Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.

Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.

Survival of Alternaria alternata during anaerobic digestion of biomass in stirred tank reactors.

The survival of Alternaria alternate during anaerobic digestion was investigated in context of a joint research project. The aim of this project was to estimate the phytosanitary risk of dissemination of pathogens by returning treated biomass as organic fertilizer to arable land. The studies were carried out in lab-scale stirred tank reactors under mesophilic conditions. After insertion of infected plant material into the reactors the influence on the viability of the fungal pathogen was studied concerning exposure time, pretreatment and storage of the digestates for four weeks or six months. The results clearly showed that anaerobic digestion leads to a complete inactivation of A. alternate already after an exposure time of six hours.

Metaproteome analysis reveals that syntrophy, competition, and phage-host interaction shape microbial communities in biogas plants.

BACKGROUND: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. RESULTS: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. CONCLUSION: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients.

Sequence comparison of the sodium-D-glucose cotransport systems in rabbit renal and intestinal epithelia.

Using an isolated 1.2 kbp PCR product as specific probe, the rabbit renal cortex sodium-D-glucose cotransport system has been found and cloned from a renal cortex cDNA library. Two overlapping cDNA clones were found, one with a length of 2.1 kbp and one of 1.1 kbp resulting in a total sequence of 2.2 kbp without the poly(A) tail. Northern blot hybridization indicated a transcript of approx. 2.4 kb in size. Tissue distribution in the rabbit kidney and intestine indicates that this sequence is present in renal cortex, renal medulla and small intestine in different proportions. It is proposed that this clone codes for a member of the sodium-D-glucose cotransporter systems.

Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E. coli.

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture. Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [(3)H]D-glucose into C(5)-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.

Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.

To investigate the role of charged intramembrane residues in the function of the rabbit Na+/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wild-type, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.

Characterization of an estradiol-stimulated mRNA in the brain of adult male rats.

Differential hybridization of a cDNA library from rat C6 glioma cells with cDNA probes from naive C6 glioma cells and from cells exposed to 17 beta-estradiol identified cDNAs of an mRNA stimulated by 17 beta-estradiol. This mRNA designated ESP1 mRNA, reached maximal levels after 8 h of treatment with 17 beta-estradiol. The stimulation was not suppressed by cycloheximide. Dexamethasone treatment of C6 glioma cells did not induce ESP1 mRNA. It codes for a 164 amino acids long peptide. The sequence is similar in part to that of CRIP protein, a probably member of the ferredoxin superfamily. The conservation of primary structure suggests a role of ESP1 peptide in oxygen consumption. ESP1 mRNA expression is sexually dimorphic in body tissue, whereas it is expressed to comparative levels in the brain of adult males and females. This suggests that 17 beta-estradiol stimulates the expression of the ESP1 gene in the brain of both gender.

Metaproteome analysis of the microbial communities in agricultural biogas plants.

In biogas plants agricultural waste and energy crops are converted by complex microbial communities to methane for the production of renewable energy. In Germany, this process is widely applied namely in context of agricultural production systems. However, process disturbances, are one of the major causes for economic losses. In addition, the conversion of biomass, in particular of cellulose, is in most cases incomplete and, hence, insufficient. Besides technical aspects, a more profound characterization concerning the functionality of the microbial communities involved would strongly support the improvement of yield and stability in biogas production. To monitor these communities on the functional level, metaproteome analysis was applied in this study to full-scale agricultural biogas plants. Proteins were extracted directly from sludge for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent identification with mass spectrometry. Protein profiles obtained with SDS-PAGE were specific for different biogas plants and often stable for several months. Differences of protein profiles were visualized by clustering, which allowed not only the discrimination between mesophilic and thermophilic operated biogas plants but also the detection of process disturbances such as acidification. In particular, acidification of a biogas plant was detected in advance by disappearance of major bands in SDS-PAGE. Identification of proteins from SDS-PAGE gels revealed that methyl CoM reductase, which is responsible for the release of methane during methanogenesis, from the order Methanosarcinales was significantly decreased. Hence, it is assumed that this enzyme might be a promising candidate to serve as a predictive biomarker for acidification.

Bioenergy from permanent grassland--a review: 2. Combustion.

The aim of this review is to summarize current knowledge on suitability and sustainability of grassland biomass for combustion. In the first section grassland management for solid biofuel as well as information on harvest, postharvest and firing technology are described. An extensive grassland management system with one late cut and low level of fertilization is favored for grass as a solid biofuel. The grass harvest usually involves drying in the field and clearing with conventional farm machinery. Pelleting or briquetting improves the biofuel quality. Grass combustion is possible as stand-alone biomass-firing or co-firing with other fuels. Firing herbaceous biomass requires various specific adaptations of the different combustion technologies. In the second section economic and environmental aspects are discussed. Costs for biomass supply mainly depend on yields and harvesting technologies, while combustion costs are influenced by the size and technical design of the plant. Market prices for grass and possible subsidies for land use are crucial for profitability. Regarding biogeochemical cycles a specific feature of combustion is the fact that none of the biomass carbon and nitrogen removed at harvest is available for return to the grassland. These exports can be compensated for by fixation from the air given legumes in the vegetation and sufficient biomass production. Greenhouse gas emissions can be considerably reduced by grass combustion. Solid biofuel production has a potential for predominantly positive impacts on biodiversity due to the extensive grassland management.

Bioenergy from permanent grassland--a review: 1. Biogas.

Grassland biomass is suitable in numerous ways for producing energy. It is well established as feedstock for biogas production. The aim of this review is to summarize current knowledge on suitability and sustainability of grassland biomass for anaerobic digestion. In the first section grassland management for biogas feedstock as well as specifics of harvest, postharvest and digestion technology are described. Methane yields from grass are influenced by many factors. While the effects of some parameters such as grass species, cutting period and management intensity can be regarded as well known, other parameters such as preservation and processing still need investigation. In the second section economic aspects and environmental impacts are discussed. Profitability can be achieved depending on grass silage supply costs and the concept of anaerobic digestion and energy use. Grassland biomass for biogas production competes with other feedstock and other forms of grassland use, in particular animal husbandry. In developed countries a growing production of milk and meat is achieved with decreasing ruminant numbers, resulting in an increasing amount of surplus grassland with a remarkable bioenergy potential. In emerging and developing countries a rapidly rising demand for and production of milk and meat induce growing pressure on grasslands, so that their use for animal feed presumably will take priority over use for bioenergy. Grasslands provide a variety of essential environmental benefits such as carbon storage, habitat function, preservation of ground and surface water quality. When producing biogas from grassland these benefits will remain or even grow, providing appropriate grassland management is implemented. In particular, greenhouse gas emissions can be considerably reduced.

Cardiac risk of coronary patients after reintegration into occupations with heavy physical exertion.

The job related reintegration of patients with coronary artery disease (CAD) is a central part of cardiac rehabilitation. However, specific occupational demands like jobs with heavy physical exertion (> 6 METs) could increase the cardiovascular risk because the relative risk for acute myocardial infarction (MI) and cardiac death is temporarily elevated after vigorous exertion ("hazard period"). Thus, in 2001 any male patient with proven CAD who performed a job with heavy exertion until the occurrence of an index event (MI/ACS, any interventional or surgical revascularization measure) received a questionnaire after an average of 20 months. Complete data were available in 108 from 119 included patients (90.8%), aged 51.8+/-7.8 years. Ejection fraction was 61.5+/-13.1% and the functional capacity at the time of hospital discharge averaged 130.1+/-31.2 W. 75% of the patients had a previous MI and 59.3% underwent bypass surgery. During follow-up the previous job with heavy exertion was performed over a cumulated time of 74 years. The aim of the study was to compare the observed and the expected incidence of MI and cardiac death with and without job performance. The expected ("basal") risk for MI and cardiac death without heavy physical exertion was determined from pooled study results and assumed to be 5.2% per year. The combined risk due to performing an occupation with strenuous exertion can be calculated from time periods with and without working hours and amounts to 11.9%. There could be expected 0.119 . 74=8.8 cardiac events related to the job. In contrast, 5 MIs (4 NSTEMI, 1 STEMI) were observed (6.8%). The relative risk for an expected event compared to the basal risk without heavy exertion was 2.3 (95% CI: 0.7-7.4). The relative risk for the observed cardiac events amounts to 1.3 (95% CI: 0.4-4.8). The lower observed risk is probably due to the high grade of physical fitness in this patient group. In spite of several limitations, our study showed no convincing evidence for increasing the cardiac risk of patients with CAD performing occupations with heavy physical exertion. Because of the importance of this prognostic finding, a representative and prospective study is strongly required.

Mapping the urea channel through the rabbit Na(+)-glucose cotransporter SGLT1.

1. The rabbit Na(+)-glucose cotransporter rbSGLT1 and its carboxy-terminal part, C5, which contains transmembrane helices 10-14 of SGLT1 and functions as a low affinity glucose uniporter, were expressed as individual proteins in Xenopus oocytes. Transport of 55 microM urea, ethylene glycol, mannitol and alpha-methyl-D-glucopyranoside (alphaMDG) by control oocytes and by oocytes expressing SGLT1 and C5 was studied by uptake measurements of the 14C-labelled substrates. 2. There was a 5- to 6-fold increase in urea transport mediated by C5, compared with control oocytes. Similar to SGLT1, the C5-urea uptake was cation independent, linear in time and with increasing urea concentration, and blocked with the same sensitivity by the inhibitor phloretin (K(i) approximately 1 mM). Like SGLT1 in choline buffer, the C5-mediated uptake was insensitive to phlorizin. 3. Mannitol was transported by C5 but not by SGLT1 or control oocytes. 4. The activation energy (E(a)) for urea transport through C5 was low (5 +/- 3 kcal mol(-1)) compared with that of non-injected oocytes (16 +/- 0.5 kcal mol(-1)) and comparable with the E(a) of passive urea or water transport through intact SGLT1. 5. The urea influx through C5 increased in the presence of alphaMDG, but not in the presence of the same concentration of mannitol. 6. We conclude that the five carboxy-terminal transmembrane helices of SGLT1 form a channel for the permeation of small molecules such as urea and water.

Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter.

The rat Na+/glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na(+)-dependent inward current was approximately 300-500 nA. The apparent affinity constants for sugar (alpha-methyl-D-glucopyranoside; alpha MDG) (K alpha MDG 0.5) and sodium (KNa0.5) at a membrane potential of -150 mV were 0.2 mM and 4 mM. The KNa0.5 increased continuously with depolarizing potentials reaching 40 mM at -30 mV, K alpha MDG 0.5 was steeply voltage dependent, 0.46 mM at -30 mV and 1 mM at -10 mV. From all tested monovalent cations only Li+ could substitute for Na+, but with lower affinity. The relative substrate specificity was D-glucose > alpha MDG approximately D-galactose > 3-O-Me-Glc >> beta-naphthyl-D-glucoside >> uridine. Phlorizin (Pz), the specific blocker of sugar transport, showed an extremely high affinity for the rat cotransporter with an inhibitor constant (KPzi) of 12 nM. SGLT1 charge movements in the absence of sugar were fitted by the Boltzmann equation with an apparent valence of the movable charge of approximately 1, a potential for 50% maximal charge transfer (V0.5) of -43 mV, and a maximal charge (Qmax) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s-1. Model simulations showed that the kinetics of the rat SGLT1 are described by a six-state ordered nonrapid equilibrium model, and comparison of the kinetics of the rat, rabbit and human cotransporters indicate that they differ mainly in their presteady-state kinetic parameters.

Sodium/D-glucose cotransporter charge movements involve polar residues.

Na(+)-dependent glucose transporters (SGLT1) exhibit transient carrier currents with a time constant (tau) of 2-20 ms, and the charge transfer (Q) fits the Boltzmann equation. There is a 60-mV negative displacement in the tau/V and Q/V curves between the human and rabbit SGLT1 proteins, and the initial goal was to identify the charges responsible for these differences in kinetics. We have focused on residue 176 in putative transmembrane helix (M4) because this is an aspartic acid in rabbit and asparagine in human. Asp-176 in rabbit SGLT1 was replaced with asparagine and alanine residues, and the wild-type and mutant proteins were expressed in Xenopus laevis oocytes. A two-electrode voltage clamp was used to measure the kinetics of charge transfer. There was no difference between the wild-type and D176N, but there was a 60-mV negative shift in the tau/V and Q/V curves with D176A. This suggests that polar residues at position 176 play an important role in determining charge transfer, probably by electrostatic bonding to a neighboring polar residue in the membrane domain of the protein. The similarity between rabbit SGLT1 and the D176N mutant further indicates that other membrane residues account for the difference between rabbit and human SGLT1. There were only modest changes in the steady-state Na+/glucose cotransport kinetics between wild-type and D176A mutant transporters in the voltage range +50 to -50 mV. Model simulations show that the mutation alters the rate constants for conformational changes of the unloaded transporter. Phlorizin, a specific competitive inhibitor of sugar transport, has a lower affinity for the D176A mutant than for SGLT1. This indicates that polar residues at position 176 hydrogen bond with the -OH group on the B-phenyl ring of the inhibitor.

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T., Yet, S. F., and Lever, J. E. (1993) J. Biol. Chem. 268, 1509-1512) encodes a 660-amino acid protein with 76% identity to SGLT1. To confirm and extend the kinetic characterization of SAAT1, we have expressed this clone in Xenopus oocytes and measured transport using both radiotracer and electrophysiological techniques. SAAT1 did not stimulate either 50 microM 2-(methylamino)isobutyrate uptake or 2-(methylamino)isobutyrate-evoked inward Na+ currents, but instead stimulated 50 microM alpha MG (alpha-methyl-D-glucopyranoside) uptake 27-fold from 2 +/- 1 pmol.h-1/oocyte (n = 9) to 55 +/- 6 pmol.h-1/oocyte (n = 9) and alpha MG-evoked inward Na+ currents (I) by up to 1000 nA/oocyte. The apparent affinity constant for alpha MG (K alpha MG 0.5) was approximately 2 mM and was independent of membrane potential from -30 to -150 mV but was voltage-sensitive between -30 and +30 mV. The relative sugar specificity for the transporter was alpha MG > or = D-glucose >> D-galactose >>> 3-O-methyl-D-glucopyranose, L-glucose. The sugar-evoked currents were Na(+)-dependent (KNa 0.5 approximately 10 mM at -50 mV) and the Hill coefficient was 1. KNa 0.5 decreased with hyperpolarization of the membrane from -50 to -150 mV. Phlorizin inhibited the alpha MG-evoked current with apparent Ki of 18 microM at -50 mV. We conclude that the SAAT1 cDNA encodes a renal low affinity Na+(1)/glucose(1) cotransporter and propose that pig SAAT1 be renamed pSGLT2.

Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 particles were heterogeneous (4.8 +/- 0.3, 7.1 +/- 1.2, and 10.3 +/- 0.8 nm) in contrast to the endogenous particles (7.6 +/- 1.2 nm). C5 increased the alpha-methyl-D-glucopyranoside (alphaMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5alphaMDG of 50 mM and a turnover of approximately 660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (alphaMDG > D-glucose > D-galactose >> L-glucose approximately D-mannose), 2) inhibited by phloretin, KiPT = approximately 500 microM, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407-648, helices 10-13), and hN13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.

Biophysical characteristics of the pig kidney Na+/glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2.

The Na+-dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na+ and alpha-methyl-D-glucopyranoside (alphaMG) concentrations were consistent with an ordered, simultaneous transport model in which Na+ binds first. Na+ binding was voltage-dependent and saturated with hyperpolarizing voltages. Phlorizin was a potent inhibitor of the sugar-evoked currents (KiPz approximately 10 microM) and blocked an inward Na+ current in the absence of sugar. SGLT2 exhibited Na+-dependent presteady-state currents with time constants 3-7 ms. Charge movements were described by Boltzmann relations with apparent valence approximately 1 and maximal charge transfer approximately 11 nC, and were reduced by the addition of sugar or phlorizin. The differences between SGLT1 and SGLT2 were that (i) the apparent affinity constant (K0.5) for alphaMG (approximately 3 mM) was an order of magnitude higher for SGLT2; (ii) SGLT2 excluded galactose, suggesting discrete sugar binding; (iii) K0.5 for Na+ was lower in SGLT2; and (iv) the Hill coefficient for Na+ was 1 for SGLT2 but 2 for SGLT1. Simulations of the six-state kinetic model previously proposed for SGLT1 indicated that many of the kinetic properties observed in SGLT2 are expected by simply reducing the Na+/glucose coupling from 2 to 1.

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.

Kinetic and specificity differences between rat, human, and rabbit Na+-glucose cotransporters (SGLT-1).

The Na+ activation and substrate specificity of human, rabbit, and rat Na+-glucose cotransporter (SGLT-1) isoforms were characterized using the Xenopus oocyte expression system and the two-electrode voltageclamp method. We find that there are differences, major and minor, in both the kinetics and substrate specificities between these isoforms; the substrate concentration at half-maximal current (K0.5) for hexoses varies from 0.2 to > 40 mM, depending on the species and sugar; the affinity constant (Ki) for phlorizin, the classic competitive inhibitor of SGLT-1, varies lover two orders of magnitude (rat Ki = 0.03 microM vs. rabbit Ki = 1.4 microM); and some glucoside inhibitors of the rabbit isoform, p-nitrophenyl glucose and beta-naphthyl glucose, are transported by the human and rat transporters. Na+ activation is more sensitive to membrane potential in the human and rat isoforms compared with rabbit. The rabbit isoform has a higher apparent affinity for alpha-methylglucose and 3-O-methylglucose by a factor of two than either human or rat. These results can be quantitatively fitted by our six-state kinetic model of SGLT-1, providing insight into the processes involved in these changes. For example, the model predicts that Na+ binding (rate constant, k12) in human and rat SGLT-1 is similar but is fourfold larger than in rabbit, whereas sugar binding (k23) in rabbit and rat is similar but double the value in human SGLT-1. The differences in the primary amino acid sequences between these three homologous proteins must account for the kinetic and substrate specificity differences, and comparisons of the functional properties and amino acid sequences of SGLT-1 isoforms provide useful information about structure/function relationships.

Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.

d-Glucose is absorbed across the proximal tubule of the kidney by two Na+/glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na+:glucose coupling ratio of 1, a K0.5 for sugar of approximately 2 mM, and a K0.5 for Na+ of approximately 1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K0.5 for sugar and Na+ of approximately 0.2 and 5 mM, respectively. We have constructed a chimeric protein consisting of amino acids 1-380 of porcine SGLT2 and amino acids 381-662 of porcine SGLT1. The chimera was expressed in Xenopus oocytes, and steady-state kinetics were characterized by a two-electrode voltage-clamp. The K0.5 for alpha-methyl-d-glucopyranoside (0.2 mM) was similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2 transports poorly D-galactose and excludes 3-O-methylglucose. The apparent K0.5Na was 3.5 mM (at -150 mV), and the Hill coefficient ranged between 0.8 and 1.5. We conclude that recognition/transport of organic substrate is mediated by interactions distal to amino acid 380, while cation binding is determined by interactions arising from the amino- and carboxyl-terminal halves of the transporters. Surprisingly, the chimera transported alpha-phenyl derivatives of D-glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and beta-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K0.5 for phlorizin was 5 microM). Thus, the pocket for organic substrate binding is increased from 10 x 5 x 5 (A) for SGLT1 to 11 x 18 x 5 (A) for the chimera.