Abstract:
:Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Tyagi NK,Goyal P,Kumar A,Pandey D,Siess W,Kinne RKdoi
10.1021/bi051377qsubject
Has Abstractpub_date
2005-11-29 00:00:00pages
15514-24issue
47eissn
0006-2960issn
1520-4995journal_volume
44pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::The phosphoramidate of CCdAp and puromycin (CCdApPuro) is a potent inhibitor of ribosomal peptidyl transferase, as assayed by the fragment reaction. Inhibition is competitive at the ribosomal A-site. CCdApPuro protects P-site-associated bases in the peptidyl transferase loop region of 23S rRNA from carbodiimide modifi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00002a001
更新日期:1995-01-17 00:00:00
abstract::3-Deoxy-d- manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the first step of lipopolysaccharide biosynthesis, namely condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P), to produce KDO8P. We have characterized Campylobacter jejuni KDO8P synthase and its inhibition by KDO8P oxime. It ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00748
更新日期:2018-09-11 00:00:00
abstract::Several phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-regulated actin-binding proteins and most phosphoinositide-specific phospholipases C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x denotes any amino acid), which was previously suggested to represent a PtdInsP2-binding site commonly present in t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00015a023
更新日期:1995-04-18 00:00:00
abstract::Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NM...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011746s
更新日期:2002-04-30 00:00:00
abstract::The inhibitory effects of urea on the normally rapid fibrillation of human calcitonin (hCT) were investigated by 1H NMR. From subtle differences in the chemical shift of hCT in the presence and absence of urea, the occurrence of weak interactions between urea and hCT was confirmed. The chemical shifts on the NH and C ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961013l
更新日期:1996-10-01 00:00:00
abstract::The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate betw...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00401a009
更新日期:1988-01-12 00:00:00
abstract::Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961648k
更新日期:1997-01-28 00:00:00
abstract::The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. T...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi8014828
更新日期:2008-11-11 00:00:00
abstract::The equilibrium unfolding reaction of Ltn, a metamorphic C-class chemokine, was monitored by tryptophan fluorescence to determine unfolding free energies. Measurements revealed that addition of 150 mM NaCl stabilized the Ltn chemokine fold by approximately 1 kcal/mol. Specific mutations involving Arg23 and Arg43 also ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300842j
更新日期:2012-11-13 00:00:00
abstract::Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has bee...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.0c00495
更新日期:2020-09-29 00:00:00
abstract::11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of 11-dehydrocorticosterone to its active form corticosterone in rodents (or cortisone to cortisol in humans). The reductive reaction of the 11-keto to 11-hydroxyl is the pivotal switch in the activation of glucocorticoids. An excess of ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi047599q
更新日期:2005-05-10 00:00:00
abstract::Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resist...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971276v
更新日期:1997-10-21 00:00:00
abstract::The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly i...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00554a012
更新日期:1980-06-24 00:00:00
abstract::Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00059a007
更新日期:1993-03-02 00:00:00
abstract::Aromatic amino acids often flank the transmembrane alpha helices of integral membrane proteins. By favoring locations within the membrane-water interface of the lipid bilayer, aromatic residues Trp, Tyr, and sometimes Phe may serve as anchors to help stabilize a transmembrane orientation. In this work, we compare the ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi500439x
更新日期:2014-06-10 00:00:00
abstract::Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the implications of oxidative stress are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with biomolecules. ROS cause activat...
journal_title:Biochemistry
pub_type: 杂志文章,评审
doi:10.1021/acs.biochem.6b00703
更新日期:2016-09-27 00:00:00
abstract::The complete sequence of rabbit antibody light-chain L 120 has been elucidated. The antibody was raised against Micrococcus lysodeikticus bacteria and is specific for the external part of the cell wall. All protein used in this work was obtained from a single 50-mL bleeding. The variable region of L 120 is compared to...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00613a031
更新日期:1978-10-03 00:00:00
abstract::Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologou...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi990896o
更新日期:1999-08-10 00:00:00
abstract::Previous studies of P-glycoprotein have demonstrated that its function can be modulated by phosphorylation. In the present study, inhibition of protein kinase C with calphostin C or stauroporine or prolonged treatment with the phorbol ester TPA decreased phosphorylation of P-glycoprotein, and impaired transport of vin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00086a022
更新日期:1993-09-07 00:00:00
abstract::The expression level of protein DR1199 is observed to increase considerably in the radio-resistant bacterium Deinococcus radiodurans following irradiation. This protein belongs to the DJ-1 superfamily, which includes proteins with diverse functions, such as the archaeal proteases PhpI and PfpI, the bacterial chaperone...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800882v
更新日期:2008-11-04 00:00:00
abstract::The reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitroanilide has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvent. Following initiation of the reaction at temperatures as low as -90 degrees C, a series of four reactions prior to the normal rate-limiting step (acylation)...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00652a031
更新日期:1976-04-06 00:00:00
abstract::The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9514774
更新日期:1996-05-28 00:00:00
abstract::Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic st...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi960239l
更新日期:1996-04-23 00:00:00
abstract::Alpha-synuclein plays a key role in the pathogenesis of many neurodegenerative diseases. To date, its cellular role has yet to be determined, although it has been proposed to be connected to calcium and G protein-mediated dopamine signaling. Alpha-synuclein is known to bind strongly to model membrane surfaces where it...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0487140
更新日期:2005-01-18 00:00:00
abstract::The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00482a018
更新日期:1990-07-31 00:00:00
abstract::A stable green heme was extracted from ferric cyanosulfmyoglobin after it had undergone an internal conversion reaction. After iron removal and conversion to the methyl ester, the resulting green porphyrin was purified by high-pressure liquid chromatography. Visible, 1H NMR, and mass spectrometric studies provided evi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00374a021
更新日期:1986-12-30 00:00:00
abstract::When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoani...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi962670g
更新日期:1997-04-08 00:00:00
abstract::A mechanistic study of rat liver alpha-(2-->6) sialyltransferase (ST) is presented that includes isotope trapping experiments and kinetic isotope effects on V/K for the ST-catalyzed reaction of isotopically labeled CMP-N-acetylneuraminate and N-acetyllactosamine. The isotope trapping experiments confirmed that the kin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971624t
更新日期:1998-01-06 00:00:00
abstract::Ribosomal protein E-L30 unfolds reversibly at pH values between 7.0 and 4.5. Unfolding of the protein involves a fast and a slow equilibrium, which depend on the degree of protonation of His19 and His33. Both the fast equilibrium between protonated and deprotonated histidines and the slow equilibrium between folded an...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00391a050
更新日期:1987-08-25 00:00:00
abstract::Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00401a059
更新日期:1988-01-12 00:00:00