Increased Substrate Affinity Research Articles

Chimeric Vesicular Monoamine Transporters Identify Structural Domains That Influence Substrate Affinity and Sensitivity to Tetrabenazine

The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. Two domains of VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 and the other from the end of TMD9 through TMD12, increase the affinity for serotonin and histamine as well as the sensitivity to tetrabenazine but only in the context of more C-terminal and more N-terminal VMAT2 sequences, respectively. In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.

AMP deaminase binding in rat skeletal muscle after high-intensity running.

Skeletal muscle deaminates a substantial fraction of its adenylate pool to inosine 5'-monophosphate (IMP) when the rate of energy expenditure exceeds supply. How AMP deaminase is activated in vivo is unclear because the substrate affinity is quite low (Michaelis constant approximately 1-2 mM) relative to estimated concentrations of free AMP in skeletal muscle (0.2-1 microM). AMP deaminase:myosin binding causes a large increase in substrate affinity; whether this binding occurs during physiological exercise is uncertain. Exhaustive high-speed (60 m/min) treadmill exercise in rats results in an extensive depletion of adenine nucleotide and a stoichiometric accumulation of IMP (1.5-2 mumol/g) in the superficial vastus lateralis muscles (predominantly fast-twitch white). We measured AMP deaminase:myosin binding after intense exercise and found the bound fraction of AMP deaminase to be increased from 9 +/- 1% at rest to 48 +/- 4% at approximately 45 s after exercise. The extent of binding lessened during recovery from exercise, falling to 32 +/- 4% after approximately 75 s and 21 +/- 2% after approximately 105 s. This postexercise dissociation of AMP deaminase from myosin appeared to be a first-order process (approximately 50 s half time). Treadmill running that leads to deamination also results in AMP deaminase:myosin binding. Binding should activate AMP deaminase and thus favor IMP formation at low physiological concentrations of AMP.

2-Oxoglutarate transport: a potential mechanism for regulating glutamate and tricarboxylic acid cycle intermediates in neurons.

2-Oxoglutarate (alpha-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na(+)-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, Vmax, and negative cooperatively. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases Vmax. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.

Effect of δ-9-tetrahydrocannabinol and theophylline on hepatic microsomal drug metabolizing enzymes

Theophylline (Th) under in vitro conditions stimulated the activities of rat liver microsomal aniline hydroxylase, N-demethylase and O-demethylase, while δ-9-tetrahydrocannabinol (δ-9-THC) inhibited the activities of these hepatic microsomal drug metabolizing enzymes under similar conditions. δ-9-THC-induced inhibition of hepatic microsomal drug metabolizing enzymes was significantly reduced in the presence of Th. Analysis of Lineweaver-Burk plots showed that Th-induced stimulation of hepatic microsomal drug metabolizing enzymes occurs due to an increase in substrate affinity (1/ K m ) and of V max. δ-9-THC-induced inhibition of N-demethylase and O-demethylase is probably due to competition of the drug with the substrates for a common intermediate in the microsomal electron transport chain. Non-competitive and mixed-type inhibition caused by δ-9-THC on aniline hydroxylation appears to be associated with a non-specific action of δ-9-THC. Blocking of δ-9-THC-induced inhibition or reduction of Th-induced stimulation of hepatic drug metabolizing enzymes with Th or δ-9-THC was due to an increase or decrease in either V max, substrate affinity (1/ K m ) or both with respect to the corresponding K m and V max observed with δ-9-THC or Th alone.

Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase.

Insulin caused a transient increase in H2O2 accumulation in human fat cell suspensions that was observed only in the presence of an inhibitor of catalase and heme-containing peroxidases, such as azide, and reached peak levels of 30 microM within 5 min. The cells contained a plasma membrane-bound NADPH oxidase, producing 1 mol H2O2/mol of NADPH oxidation, that was activated on exposure of intact cells to insulin at contrations that are physiologically relevant (0.1-10 nM). The hormone effect was rapid and was due to a selective increase in substrate affinity. The enzyme was magnesium dependent, required a flavine nucleotide for optimal activity, and was most active at pH 5.0-6.5. In contrast to all other hormone- or cytokine-sensitive NADPH oxidases that have been characterized in sufficient detail, the human fat cell oxidase retained its hormone responsiveness after cell disruption, and only Mn2+, but no ATP, was required for a ligand-induced activation in crude plasma membranes. The results demonstrate that insulin utilizes tyrosine kinase-independent pathways for receptor signaling and strongly support the view that H2O2 contributes to the intracellular propagation of the insulin signal.

Maximal proton secretory rate of rat distal tubules is higher during chronic metabolic alkalosis.

In vivo microperfusion studies show augmented proton secretion in the distal tubule of rats with chronic metabolic alkalosis. The present studies used the same technique to determine whether this augmented proton secretion is due predominantly to an increase in substrate affinity or alternatively to a predominant increase in the maximal proton secretory rate. Surface distal tubules of alkalotic and control rats were microperfused in vivo with solutions containing increasing concentrations of HCO3. Proton secretion was determined as the difference between measured net HCO3 reabsorption and passive HCO3 transport calculated by use of the permeability derived from perfusing with a HCO3-free solution. Proton secretion was expressed as a function of the initial luminal HCO3 concentration and was assumed to follow saturable Michaelis-Menten kinetics. Alkalotic animals had a significantly higher Km (33.9 vs. 21.6 mM, P less than 0.03) and Vmax (223.8 vs. 99.1 pmol.mm-1.min-1, P less than 0.001) compared with control. These data are consistent with the augmented proton secretion in the distal tubule of alkalotic animals as predominantly due to an increased maximal proton secretory rate rather than to increased substrate affinity.

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.

Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein

Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of G sα, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of G sα is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of G sα and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment (‘A 1’), as a result of an increase in substrate affinity with no significant effects on V max. Activation of toxin was independent of G sα and was stimulated 4-fold sodiuim dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A 1 protein and may thus amplify the pathological effects of cholera toxin.

Insecticide-insensitive acetylcholinesterase from a laboratory selected and a field strain of housefly ( Musca domestica) (L.)

1. 1. Acetylcholinesterase from the heads of a strain of houseflies selected for resistance to the carbamate insecticide methomyl, and from a methomyl-resistant field strain was found to be less sensitive to inhibition by methoml than that from a susceptible strain. 2. 2. The enzyme from resistant insects was also more tolerant to malaoxon, dichlorvos and bomyl but not to azamethiphos. 3. 3. The decrease in sensitivity to inhibition appeared to be due to an increase in affinity for substrate.

Enzymatic and transport studies in cholesterol-fed guinea pigs using intestinal brush border membrane vesicles.

The intestinal absorptive and digestive functions using the brush border membrane (BBM) vesicles were evaluated in guinea pigs receiving cholesterol-supplemented diet for 12 weeks. The Na+-gradient-dependent transport of D-glucose (p less than 0.001), L-alanine and L-phenylalanine (p less than 0.01) was decreased significantly the BBM of cholesterol-fed animals. The maximal velocity (Vmax) value of the sucrase and leucine aminopeptidase was decreased without any change in the affinity constant (Km) value, demonstrating that the enzyme contents were reduced in response to cholesterol-rich diet. However, both the Km and Vmax values of the alkaline phosphatase decreased markedly, suggesting that a new enzyme of increased substrate affinity had been formed due to intestinal adaptation of cholesterol load in diet. The present study demonstrated that cholesterol feeding caused a significant alteration in nutrients absorption, membrane enzymes and chemical composition of the small intestine.

Selective expression of phosphoribosylpyrophosphate synthetase superactivity in human lymphoblast lines.

Phenotypic expression of 5-phosphoribosyl 1-pyrophosphate (PRPP) synthetase superactivity was examined in lymphoblast lines derived from six unrelated male patients. Fibroblasts from these individuals have increased rates of PRPP and purine nucleotide synthesis and express four classes of kinetic derangement underlying enzyme superactivity: increased maximal reaction velocity (catalytic defect); inhibitor resistance (regulatory defect); increased substrate affinity (substrate binding defect); and combined catalytic and regulatory defects. Lymphoblast lines from three patients with catalytic defects and from three normal individuals were indistinguishable with respect to enzyme activities, PRPP concentrations and generation, and rates of purine synthesis. Enzyme in lymphoblasts from a patient with combined defects also showed normal maximal reaction velocity but expressed purine nucleotide inhibitor resistance. A second regulatory defect and a substrate binding defect were also demonstrable in lymphoblasts and were identical to the enzyme defects in fibroblasts from the respective patients. Regulatory and substrate binding defects in lymphoblasts were accompanied by increased rates of PRPP and purine nucleotide synthesis. Among explanations for selective expression of enzyme superactivity, reduced concentrations of catalytically superactive enzymes seemed unlikely: immunoreactive PRPP synthetase was comparable in normal-derived and patient-derived cells. Activation of normal enzyme in transformed lymphocytes was also unlikely because absolute specific activities of lymphoblast PRPP synthetases corresponded to those of normal fibroblast and erythrocyte enzymes. Abnormal electrophoretic mobilities and thermal stabilities, identified in certain catalytically superactive fibroblast PRPP synthetases, were not found in the corresponding lymphoblast enzymes. Thus, lymphoblast PRPP synthetases from patients with catalytic superactivity appeared to differ structurally and functionally from their fibroblast counterparts.

Effects of insulin, glucagon and dexamethasone on pyruvate kinase in cultured hepatocytes

Long-term (24–48 h) and short-term (10–30 min) regulation by hormones of hepatic pyruvate kinase activity was investigated in adult rat hepatocytes cultured under serum-free conditions. In the absence of hormones, pyruvate kinase total activity decreased to 83%, 67% and 39% of the initial level at 24, 48 and 72 h of culture. Insulin (100 nM) maintained total activity significantly above control levels throughout this period. In contrast, glucagon (100 nM) and dexamethasone (100 nM) accelerated the gradual decrease within 24 h (glucagon) or 48 h (dexamethasone) of culture. In these long-term experiments, activity at non-saturating concentrations of phospho enolpyruvate was decreased by glucagon and dexamethasone but not directly modulated by insulin. However, insulin increased the cellular content of the pyruvate kinase activator fructose-1,6-diphosphate. In short-term experiments on cells cultured under serum- and hormone-free conditions for 48 h, both glucagon and dexamethasone independently caused a rapid, dose-dependent increase of the K 0.5 for phosphoenolpyruvate within 10 min, while V max was not affected. Insulin inhibited this action of glucagon and dexamethasone and, in their absence, significantly increased substrate affinity for phospho enolpyruvate within 30 min. Cellular fructose-1,6-diphosphate contents remained unchanged under these conditions. The data identify glucocorticoids and insulin - in addition to glucagon - as short-term regulators of the catalytic properties of pyruvate kinase. All three hormones are effective in the long-term control of total enzyme activity.

Regulatory Properties of Pyruvate Kinase Isoenzymes from a Chlorophyll-free Chlorella Mutant in Darkness or Blue-Light

Summary In analysing the stimulating effect of blue-light on respiration and carbohydrate degradation of green algae, a dual light effect on pyruvate kinase, one of the regulatory enzymes of glycolysis, was demonstrated in the chlorophyll-free, carotenoid-containing mutant no. 20 of Chlorella vulgaris . It consists of an increase in the amount of active enzyme (V max ) and of an increase in substrate affinity, the latter probably resulting from an interconversion of two iso-enzymes with different substrate affinities. Since a thorough comparison of the respective time courses revealed no agreement between the blue-light action on respiration and pyruvate kinase, however, other mechanisms of regulation may be involved. Indeed, kinetic and regulatory properties of the two pyruvate kinase isoenzymes lead to the conclusion that respiration and carbohydrate degradation may be regulated by a light-influenced fine control, i.e. by a blue-light-controlled supply of respirable substrate and/or allosteric effectors and cofactors.

Limiting nutrient patchiness and its rôle in phytoplankton ecology

Limiting nutrient patchiness is examined as a factor affecting the community structure and species succession of natural phytoplankton communities held in ammonia limited continuous culture at a dilution rate of 0.3 day −1. It was found that under a homogeneous distribution of the limiting nutrient members of genus Chaetoceros dominated and when ammonia was added daily (patchy distribution), Skeletonema dominated. Intermediate patchiness gave rise to an assemblage dominated by both Chaetoceros and Skeletonema. The nutrient uptake ability of each assemblage was determined three weeks after experiment initiation. Each assemblage was best able to optimize uptake of ammonia under its particular patchy nutrient regime. Optimization of a patchy environment took place by an increased maximal uptake rate ( V max ) while optimization of a homogeneous environment appeared to take place by increased substrate affinity (i.e., low K s ). This study demonstrates that limiting nutrient patchiness can alter the relative abundance of populations within a community based on each population's ability to exploit the limiting resource under a particular degree of patchiness. We also show that coexistence of two populations might be expected due to the patchiness of a single limiting nutrient. The importance of patchiness in relation to other factors which determine community structure is discussed.

Boar malpha-acrosin. Purification and characterization of the inital active enzyme resulting from the conversion of boar proacrosin to acrosin.

The preparation of highly purified malpha-acrosin is described. Purification was achieved by controlled activation of partially purified proacrosin, followed by gel chromatography over Sephadex G-100 at pH 3.0. The final malpha-acrosin preparation resulted in a single protein band with a molecular weight of 49,000 as determined by sodium dodecyl sulfate-disc gel electrophoresis. Disc arginine naphthylamide hydrolyzing band with a relative migration of 0.39 malpha-acrosin catalyzed the hydrolysis of synthetic substrates containing arginine and lysine, but not phenylalanine. Although calcium ions were not required for enzymatic activity, the addition of calcium chloride stimulated the activity through an increased substrate affinity and an increased maximal velocity. Polyamines stimulated the maximal velocity of the reaction, but were without effect on the substrate affinity. malpha-Acrosin was inhibited by lima bean, ovo-mucoid, and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and 1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an irreversible inhibition, while L-arginine, benzamidine, and p-aminobenzamidine were competitive inhibitors with respect to substrate. These properties of malpha-acrosin are very similar to those previously reported for mbeta-acrosin and suggest that the portion of the molecule lost during the conversion of malpha-acrosin to mbeta-acrosin contributes little to the topography of either the active site or regulatory sites of the enzyme.

The effect of fructose 1,6-diphosphate on pyruvate kinase from white muscle of three species of Mediterranean fish

1. 1. The kinetics of pyruvate kinase in the absence and presence of fructose 1,6-diphosphate was investigated in preparations from white muscle of Boops boops (L.), Coryphaena hippurus (L.) and Mugil cephalus (L.). 2. 2. Mugil cephalus pyruvate kinase showed sigmoidal kinetics which changed to hyperbolic in the presence of fructose 1,6-diphosphate with increase in substrate affinity and maximum velocity. 3. 3. Coryphaena hippurus and Boops boops pyruvate kinase showed hyperbolic kinetics with increase in substrate affinity and maximum velocity in the presence of fructose 1,6-diphosphate, but the Boops boops enzyme was inhibited in the presence of the effector at phosphoenolpyruvate concentrations above 0.375 mM.

Effects of amino acids on the kinetic properties of three noninterconvertible rat pyruvate kinases

Three apparent isozymes of pyruvate kinase respond differentially to amino acids. The kidney cortex enzyme (K isozyme) was inhibited by the greatest number of amino acids, alanine and phenylalanine being the most effective. As the nonpolar side chain was increased in bulk relative to alanine or decreased relative to phenylalanine, the amino acids progressively became less effective inhibitors. Polarity also decreased inhibitory potential and serine (3-OH alanine) was a potent activator, able to increase substrate affinity and to reverse the inhibition induced by other amino acids. The amino acids having an effect on the muscle enzyme (M isozyme) show a similar relationship between structure and potential activity, except amino acids with less bulky side chains activate. Again hydroxylation appears to increase the potency of the amino acid as an activator. The liver enzyme (L isozyme) shows a pattern of inhibition similar to the kidney cortex enzyme except that it is much less sensitive to the amino acids with bulky side chains and serine remains an inhibitor, albeit less effective than alanine. These data, plus observed differences in the alanine and phenylalanine inhibition under varied conditions, support a hypothesis that there are two different types of amino acid binding sites with each isozyme having both. However, the response to the bound amino acid varies from isozyme to isozyme. It also was concluded that fatty acids were not modulators of pyruvate kinase activity.

Synergistic activation of β-galactosidase by Na + and Cs +

The rate of hydrolysis of ortho-nitropheno-β- d-galactopyranoside ( o-NPG) by β-galactosidase from Escherichia coli was measured in the presence of varying concentrations of substrate, Na + and Cs +. The results were analyzed by means of Dixon plots. Na + increases both the affinity of the enzyme for the substrate and the maximal rate of hydrolysis at substrate saturation. The substrate increases the affinity of the enzyme for Na + by an amount proportional to the increase in substrate affinity produced by the Na +. It is shown that this mutual effect of activator and substrate on each other's affinities is a thermodynamic necessity. Cs + increases the rate of hydrolysis at concentrations lower than 0.01 m; the extent of this activation depends on the Na + concentration. Above 0.01 m Cs + becomes inhibitory. A model based on inductive changes in the active site which result from the binding of substrate and ions is proposed to explain the observed facts.