Biomedical subjects

H M Said

Publications and source records attributed to H M Said.

At least 55 records · Page 3Linked to original sources

PubMed1994

Intestinal uptake of uridine in suckling rats: mechanism and ontogeny.

Nucleosides, essential substrates for a variety of intracellular metabolic reactions, are obtained from dietary and endogenous sources. Nucleotides (which dephosphorylate to nucleosides prior to intestinal absorption) are present in milk and have trophic effects on the developing gastrointestinal tract. The mechanism of transport of nucleosides in the developing intestine of suckling rats is unknown. To address this issue, we therefore examined uridine uptake in rat everted intestinal sacs. In suckling rats (15-17 days old), tissue uptake of low (5-microM) and high (60 microM) concentrations of [3H]-uridine was linear for up to 2 min of incubation. Initial rate of uptake of [3H]-uridine was (i) not significantly different in the jejunum and the ileum; (ii) greater in the presence of Na+, than other cations; (iii) saturable as a function of concentration with a Vmax of 21,044 +/- 2,302 pmol/g tissue wet wt/30 sec and an apparent Km of 33.8 +/- 10.1 microM; (iv) inhibited by high concentration (500 microM) of unlabeled uridine and other nucleosides; (v) temperature-dependent; (vi) energy-dependent; and (vii) pH-sensitive. Developmental maturation was associated with a progressive decrease in the Vmax of the uridine transport process (21,044 +/- 2,302, 14,651 +/- 1,679, and 8,461 +/- 1,369 pmol/g tissue wet wt/30 sec for suckling, weanling, and adult rats, respectively) and a progressive increase in the apparent Km of the uptake system (33.8 +/- 10.1, 55.6 +/- 13.1, and 61.7 +/- 14.5 microM for suckling, weanling, and adult rats, respectively). We concluded that uptake of uridine by the developing intestine of suckling rats involves a carrier-mediated system, which is energy- and temperature-dependent, and requires extracellular sodium. Furthermore, the uptake process was found to undergo clear ontogenic changes with maturation.

2,4-Dinitrophenol↗

PubMed1993

Uptake of riboflavin by intestinal basolateral membrane vesicles: a specialized carrier-mediated process.

The mechanism of riboflavin (RF) uptake by intestinal basolateral membrane vesicles (BLMV) was examined in this study. BLMV were isolated by an established Percoll-gradient methodology from rabbit small intestine. Uptake of riboflavin was mainly the result of transport of the substrate into an osmotically active intravesicular space with less binding to membrane surfaces. Uptake of RF with time was similar in the presence of a Na+ and a K+ gradient (out > in) and was not significantly influenced by changes in incubation buffer pH. The initial rate of uptake of riboflavin as a function of concentration was saturable in both jejunal and ileal BLMV and occurred with apparent Km values of 5.0 microM and 4.4 microM and Vmax values of 91.6 and 60.8 pmol/mg protein per 5 s, respectively. Unlabeled riboflavin and the structural analogues lumiflavin, isoriboflavin and 8-aminoriboflavin all caused significant inhibition (but to different degrees) in the uptake of [3H]riboflavin. On the other hand, 8-hydroxyriboflavin, lumichrome, lumazine and D-ribose failed to inhibit [3H]riboflavin uptake. Trans-stimulation of [3H]riboflavin efflux from preloaded BLMV by unlabeled riboflavin or lumiflavin was also observed. Altering transmembrane electrical potential by anion substitution and valinomycin-induced K+ diffusion did not affect the riboflavin uptake process. These results demonstrate the existence of a specialized carrier-mediated mechanism for riboflavin uptake by intestinal BLMV. Furthermore, the system appears to transport the vitamin by a process which is Na(+)- and pH-independent, and electroneutral in nature.

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

PubMed1993

Folate transport in intestinal brush border membrane: involvement of essential histidine residue(s).

We examined the possible existence of histidine residue(s) in the folate transporter of rabbit intestine. This was done with use of the histidine-specific reagent diethyl pyrocarbonate (DEPC) and purified intestinal brush-border-membrane vesicles. DEPC caused significant concentration- and time-dependent inhibition of folic acid transport. The inhibition was only seen when transport was examined in vesicles incubated in buffer at pH 5.2 and not in those incubated in buffer at pH 7.4. The addition of unlabelled folic acid to vesicle suspension before treatment with DEPC (2.5 mM) led to a significant (P < 0.01) protection (84%) against the inhibition of folic acid transport. Treating vesicles pretreated with DEPC (2.5 mM) with reducing reagents (dithiothreitol, 2-mercaptoethanol and 2,3-dimercaptopropanol, all at a final concentration of 10 mM) did not reverse the inhibitory effect of DEPC on folic acid transport. On the other hand, treating the DEPC-pretreated vesicles with hydroxylamine (140 mM) led to a significant reversal (P < 0.01) (54%) of the inhibition of folic acid transport. The inhibitory effect of DEPC on carrier-mediated folic acid transport was found to be mediated through a decrease in the Vmax. (i.e. a decrease in the number and/or activity) of the carriers and an increase in the apparent Km (i.e. a decrease in their affinity), classifying the effect as a mixed-type inhibition. These results demonstrate the existence of critical histidine residue(s) in the intestinal brush-border-membrane folate transporter which is essential for its interaction with, and transport of, the vitamin. These findings also suggest that the histidine residue(s) is located at (or near) the substrate-binding site.

Animals↗

PubMed1993

Uptake of riboflavin across the brush border membrane of rat intestine: regulation by dietary vitamin levels.

BACKGROUND: Possible regulation of riboflavin (RF) intestinal uptake process by dietary substrate level is not known and was examined in this study. METHODS: RF uptake was examined using intestinal brush border membrane vesicles (BBMV). RESULTS: Oversupplementation with RF caused significant decrease (P < 0.05; down-regulation) in the uptake of 3 mumol/L RF compared with control. This effect was not due to differences in the relative purity of the intestinal BBMV preparations and appeared to be specific for RF. This down-regulation was mediated through a significant (P < 0.01) decrease in the Vmax of the RF uptake process with no significant change in the apparent Michaelis constant (Km). In contrast, RF deficiency caused a significant (P < 0.01) enhancement (up-regulation) in the uptake of 3 mumol/L RF compared with pair-fed control. Again the enhancement was not due to differences in the relative purity of the BBMV preparations and appeared to be specific for RF. This up-regulation was mediated via a significant (P < 0.01) increase in the Vmax of the RF uptake process with no significant change in the apparent Km. CONCLUSIONS: These findings show that the RF intestinal uptake process is regulated by the level of the vitamin in the diet (and/or body stores) and that the regulation is mediated via changes in the number (and/or activity) of the RF uptake carriers with no change in their affinity.

Animals↗

PubMed1993

Transport of the biotin dietary derivative biocytin (N-biotinyl-L-lysine) in rat small intestine.

BACKGROUND: Biocytin is an important end product of intraluminal digestion of dietary protein-bound biotin. Limited studies are available regarding the ability of the small intestine to transport biocytin and about the mechanism involved. The aim of the present study was to delineate these issues. METHODS: Transport of [3H]-biocytin was examined using everted sacs from rat intestine. RESULTS: Mucosal-to-serosal transport of low (0.022 mumol/L) and high (5 mumol/L) concentrations of biocytin were linear for up to 20 minutes of incubation. Transport of biocytin as a function of concentration (0.022-5 mumol/L) was linear (r = 0.99) and occurred at a rate of 22,062 fmol.g tissue (wet wt)-1.15 min-1. Addition of high concentrations of unlabeled biocytin, biotin, biotin methyl ester, and lysine did not cause a significant inhibition of the transport of [3H]-biocytin. Furthermore, transport of biocytin was independent of Na+ concentration, pH, energy, and temperature. Compared with transport of equimolar concentrations of free biotin, transport of biocytin (0.022 mumol/L) was significantly lower in both the jejunum and the ileum. CONCLUSIONS: (1) Biocytin transport in rat intestine is lower than that of free biotin and occurs via simple physical diffusion. (2) In the rat, efficient absorption and optimal bioavailability of dietary protein-bound biotin necessitates its conversion to free biotin.

Animals↗

PubMed1993

5-Methyltetrahydrofolate transport in basolateral membrane vesicles from human liver.

Transport of 5-methyltetrahydrofolate was studied in vesicles isolated from the basolateral membrane (BLM) of human liver. Uptake was mostly via transport into an osmotically active intravesicular space, with some membrane binding (approximately 20%). Transport was more rapid with an imposed pH gradient (pHout = 5.0, pHin = 7.5) as compared with either pHout = pHin = 5.0 or pHout = pHin = 7.5. Transport under the influence of a pH gradient showed a transient overshoot; uptake at 60 s was 4.2 times higher than at equilibrium (60 min). Transport in the presence of a pH gradient was saturable; apparent Km = 0.55 mumol/L and Vmax = 1.98 nmol.g protein-1.10 s-1. Transport was not saturable at pHout = pHin = 7.5. Transport was inhibited by the structural analogs 5-formyltetrahydrofolate, folic acid, and methotrexate, and was electroneutral in nature. These results suggest that 5-methyltetrahydrofolate transport in human BLM vesicles is via carrier-mediated cotransport with hydrogen ions.

Biological Transport↗

PubMed1993

Mechanism of transport of riboflavin in rabbit intestinal brush border membrane vesicles.

Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (> 76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All 3H radioactivity that appeared in the intravesicular space after incubation with [3H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na+ or a K+ gradient (out > in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent Km of 7.24 +/- 1.06 and 8.88 +/- 0.90 microM and Vmax of 24.31 +/- 1.48 and 34.24 +/- 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [3H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and D-ribose all failed to inhibit [3H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [3H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K(+)-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na+ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.

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

PubMed1993

Effect of chronic portal hypertension on glutamine transport across rat intestinal brush border and basolateral membranes.

The present study examined the effect of chronic portal hypertension on the intestinal transport of glutamine across the brush border membrane (BBM) and the basolateral membrane (BLM) of the rat enterocyte. Chronic portal hypertension was induced by partial portal vein ligation, and the results were compared with results in sham-operated rats. Transport studies were performed by using established techniques of isolated membrane vesicles. In chronic portal hypertensive rats, transport of glutamine in BBM vesicles (BBMVs) exhibited significant inhibition in the maximal velocity (Vmax) of both the Na(+)-dependent and the Na(+)-independent transport processes, with no significant changes in the apparent Michaelis-Menten constant (Km) of the carrier systems. Similarly, chronic portal hypertension caused a significant decrease in the Vmax of the Na(+)-dependent and the Na(+)-independent uptake processes in intestinal BLM vesicles (BLMVs), with no change in the apparent Km of the uptake systems. These changes in glutamine transport in BBMVs and BLMVs brought about by chronic portal hypertension are not due to differences in the relative purity of the vesicular preparations between the portal hypertensive and sham-operated rats, because similar degrees of enrichment and depurification of marker enzyme activities were seen in final vesicular preparations in the two rat groups. These results demonstrate that in chronic portal hypertension, glutamine transport across both poles of the enterocyte is impaired. At both the BBM and BLM domains, the impairment appears to be mediated via a decrease in the number (and/or activity) of the glutamine carrier systems, with no changes in their affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane.

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.

Animals↗

PubMed1992

Biotin transport in human liver basolateral membrane vesicles: a carrier-mediated, Na+ gradient-dependent process.

The characteristics of biotin transport into human liver were examined using purified liver basolateral membrane vesicle (BLMV) preparations. Biotin uptake by BLMVs was mostly due to transport of the vitamin into the inside of vesicles. In the presence of an Na+ gradient (out greater than in), biotin transport with time was significantly higher than that in the presence of a K+ gradient and showed transient accumulation (overshoot). High concentrations of unlabeled biotin and related compounds caused significant cis inhibition in biotin transport in the presence of an Na+ (but not a K+) gradient. Transport of biotin as a function of concentration in the presence of an Na+ gradient included a saturable component, while it was lower and linear in the presence of a K+ gradient. Apparent Km and Vmax of the saturable Na+ gradient-dependent component were 1.22 mumol/L and 4.76 pmol.mg protein-1 x 10 s-1, respectively. Induction of a negative or positive intravascular potential using valinomycin-K diffusion methodology did not affect biotin transport into BLMVs. Also, neither the anion-exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid nor 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid caused significant inhibition in biotin transport. These results indicate that biotin transport into human liver occurs via a specialized, carrier-mediated transport system. This system is Na(+)-gradient dependent and transports the vitamin via an electroneutral process.

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

PubMed1992

Uptake of 5-formyltetrahydrofolate in isolated rat liver mitochondria is carrier-mediated.

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 mumol.L-1 and Vmax = 3.49 pmol.min-1.mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.

Animals↗

PubMed1992

Transport of 5-methyltetrahydrofolate in basolateral membrane vesicles of rat liver.

We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5-methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5-methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Uptake of biotin by isolated rat liver mitochondria.

This study examined the mechanism of biotin uptake by liver mitochondria. Mitochondria were isolated from rat liver by an established procedure and demonstrated normal respiratory control ratios. Uptake of biotin with time at incubation buffer pH 6.1 was rapid and linear and occurred with a distinct "overshoot" phenomenon that peaked at approximately 1 min of incubation. At incubation buffer pH 7.4, however, uptake of biotin with time was significantly slower and no overshoot was observed. Gradual lowering of incubation buffer pH from 7.9 to 6.1 caused a similar pattern of increase in uptake of low (0.024 microM) and high (8 and 30 microM) concentrations of biotin. At incubation buffer pH 6.1 and 7.4, uptake of biotin as a function of concentration (0.012-30 microM) was linear and occurred at rates of 3.62 and 1.90 pmol.mg protein-1.5 s-1, respectively. Addition to the incubation medium of high concentrations of unlabeled biotin, its related compounds (biocytin, desthiobiotin, biotin methyl ester, thioctic acid, and thioctic amide), or substrates of known mitochondrial transporters (succinate, pyruvate, glutamate, malate, and phosphate) failed to cause any significant inhibition in uptake of [3H]biotin by mitochondria incubated in buffer pH 6.1 and 7.4. Furthermore, preloading mitochondria with unlabeled biotin, biocytin, malate, or aspartate failed to cause any significant stimulation in biotin uptake. At incubation buffer pH 6.1, treatment of mitochondria with the protonophore FCCP caused significant inhibition in pH-dependent overshoot of biotin uptake. However, treatment of mitochondria with the potassium ionophore valinomycin caused significant stimulation in the pH-dependent overshoot of biotin uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effect of chronic renal failure on intestinal transport of biotin in the rat.

The present study examined the effect of chronic renal failure (RF) on biotin transport in rat intestine. Chronic RF was induced by subjecting rats to right nephrectomy and left two-thirds nephrectomy. Control rats underwent sham operation but without tissue removal and were pair fed. Transport studies were performed 5 to 6 weeks after operation. Chronic RF was found to cause a marked decrease in the maximal velocity (Vmax) of biotin mucosal-to-serosal transport in jejunal everted sacs with minimal change in the apparent Michaelis-Menten constant (Km) of the transport process. This impairment in biotin transport was found to involve the transport process of the vitamin across the brush border membrane (BBM) domain of the enterocyte as shown by studies with BBM vesicles (BBMVs). These studies with BBMV similarly showed a decrease in the Vmax of biotin transport with minimal change in the apparent Km. The impairment of biotin transport in intestinal BBMVs was not due to dissipation of the Na+ gradient across the BBM (which is the driving force of biotin movement across this membrane) because transport of sodium 22 was found to be similar in BBMVs prepared from the RF rats and the sham-operated PF control rats. These results demonstrate that chronic RF in rats causes impairment in biotin intestinal transport. This impairment is due to a decrease in the number (and/or activity) of the biotin transport carriers and involves the transport process of biotin at the BBM.

Animals↗

PubMed1991

An endogenous carrier-mediated uptake system for folate in oocytes of Xenopus laevis.

We investigated the existence of an endogenous uptake system for folate in Xenopus laevis oocytes. This was done by performing uptake measurements using [3H]folic acid. Uptake of folic acid was linear with time for 4 h of incubation, and was similar in collagenase-treated and non-treated oocytes. The uptake process was carrier-mediated, as suggested by the saturation of folic acid uptake with concentration, and by the ability of unlabelled folic acid and its related compounds to significantly inhibit the uptake of [3H]folic acid. The apparent Km and Vmax of the uptake process were 42 +/- 7 nM and 10.56 +/- 0.46 fmol per oocyte per 2 h, respectively. The uptake of folic acid was independent of the presence of Na+ in the incubation medium, but was highly pH dependent with severe inhibition occurring at pH lower than 6.5. Folic acid uptake was energy- and temperature-dependent, and was significantly inhibited by the anion transport inhibitors DIDS and SITS. These results demonstrate the existence of an endogenous carrier-mediated system for folic acid uptake in Xenopus oocytes. Further characterization of the molecular mechanism of folic acid uptake and its regulation in this non mammalian in vitro unicellular system may prove useful in furthering our understanding of folate movement across biological membranes.

Animals↗

PubMed1991

Movement of biotin across the rat intestinal basolateral membrane. Studies with membrane vesicles.

The characteristics of the transport process of the water-soluble vitamin biotin across the basolateral membrane (BLM) of rat intestine, i.e. the exit of biotin from rat enterocyte, were examined using established BLM vesicles (BLMV) techniques. Results of osmolarity and temperature studies have indicated that the majority of biotin taken up by these vesicles is the result of transport of the vitamin into the intravesicular spaces, with little membrane binding. Transport of biotin in these vesicles was found to be: (1) higher in BLMV of the jejunum than those isolated simultaneously from the ileum, (2) Na(+)-independent in nature, (3) saturable as a function of concentration, with an apparent Km of 3.51 microM and a Vmax. of 16.49 pmol/10 s per mg of protein, (4) inhibited by high concentrations of unlabelled biotin and its related compounds, but not by the unrelated taurocholate, (5) sensitive to the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2'-disulphonic acid (SITS), and (6) not affected by imposing a positive or a negative intravesicular space with the use of a valinomycin-induced K+ diffusion potential. Based on these findings, it is concluded that biotin transport across the rat intestinal BLM is mediated via a specialized carrier system which is Na(+)-independent, transports the vitamin by a process which is sensitive to the effects of anion transport inhibitors, and is electroneutral in nature.

Animals↗

PubMed1991

Transport of riboflavin in human intestinal brush border membrane vesicles.

The transport of riboflavin across the brush border membrane of human intestine was examined using the established brush border membrane vesicle technique. Both osmolarity and temperature studies have concluded that the uptake of riboflavin by these vesicles is mostly the result of transport of riboflavin into an active intravesicular space with less binding to membrane surfaces. When an inwardly directed Na+ gradient was imposed, transport of riboflavin was linear with time for approximately 20 seconds of incubation and was significantly higher than in the presence of an identical K+ gradient. Initial rate of transport of riboflavin as a function of concentration was found to include a saturable component in the presence of an inwardly directed Na+ gradient but was linear in the presence of an identical K+ gradient. The apparent Km and Vmax of the Na+ stimulated transport process were found to be 7.26 mumols/L and 0.97 pmol/mg protein per 10 seconds, respectively. The addition of high concentrations of unlabeled riboflavin and its structural analogue lumiflavin to the incubation medium caused significant inhibition in the transport of 3H-riboflavin in the brush border membrane vesicle incubated in the presence of an inwardly directed Na+ gradient but not in vesicles incubated in the presence of an identical K+ gradient. Inducing a relatively positive intravesicular space with the use of valinomycin and an inwardly directed K+ gradient caused significant inhibition in the transport of riboflavin. On the other hand, inducing a relatively negative intravesicular space with the use of anions of different lipid permeabilities caused significant stimulation in the transport of riboflavin. These results demonstrate that riboflavin transport in human intestinal brush border membrane vesicle is through a carrier-mediated system. This system functions in the presence of a Na(+)-gradient and seems to transport the substrate by an electrogenic process.

Biological Transport↗

PubMed1991

An Na(+)-dependent and an Na(+)-independent system for glutamine transport in rat liver basolateral membrane vesicles.

In the present study the transport of glutamine across rat liver basolateral membrane was examined with special emphasis on the existence of an Na(+)-independent system and on the characteristics of the Na(+)-dependent system with respect to stoichiometry of glutamine to Na+. Well-validated and purified liver basolateral membrane vesicles were used in the study. Results of studies on the effect of incubation medium osmolarity and incubation temperature indicated that glutamine uptake by liver basolateral membrane vesicles is largely the result of transport of the substrate into the intravesicular compartment with little binding to basolateral membrane vesicles. Transport of glutamine with time was Na+ gradient dependent (out greater than in) with a distinct "overshoot" phenomenon. Replacing Na+ with an equivalent concentration of K+, NH4+, choline, or mannitol caused significant inhibition of the initial rate of glutamine transport; on the other hand, Li+ could partially substitute for Na+. The initial rate of transport of glutamine as a function of concentration (0.05-12 mmol/L) was saturable both in the presence and in the absence of an inwardly directed Na+ gradient. Apparent Km values of 2.95 and 3.35 mmol/L and Vmax values of 11,565 and 6663 pmol.mg protein-1.10s-1 were calculated in the presence and absence of a Na+ gradient, respectively. Both in the presence and absence of an Na+ gradient (out greater than in), transport of [3H]glutamine was significantly inhibited by the addition to the incubation medium of unlabeled glutamine as well as histidine, asparagine, and serine. Transport of glutamine by the Na(+)-dependent process was significantly inhibited or stimulated, respectively, by inducing a relatively positive or negative intravesicular space. On the other hand, glutamine transport by the Na(+)-independent process was not affected by changes in transmembrane electrical potential. Using the "activation method," the stoichiometry of glutamine Na+ transport was found to be 1:1. These results show that glutamine transport in rat liver basolateral membrane vesicles is carrier mediated both in the presence and absence of an Na+ gradient. Furthermore, the Na(+)-dependent process is electrogenic in nature (net positive) and cotransports one glutamine molecule with one Na+. Transport of glutamine by the Na(+)-independent system, on the other hand, is electroneutral in nature.

Animals↗