Biomedical subjects

J M May

Publications and source records attributed to J M May.

At least 73 records · Page 4Linked to original sources

PubMed1990

Localization of a reactive exofacial sulfhydryl on the glucose carrier of human erythrocytes.

Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacial sulfhydryl with bis(maleimidomethyl) ether-L-[35S]cysteine was chemically cleaved, either at tryptophans by N-bromosuccinimide or at nonalkylated cysteines by 2-nitro-5-thiocyanobenzoic acid. The resulting fragments were separated by linear gradient polyacrylamide gel electrophoresis, and the labeled fragments were identified by their apparent molecular weight, and by immunoblotting with antibodies to specific regions of the carrier protein. All of the labeled fragments were recognized by an antibody to the carboxy terminus of the carrier, but not by an antibody to a cytoplasmic loop on the C-terminal half of the carrier. The labeled exofacial sulfhydryl was assigned to Cys429, since this is the only residue of the three possibilities which is beyond the expected cleavage sites of the two reagents in the carrier sequence. These results concur with the predictions of hydropathy analysis and will be relevant for studies of how modification of this sulfhydryl affects carrier function, particularly since several other known carrier isoforms lack a corresponding cysteine.

Binding Sites↗

PubMed1990

Thiol-fatty acylation of the glucose transport protein of human erythrocytes.

Incubation of intact human erythrocytes with [3H]palmitate labeled a protein with electrophoretic characteristics of the glucose transporter. This labeling occurred via a thioester linkage, since it was unaffected by organic solvent extraction, but was substantially removed as the hydroxamate upon treatment with neutral hydroxylamine. Immunoprecipitation of the labeled protein with a monoclonal antibody to the glucose transporter confirmed its identity.

Acylation↗

PubMed1989

Differential labeling of the erythrocyte hexose carrier by N-ethylmaleimide: correlation of transport inhibition with reactive carrier sulfhydryl groups.

Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.

3-O-Methylglucose↗

PubMed1989

Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism.

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.

Biological Transport↗

PubMed1989

Selective labeling of the erythrocyte hexose carrier with a maleimide derivative of glucosamine: relationship of an exofacial sulfhydryl to carrier conformation and structure.

Sulfhydryl-reactive derivatives of glucosamine were synthesized as potentially transportable affinity labels of the human erythrocyte hexose carrier. N-Maleoylglycyl derivatives of either 6- or 2-amino-2-deoxy-D-glucopyranose were the most potent inhibitors of 3-O-methylglucose uptake, with concentrations of half-maximal irreversible inhibition of about 1 mM. Surprisingly, these derivatives were very poorly transported into erythrocytes. They reacted rather with an exofacial sulfhydryl on the carrier following a reversible binding step, the latter possibly to the exofacial substrate binding site. However, their reactivity was determined primarily by access to the exofacial sulfhydryl, which, as predicted by the one-site model of transport, required a carrier conformation with the exofacial substrate binding site exposed. Once reacted, the carrier was "locked" in a conformation unable to reorient inwardly and bind cytochalasin B. In intact erythrocytes the N-maleoylglycyl derivative of 2-[3H]glucosamine labeled predominantly an Mr 45,000-66,000 protein on gel electrophoresis in a quantitative and cytochalasin B inhibitable fashion. By use of changes in carrier conformation induced by competitive transport inhibitors in a "double" differential labeling method, virtually complete selectivity of labeling of the carrier protein was achieved, the latter permitting localization of the reactive exofacial sulfhydryl to an Mr 18,000-20,000 tryptic fragment of the carrier.

3-O-Methylglucose↗

PubMed1989

Inhibition of hexose transport in the human erythrocyte by 5, 5'-dithiobis(2-nitrobenzoic acid): role of an exofacial carrier sulfhydryl group.

The sulfhydryl reagent 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1 mM DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10 mM cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120 mM accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 microM cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfhydryl, but reaction of this group may not always "lock" the carrier in an outward-facing conformation.

3-O-Methylglucose↗

PubMed1988

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes.

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.

3-O-Methylglucose↗

PubMed1988

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport.

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.

3-O-Methylglucose↗

PubMed1988

Inhibition of hexose transport and labelling of the hexose carrier in human erythrocytes by an impermeant maleimide derivative of maltose.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.

Biological Transport↗

PubMed1988

Photolabeling of the human erythrocyte glucose carrier with androgenic steroids.

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.

3-O-Methylglucose↗

PubMed1988

Inhibition of hexose transport by adenosine derivatives in human erythrocytes.

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.

3-O-Methylglucose↗

PubMed1987

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter.

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Glucose utilization in islets of hyperglycemic rat models with impaired glucose-induced insulin secretion.

Under most experimental conditions islet glucose metabolism is well-correlated with short-term glucose-induced insulin secretion. Two hyperglycemic rat models (neonatal streptozotocin and glucose infusion) have been previously found to have markedly impaired insulin responses to glucose, and the glucose utilization of islets isolated from these models was therefore studied to see if reduced glucose metabolism might be related to the secretory abnormalities. It was found that glucose utilization in the islets of the two models was similar or higher than in comparable control islets. These data suggest that the secretory defect of these models, which is presumably induced by chronic hyperglycemia, is at a step in the secretion process distal to glucose metabolism.

Animals↗

PubMed1987

Photolabeling of the adipocyte hexose carrier with an aryl azide derivative of maltose.

A nitrophenyl azide derivative of maltose, N-(4-azido-2-nitrophenyl)-maltosylamine (NAP-maltosylamine), was synthesized as a potential photoaffinity label for the hexose carrier of the rat adipocyte. This derivative inhibited 3-O-methylglucose uptake with a Ki of 1.3 mM in the dark, while that of maltose was 10.0 mM. Carbon-14-labeled maltose and NAP-maltosylamine entered adipocytes via the hexose carrier, the latter in a concentrative fashion. Photolysis of NAP-[14C]maltosylamine in the presence of an adipocyte low density microsomal membrane fraction labeled several electrophoretic bands. Among these are a 45 kDa band which showed features expected of the hexose carrier: its labeling was decreased 40% by D- but not L-glucose and pretreatment of intact adipocytes with insulin decreased labeling of the 45 kDa band by 10-40%, as predicted by the translocation theory of insulin-stimulated transport activation. These studies show the suitability of using carbon-1-modified sugar photoaffinity labels as probes for the hexose carrier and possibly of its regulation in rat adipocytes.

Adipose Tissue↗

PubMed1986

Regulation of beta-adrenoceptor density and function in rat vas deferens.

beta-Adrenoceptor density and responsiveness were examined in rat vas deferens following surgical and pharmacological treatments. Receptor density was measured by Scatchard analysis of saturation isotherms of specific [125I]pindolol ([125I]PIN) binding in membrane homogenates. Functional responsiveness was measured by isoprenaline-induced inhibition of field stimulated (60 V, 1 ms, 0.1 Hz) or 40 mM K+-induced contractions. Four days following surgical denervation of vas deferens there was no change in the density of [125I]PIN binding sites, suggesting that these sites are not located on prejunctional neurons. Neither 7 day bilateral adrenalectomy, 21 day denervation, nor 7 days treatment with 10 mg/kg per day desmethylimipramine caused changes in either the potency of isoprenaline in inhibiting contraction or the density of [125I]PIN binding sites compared to controls. Infusion of 3 mg/kg per day isoprenaline for 8 days significantly reduced the potency of isoprenaline in inhibiting field stimulated contractions, reduced the maximum degree of inhibition, and reduced the density of [125I]PIN binding sites. These results suggest that beta-adrenoceptor density and responsiveness in rat vas deferens are not affected by removal of adrenal hormones or neuronal stimulation, but that receptor density and responsiveness can be decreased by increasing the concentration of beta-adrenoceptor agonists at the receptor. Therefore, beta-adrenoceptors in rat vas deferens probably receive little tonic stimulation under normal circumstances.

Adrenalectomy↗

PubMed1986

Photoaffinity labeling of glyceraldehyde-3-phosphate dehydrogenase by an aryl azide derivative of glucosamine in human erythrocytes.

An aryl azide derivative of glucosamine, N-(4-iodoazidosalicyl)-2-amido-2-deoxy-D-glucopyranose (GlcNAs), was synthesized as a potential photoaffinity label for the facilitative hexose carrier. The derivative inhibited hexose uptake into intact human erythrocytes half-maximally at 3.5 mM and was itself slowly transported into cells. However, photolysis of iodinated GlcNAs with leaky erythrocyte ghosts produced appreciable labeling on gel electrophoresis only of Band 6, which is glyceraldehyde-3-phosphate dehydrogenase. Band 6 photolabeling in leaky ghosts by GlcNAs was: saturable, due mostly to the aryl azide moiety, inhibited by agents with known affinity for the enzyme including sulfhydryl reagents and the enzyme substrate glyceraldehyde-3-phosphate, and not inhibited by the free-radical scavenger p-aminobenzoic acid. Moreover, GlcNAs also inhibited erythrocyte glyceraldehyde-3-phosphate dehydrogenase activity in a dose-dependent fashion in the dark and more potently following irradiation. In resealed ghosts, Band 6 labeling was decreased by D-glucose, reflecting inhibition of carrier-mediated uptake of the agent. GlcNAs appears to be a specific photoaffinity label for erythrocyte glyceraldehyde-3-phosphate dehydrogenase, and therefore potentially useful for studies of enzyme activity, compartmentation, or membrane association.

Affinity Labels↗

PubMed1986

Cognitive processes and violent behaviour in young people.

Forty-three subjects from secondary school took part in a correlation study investigating the nature of cognitive processes involved in the presentation of violent behaviour. Measures of violence were scores on "aggression items" of a self-report questionnaire. The experimental procedure involved binocular tachistoscopic presentation of neutral and violent slide pairs. Descriptions of the composite stimuli were scored for violent content. The main finding was that subjects who had reported more involvement in violent acts also reported seeing more violence in the stimulus array. This association held irrespective of age, IQ, socio-economic status and starting mood. It is argued that these findings indicate a perceptual, rather than a response, bias. A role for this bias as a possible maintaining condition in the presentation of aggressive behaviour is presented. The implications of the present findings for interventions with young people are discussed. It is suggested that cognitive techniques may prove more effective than traditional behavioural programmes.

Adolescent↗

PubMed1986

Potentiation by glucose of lipolytic responsiveness of human adipocytes.

The effect of glucose on lipolytic regulation was studied in isolated human adipocytes. Glucose enhanced adipocyte glycerol release in the presence and absence of the beta-adrenergic agent ritodrine by 150-200% of control rates. The glucose effect was maximal at just greater than 1 mM glucose and could not be attributed to prevention of a time-dependent decline in lipolysis. Glucose not only increased lipolytic stimulation at each of several concentrations of ritodrine but also enhanced the sensitivity to stimulation at low concentrations of the agent. Ritodrine-stimulated lipolysis was inhibited by insulin by 50-60%; although glucose increased absolute rates of lipolysis, it did not affect the relative inhibition of lipolysis by insulin or the sensitivity to the hormone. In investigating a possible cause of the glucose effect on lipolysis, it was found that the addition of adenosine deaminase increased lipolytic rates in the absence of glucose and blunted the relative stimulation of lipolysis by glucose, the latter implicating extracellular adenosine in the mechanism of the glucose effect.

Adenosine Deaminase↗