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1.
Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley ( Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U- 14C]Phe indicated a rapid distribution of tracer, i.e. [U- 14C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly
glycine
- Leu
leucine
- Phe
phenylalanine
- Pro
proline 相似文献
2.
Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7’s unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells. 相似文献
3.
In this study we investigated the role of active site residues in the peroxidase activity of Orp1 (GPx3) using three different peroxide substrates. Using a structural homology model of the reduced form of Orp1, we identified Asn126 and Phe127 as evolutionarily conserved residues that line the back of the Orp1 active site and which are likely to affect the peroxidase activity of Orp1. Additionally, we identified Phe38 as a surface residue that could influence substrate specificity as it is located adjacent to Cys36, in the same position occupied by similar hydrophobic amino acids in many Orp1 homologs. We individually mutated these residues to alanine and examined the effect of each mutation in vitro and in vivo. Chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to identify Cys-SOH modification of Cys36 in response to H 2O 2, tert-butyl-hydroperoxide ( tert-BHP), and cumene hydroperoxide (CHP) in Orp1 WT. Mutation of Asn126 and Phe127 eliminate Cys-SOH formation and peroxidase activity in response to H 2O 2, tert-BHP and CHP. Furthermore, the p Ka of Cys36 is elevated closer to that of free cysteine compared to Orp1 WT. Mutation of Phe38 does not affect the peroxidase activity of Orp1 upon exposure to H 2O 2. The Phe38 mutation decreases Orp1 peroxidase activities in response to either tert-BHP or CHP. The in vivo sensitivity of the Phe38 mutant to both tert-BHP and CHP is increased, while the H 2O 2 sensitivity is unchanged. The p Ka of Cys36 in the Phe38 mutant is 5.0, which is the same as Orp1 WT. Taken together, these results suggest that Phe38 does not play a role in the reactivity of Cys36, but does modulate the affinity of Orp1 for alkyl hydroperoxides. 相似文献
4.
The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids. 相似文献
5.
Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine,
and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic
acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates
by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were
determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl
group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate
affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts
strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated
in this study will be useful for the development of prodrugs. 相似文献
6.
Scutella separated from germinating grains of barley ( Hordeum vulgare L. cv Himalaya) took up [ 14C]glutamine at an initial rate of about 10 micromoles·gram −1·hour −1 in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent Km of about 2 millimolar and a V max of about 25 micromoles·gram −1·hour −1. The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine. Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella. 相似文献
7.
The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations. 相似文献
8.
The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein. 相似文献
9.
1. The capacity of various amino acids to convert the pyridoxal form of aspartate aminotransferase into the pyridoxamine form has been investigated. 2. Glutamate has the highest converting capacity; aspartate, α-aminopimelate, α-aminoadipate and other amino acids follow. 3. The converting capacity of the various amino acids assayed is connected with their structural features. 4. A possible role of amino acids as secondary substrates of aspartate aminotransferase is suggested. 相似文献
10.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. 相似文献
11.
Three highly conserved aromatic residues in RNA recognition motifs (RRM) participate in stacking interactions with RNA bases upon binding RNA. We have investigated the contribution of one of these aromatic residues, Phe56, to the complex formed between the N-terminal RRM of the spliceosomal protein U1A and stem–loop 2 of U1 snRNA. Previous work showed that the aromatic group is important for high affinity binding. Here we probe how mutation of Phe56 affects the kinetics of complex dissociation, the strength of the hydrogen bonds formed between U1A and the base that stacks with Phe56 (A6) and specific target site recognition. Substitution of Phe56 with Trp or Tyr increased the rate of dissociation of the complex, consistent with previously reported results. However, substitution of Phe56 with His decreased the rate of complex association, implying a change in the initial formation of the complex. Simultaneous modification of residue 56 and A6 revealed energetic coupling between the aromatic group and the functional groups of A6 that hydrogen bond to U1A. Finally, mutation of Phe56 to Leu reduced the ability of U1A to recognize stem–loop 2 correctly. Taken together, these experiments suggest that Phe56 contributes to binding affinity by stacking with A6 and participating in networks of energetically coupled interactions that enable this conserved aromatic amino acid to play a complex role in target site recognition. 相似文献
12.
Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 ( Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with K D values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (k a) in the range of 10 3 M -1s -1 and an even slower dissociation rate (k d) in the range of 10 −4 s -1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not entirely clear from the published literature whether OCTs function as major prostaglandin transporters, the tight binding of the naturally occurring PGE2, as well as the slow dissociation rate, could conceivably affect the transport of lower affinity substrates such as drugs and metabolites by SLC22 transporters. More research is necessary to establish the extent to which individual SLC22 family members actually function as PG transporters in vitro and in vivo and to investigate whether PGs can, independent of being directly transported, alter the ability of SLC22 transporters to handle drugs and other substrates. 相似文献
13.
For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides. 相似文献
14.
Mixed ruminal bacteria, isolated from sheep (Q and W) fed a concentrate and hay diet, were anaerobically incubated with either 14C-peptides or 14C-amino acids. Experiment 1 showed that uptake of both 14C-labeled substrates was rapid, but the rate for amino acids was twofold greater than for peptides (molecular weight, 1,000 to 200) initially but was similar after 10 min. Experiment 2 demonstrated that metabolism was also rapid; at least 90% of either 14C-labeled substrate was metabolized by 3 min. Of the radioactivity remaining in bacteria, approximately 30% was in the form of 14C-amino acids, but only in leucine, tyrosine, and phenylalanine. Supernatant radioactivity was contained only in tyrosine, phenylalanine, and mostly proline for incubations with 14C-amino acids but in up to 10 amino acids when 14C-peptides were the substrates. Short-term incubations (< 5 min; experiment 3) confirmed previous uptake patterns and showed that the experimental system was responsive to substrate competition. Experiment 4 demonstrated that bacteria from sheep Q possessed initial and maximum rates of 14C-amino acid uptake approximately fourfold greater (P < 0.01) than those of 14C-peptides, but with no significant differences (P > 0.1) between four 14C-peptide substrate groups with molecular weights of 2,000 to < 200. By contrast, bacteria from sheep W showed no such distinctions (P > 0.1) between rates for 14C-peptides and 14C-amino acids. Calculations suggested that peptides could supply from 11 to 35% and amino acids could supply from 36 to 68% of the N requirements of mixed ruminal bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic or/and aliphatic–aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small‐molecule model systems at the ab‐initio level, predicts that Met–Met interactions are stronger than Met–Cys ≈ Met–Phe ≈ Cys–Phe interactions, stronger than Phe–Phe ≈ Phe–Leu interactions, stronger than the Met–Leu interaction, and stronger than Leu–Leu ≈ Cys–Leu interactions. These results show that sulfur‐containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity. 相似文献
16.
CodB is a cytosine transporter from the Nucleobase‐Cation‐Symport‐1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5‐fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium‐dependent uptake of cytosine and can bind 5‐fluorocytosine. Comparison of the substrate‐bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane. 相似文献
17.
Amino acids are available to plants in some soils in significant amounts, and plants frequently make use of these nitrogen sources. The goal of this study was to identify transporters involved in the uptake of amino acids into root cells. Based on the fact that high concentrations of amino acids inhibit plant growth, we hypothesized that mutants tolerating toxic levels of amino acids might be deficient in the uptake of amino acids from the environment. To test this hypothesis, we employed a forward genetic screen for Arabidopsis thaliana mutants tolerating toxic concentrations of amino acids in the media. We identified an Arabidopsis mutant that is deficient in the amino acid permease 1 (AAP1, At1g58360) and resistant to 10 mm phenylalanine and a range of other amino acids. The transporter was localized to the plasma membrane of root epidermal cells, root hairs, and throughout the root tip of Arabidopsis. Feeding experiments with [(14)C]-labeled neutral, acidic and basic amino acids showed significantly reduced uptake of amino acids in the mutant, underscoring that increased tolerance of aap1 to high levels of amino acids is coupled with reduced uptake by the root. The growth and uptake studies identified glutamate, histidine and neutral amino acids, including phenylalanine, as physiological substrates for AAP1, whereas aspartate, lysine and arginine are not. We also demonstrate that AAP1 imports amino acids into root cells when these are supplied at ecologically relevant concentrations. Together, our data indicate an important role of AAP1 for efficient use of nitrogen sources present in the rhizosphere. 相似文献
18.
Non-Hodgkin lymphoma represents a diverse group of blood malignancies, of which follicular lymphoma (FL) is a common subtype. Previous genome-wide association studies (GWASs) have identified in the human leukocyte antigen (HLA) class II region multiple independent SNPs that are significantly associated with FL risk. To dissect these signals and determine whether coding variants in HLA genes are responsible for the associations, we conducted imputation, HLA typing, and sequencing in three independent populations for a total of 689 cases and 2,446 controls. We identified a hexa-allelic amino acid polymorphism at position 13 of the HLA-DR beta chain that showed the strongest association with FL within the major histocompatibility complex (MHC) region (multiallelic p = 2.3 × 10 −15). Out of six possible amino acids that occurred at that position within the population, we classified two as high risk (Tyr and Phe), two as low risk (Ser and Arg), and two as moderate risk (His and Gly). There was a 4.2-fold difference in risk (95% confidence interval = 2.9–6.1) between subjects carrying two alleles encoding high-risk amino acids and those carrying two alleles encoding low-risk amino acids (p = 1.01 × 10 −14). This coding variant might explain the complex SNP associations identified by GWASs and suggests a common HLA-DR antigen-driven mechanism for the pathogenesis of FL and rheumatoid arthritis. 相似文献
19.
In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H + symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence 379NXX[S/T] HX[S/T] QDXXX T391. Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H + symporter subfamily. 相似文献
20.
The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity ( KD ∼0.4 μ m) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. 相似文献
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