCs. In cells treated with 2,5-di-t-butylhydroquinone (DBHQ) or vasopressin to deplete the endoplasmic reticulum Ca2+ stores, then washed to remove DBHQ or vasopressin, the addition of Ca2+ caused a substantial increase in the concentration of Ca2+ in the endoplasmic reticulum and cytoplasmic space due to the activation of SOCs. These increases were inhibited 80% by 2-APB, indicating that Ca2+ inflow is predominantly through SOCs. In the presence of 2-APB (to block SOCs), maitotoxin induced a substantial increase in [Ca2+](cyt), but only a modest and slower increase in [Ca2+](er). Under these conditions, Ca2+ inflow is predominantly through maitotoxin-activated NSCCs. It is concluded that SOCs are more effective than maitotoxin-activated NSCCs in refilling the endoplasmic reticulum Ca2+ stores. The previously developed concept of a specific role for SOCs in refilling the endoplasmic reticulum is consistent with the results reported here.

Animals↗
PubMed2001

Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors.

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P(3) receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P(3) receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647-1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10 microM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+](cyt)) had been established, the kinetics of the decrease in [Ca2+](cyt) were identical with those induced by the addition of 50 microM Gd(3+) (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P(3) to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P(3)-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P(3) receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P(3) receptors in the activation of SOCs in other cell types is briefly discussed.

Animals↗
PubMed2000

Regulation of F-actin and endoplasmic reticulum organization by the trimeric G-protein Gi2 in rat hepatocytes. Implication for the activation of store-operated Ca2+ inflow.

The roles of the heterotrimeric G-protein, G(i2), in regulating the actin cytoskeleton and the activation of store-operated Ca(2+) channels in rat hepatocytes were investigated. Galpha(i2) was principally associated with the plasma membrane and microsomes. Both F-actin and Galpha(i2) were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant co-precipitated with Galpha(i2) using either anti-Galpha(i2) or anti-actin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some Galpha(i2) co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70-80% of Galpha(i2) in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca(2+) inflow. It is concluded that (i) a significant portion of hepatocyte Galpha(i2) associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca(2+) inflow.

Actins↗
PubMed2000

Store-operated Ca(2+) inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca(2+) channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein.

The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2, 5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca(2+) inflow (assayed using intracellular fluo-3 and a Ca(2+) add-back protocol). No stimulation of Mn(2+) inflow by thapsigargin was detected. Thapsigargin-stimulated Ca(2+) inflow was inhibited by Gd(3+) (maximal inhibition at 2 microM Gd(3+)), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca(2+) channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca(2+) inflow and release of Ca(2+) from intracellular stores. No inhibition of either Ca(2+) inflow or Ca(2+) release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca(2+) inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca(2+) inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of G(i2(alpha)) with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of G(i2(alpha)) and substantially less total G(i2(alpha)) than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca(2+) channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca(2+) channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require G(i2(alpha)) for activation. Possible explanations for differences in the requirement for G(i2(alpha)) in the activation of Ca(2+) inflow are briefly discussed.

Animals↗
PubMed1999

Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes.

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-alpha-d-glucopyranoside 2,3',4'-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4, 5)P3P4(5)-1-(2-nitrophenyl)ethyl ester ['caged' Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

Animals↗
PubMed1998

Protein kinase A regulates the disposition of Ca2+ which enters the cytoplasmic space through store-activated Ca2+ channels in rat hepatocytes by diverting inflowing Ca2+ to mitochondria.

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and AlF4(-) inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3',5'-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

Adenosine Triphosphate↗
PubMed1997

Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes.

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

ADP-Ribosylation Factors↗
PubMed1996

Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion.

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+O,-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trp1 cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2-17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and A1F4-, but not guanosine 5'-[beta-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] of FA1F4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.

Aniline Compounds↗
PubMed1996

Effects of thyroid status on glucose cycling by isolated rat hepatocytes.

The effects of alterations in thyroid status on glucose metabolism have been investigated in rat hepatocytes. Addition of 10 or 40 mmol/L glucose induced increases in respiration rate that were significantly larger in cells from hyperthyroid rats than from hypothyroid animals. The responses of hepatocytes from euthyroid rats were intermediate. In cells from hyperthyroid rats, most of the increase occurred upon addition of 10 mmol/L glucose, with only a further small stimulation resulting when glucose concentration was increased to 40 mmol/L. For a given glucose concentration, glycolytic rates, determined by measuring release of tritium from [6-3H]glucose, were comparable in all thyroid states. Studies with 10 mmol/L [2-3H]glucose showed that cycling between glucose-6-phosphate and glucose was almost twofold higher in euthyroid and hyperthyroid states as compared with the hypothyroid state, although the magnitude of the increase in cycling rate was only approximately 0.2 mumol glucose.min-1.g-1. When 40 mmol/L [2-3H]glucose was added, over 44% of the glucose that was phosphorylated to glucose-6-phosphate was cycled back to glucose, but this cycling was independent of thyroid status. Cycling between fructose-1,6-bisphosphate and fructose-6-phosphate was negligible in all thyroid states. Rates of glycogen synthesis were comparable in hypothyroid and hyperthyroid states and slightly less than in the euthyroid state. Glycolytically formed pyruvate was cycled back to glucose in hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats. During a 60-minute incubation period, cycling to glucose in the presence of 10 mmol/L or 40 mmol/L glucose was up to twofold higher in cells from euthyroid and hyperthyroid rats than in hepatocytes from hypothyroid animals. The measured increases in cycling rates induced by thyroid hormone were small and in theory could have been satisfied by a much smaller increase in respiration rate than was observed.

Animals↗
PubMed1995

The influence of thyroid state on hepatic glycolysis.

The effects of thyroid status on glycolysis using 10, 20, and 40 mM glucose have been examined in hepatocytes derived from hypothyroid, euthyroid, and hyperthyroid rats. For any given concentration of added glucose, total glycolytic rates, as measured by the release of tritium from [6-3H]glucose, were similar in all thyroid states. The aerobic component of glycolysis, where cytoplasmically generated reducing equivalents are transferred to the mitochondria for oxidation, was the major component in the hyperthyroid state, at all concentrations of glucose. In contrast, the aerobic proportion of glycolysis in the hypothyroid and euthyroid states decreased with increasing concentration of added glucose and the anaerobic component became dominant above 20 mM glucose. Cytoplasmic reducing equivalents generated during aerobic glycolysis were transferred to the mitochondria via both the glycerol 1-phosphate and malate/aspartate shuttles in each thyroid state, even though the former shuttle was considerably depressed in the livers of hypothyroid rats. Both asparagine and aminooxyacetate had only minor effects on the rate of glycolysis, but aminooxyacetate depressed the contribution of aerobic glycolysis whereas asparagine had relatively little influence. The respiration rate in the presence of 40 mM glucose was twice as high in hepatocytes from hyperthyroid rats as in cells from hypothyroid animals, and 1.4 times as high as in hepatocytes from euthyroid rats. Smaller stimulations were observed with lower concentrations of added glucose. Furthermore, the increase in respiratory rate over the endogenous value, induced by 10 mM glucose, was six times higher in cells from hyperthyroid rats than in hepatocytes from hypothyroid animals and 2.7 times higher than that observed with cells from euthyroid rats. The insensitivity of glycolysis to thyroid status in contrast to the marked response of respiration provides additional support for the view that the stimulation of metabolism by thyroid hormone is mediated primarily by its action on mitochondrial processes.

Aerobiosis↗
PubMed1994

The capacity of reducing-equivalent shuttles limits glycolysis during ethanol oxidation.

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing-equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.

Animals↗
PubMed1993

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-NMR spectroscopy.

13C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin-lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about 0.1-0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups.

Animals↗
PubMed1993

The influence of hydration on the conformation of bovine serum albumin studied by solid-state 13C-NMR spectroscopy.

13C proton-decoupled cross-polarization magic-angle spinning nmr spectra of bovine serum albumin are reported as a function of hydration. Increases in hydration level enhance the resolution of the peak centered at about 40 ppm but has little or no effect on the other spectral peaks. Hydration has little effect on either the rotating frame proton spin-lattice relaxation time or the cross-relaxation time for any of the peaks, suggesting that the efficiency of dipolar coupling is largely preserved on hydration of the protein. Resolution enhancement of the peak at 40 ppm is not understood, but possible sources of the behavior include a decrease in the line width of contributing resonances from lysine epsilon carbons due to increased motional averaging on hydration, reordering of disulfide bridges, and titration shifts induced by hydration. Hydration of bovine serum albumin appears to have little effect on the distribution of conformations sampled by the protein so that the broad distribution of conformations observed in the dry state is also observed in the fully hydrated state. This is in contrast to lysozyme where significant ordering of the conformation is seen on hydration.

Animals↗
PubMed1992

Reducing-equivalent transfer to the mitochondria during gluconeogenesis and ureogenesis in hepatocytes from rats of different thyroid status.

Isolated hepatocytes from hypothyroid, euthyroid and hyperthyroid rats have been employed to investigate the relative importance of reducing-equivalent shuttles for the transfer of hydrogen between cytoplasm and mitochondria during simultaneous ureogenesis and gluconeogenesis. In cells from hypothyroid animals, a 58% depression of glucose formation and 68% reduction in ureogenesis were induced by n-butylmalonate, an inhibitor of the malate shuttle. A more reduced state of the cytoplasmic compartment and a substantial fall in the concentrations of pyruvate, aspartate, alanine and glutamate accompanied this inhibition. Preincubation of cells with n-butylmalonate yielded greater inhibitory effects than observed in the absence of preincubation. The inhibitory effects on gluconeogenesis and ureogenesis were less in cells from euthyroid rats and were very much reduced in the case of glucose synthesis and absent in the case of ureogenesis, in cells from hyperthyroid rats. It is inferred that both the malate-aspartate and alpha-glycerophosphate shuttles may function in the transfer of reducing equivalents from cytoplasm to mitochondria during ureogenesis in hepatocytes. The major inhibition by n-butylmalonate of glucose and urea synthesis in hepatocytes from hypothyroid rats is due to the diminished activity of the alpha-glycerophosphate shuttle in these cells. Moreover, it follows that the NADH arising from the cytoplasmic malate dehydrogenase-catalysed reaction is accessible to both the malate-aspartate shuttle and the alpha-glycerophosphate shuttle.

Alanine↗
PubMed1992

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes.

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.

Ammonia↗