High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.

Erythrocytes from several mammalian species contain mercurial-sensitive water transporters. By a stopped-flow light scattering technique, osmotic water permeability (Pf) was exceptionally high in rabbit erythrocytes (0.053 +/- 0.002 cm/s) and reversibly inhibited by 98% by p-(chloromercuri)benzenesulfonate (pCMBS). The activation energy (Ea) was 4.6 kcal/mol (15-37 degrees C). pCMBS inhibition was half-maximal at 0.1 mM (60-min incubation); at 1 mM pCMBS, half-maximal inhibition occurred in 8 min. Pf was also inhibited by HgCl2 and pCMB with greater than 90% inhibition in 5 min. There was no inhibition by high concentrations of phloretin, DNDS, cytochalasin B, amiloride, ouabain, furosemide, and several proteases. In defolliculated Xenopus oocytes microinjected with 50 nL of water or unfractionated mRNA (1 mg/mL) from rabbit reticulocytes, oocyte Pf assayed at 10 degrees C after 72-h incubation increased from (4 +/- 1) X 10(-4) cm/s (water injected) to (18 +/- 2) X 10(-4) cm/s (mRNA injected). Pf increased linearly with [mRNA] (0-75 ng/oocyte) and was inhibited slowly and reversibly by pCMBS and immediately by HgCl2 but not by cytochalasin B, phloretin, or DNDS. Ea was 9.6 kcal/mol (water injected) and 2.6 kcal/mol (mRNA injected). These results demonstrate that rabbit erythrocytes have the highest Pf and the greatest percentage inhibition of Pf by mercurials of any mammalian erythrocyte studied. The characteristics of the expressed and native water channels were similar, suggesting that the erythrocyte water channel is a membrane protein suitable for expression cloning.     

Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter

The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.

BACKGROUND INFORMATION: Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys(189) as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys(189) of AQP1. Indeed, the osmotic water permeability (P(f)) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. RESULTS: The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys(178), which is located at loop D facing the intracellular side, is a target responding to Hg2+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. CONCLUSIONS: Our results suggest that mercury inhibits the P(f) of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.     

Differential effects of methylmercuric chloride and mercuric chloride on the L-glutamate and potassium evoked release of [3H]dopamine from mouse striatal slices

The effects of CH3HgCl and HgCl2 on the evoked release of 3H from mouse striatal slices prelabelled with [3H]dopamine have been examined. CH3HgCl (10 microM) was observed to increase the L-glutamate-evoked release of [3H]dopamine, while HgCl2 (10 microM) had no effect. In contrast, CH3HgCl at concentrations up to 100 microM had no effect on the 25 mM K+-stimulated release of [3H]dopamine, whereas HgCl2 (100 microM) significantly reduced the 25 mM K+-stimulated release of [3H]dopamine. Thus CH3HgCl and HgCl2 have differential effects on the L-glutamate- and K+-stimulated release of [3H]dopamine from mouse striatal slices, suggesting that these compounds may have different sites and (or) mechanisms of action in altering neurotransmitter release. It is suggested that CH3HgCl may act predominantly at intracellular sites or at the level of the L-glutamate receptor, whereas the major site of action of HgCl2 may be the voltage-operated calcium channel.     

The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.     

Phloretin - an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation

The effect of phloretin on respiration by isolated mitochondria and submitochondrial particles was studied. In submitochondrial particles, both NADH- and succinate-dependent respiration was inhibited by phloretin. 50% maximum inhibition was reached at phloretin concentrations of 0.1 mM (NADH oxidation) and 0.7 mM (succinate oxidation). In isolated mitochondria, phloretin inhibited glutamate oxidation in both State 3 and State 4; 50% maximum inhibition occurred at about 30 microM. Succinate oxidation is inhibited in State 3 by phloretin, inhibition being half its maximum value at 0.5 mM, but in State 4 it is stimulated about 2-fold by phloretin at a concentration of 0.6 mM. Ascorbate oxidation is stimulated in both State 3 and State 4, maximum stimulation being equal to that obtained with an uncoupler of oxidative phosphorylation. Under all circumstances, phloretin lowered the transmembrane electrical potential difference in isolated mitochondria. These results are discussed in terms of mosaic non-equilibrium thermodynamics. We conclude that phloretin is both an uncoupler and an inhibitor of oxidative phosphorylation.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.     

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.     

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.     

Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride

Effective protocols for oocyte activation are crucial for study of parthenogenetic development and to produce nuclear transfer reconstructed embryos. This study investigated the use of ionomycin (ION) and strontium chloride (Sr(2+)) in the activation of parthenogenetic and nuclear transfer porcine oocytes. In-vitro-matured oocytes with a polar body were treated with varying concentrations of ION, Sr(2+) or its combinations, and then fixed or cultured to assess activation and development rates, respectively. Ionomycin concentrations of 10 and 15 microM resulted in more frequent oocyte activation and the 15 microM in advanced development compared to 5 microM (71.8 and 70%vs. 47.5%; P=0.04, and 43.7%vs. 19.3%; P=0.008, respectively). Oocytes treated with 10, 20 or 30 mM of Sr(2+) for 2 or 4h displayed a pronuclear formation rate ranging from 46.7 to 70%. When employed after a 5 min treatment with 10 or 15 microM ION, exposure to 10 mM Sr(2+) for 4 h resulted in higher pronuclear formation than did the 20 mM concentration (82 and 88.6%vs. 63.3 and 73.2%; P=0.03). Nuclear transfer reconstructed oocytes treated with 15 microM/5 min ION followed by 10 mM/4 h Sr(2+) resulted in a higher development to blastocyst stage compared to those treated with 15 microM ION alone (17.7 vs. 11.3%; P=0.06). In conclusion, we inferred that the inclusion of Sr(2+) in the activation protocol can benefit the development of nuclear transfer reconstructed porcine oocytes.     

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.     

Are pacemaker properties required for respiratory rhythm generation in adult turtle brain stems in vitro?

The role of pacemaker properties in vertebrate respiratory rhythm generation is not well understood. To address this question from a comparative perspective, brain stems from adult turtles were isolated in vitro, and respiratory motor bursts were recorded on hypoglossal (XII) nerve rootlets. The goal was to test whether burst frequency could be altered by conditions known to alter respiratory pacemaker neuron activity in mammals (e.g., increased bath KCl or blockade of specific inward currents). While bathed in artificial cerebrospinal fluid (aCSF), respiratory burst frequency was not correlated with changes in bath KCl (0.5-10.0 mM). Riluzole (50 microM; persistent Na(+) channel blocker) increased burst frequency by 31 +/- 5% (P < 0.05) and decreased burst amplitude by 42 +/- 4% (P < 0.05). In contrast, flufenamic acid (FFA, 20-500 microM; Ca(2+)-activated cation channel blocker) reduced and abolished burst frequency in a dose- and time-dependent manner (P < 0.05). During synaptic inhibition blockade with bicuculline (50 microM; GABA(A) channel blocker) and strychnine (50 muM; glycine receptor blocker), rhythmic motor activity persisted, and burst frequency was directly correlated with extracellular KCl (0.5-10.0 mM; P = 0.005). During synaptic inhibition blockade, riluzole (50 microM) did not alter burst frequency, whereas FFA (100 microM) abolished burst frequency (P < 0.05). These data are most consistent with the hypothesis that turtle respiratory rhythm generation requires Ca(2+)-activated cation channels but not pacemaker neurons, which thereby favors the group-pacemaker model. During synaptic inhibition blockade, however, the rhythm generator appears to be transformed into a pacemaker-driven network that requires Ca(2+)-activated cation channels.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Sulfhydryl groups are essential for organic anion exchange in canine renal brush-border membranes

The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.     

Monosaccharide transport into lactating-rat mammary acini.

The uptake and release of 3-O-methyl-D-[3H]glucose at 37 degrees C by acini, prepared from lactating-rat mammary gland with collagenase, was inhibited by glucose, phloretin, cytochalasin B, HgCl2 and low temperature. Uptake and phosphorylation of 2-deoxy-D-[3H]glucose, studied in greater detail, could be ascribed to a specific, saturable, inhibitable, process of apparent Km 16 mM and Vmax. approx. 56 nmol/min per mg of protein, plus a non-specific, non-inhibitable process that was monitored with [14C]fructose. The mean rate of uptake of 5 mM-2-deoxyglucose (16 nmol/min per mg of protein) was similar to the rate of consumption of 5 mM-glucose, suggesting that transport was a rate-limiting step in the overall metabolism of glucose. This accords with evidence for a glucose gradient across the plasma membrane.