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1.
The β-subunit of the voltage-sensitive K+ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of Kvβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The KD values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the KD for NADH and NAD were between 1–3 μM whereas the KD for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of Kvβ2.1 for NADPH over NADP may be significant in regulating the K+ channels as a function of the cellular redox state. 相似文献
2.
The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding
Barbara E. Kream Mimi J.L. Jose Hector F. DeLuca 《Archives of biochemistry and biophysics》1977,179(2):462-468
The structural features of 1,25-dihydroxyvitamin D3 that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D3 molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D2) did not alter the binding of 1,25-dihydroxyvitamin D3, but significantly diminished the binding of 25-hydroxyvitamin D3. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein. 相似文献
3.
Emma Smith Rina Vekaria Katherine A. Brown Colin Longstaff 《Molecular and cellular biochemistry》2013,382(1-2):193-201
A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K D in the 10?8 M range and (ii) a kinetically rapid, low-affinity interaction with K D in the 10?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface. 相似文献
4.
Synthetic [125I]-Tyr23, Phe2, Nle4-adrenocorticotropin (ACTH)-(1–38) ([125I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 ± 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [125I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent KD = 170 ± 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent KD (143 ± 16.5 pm). The number of receptors per adipocyte was quite low (521–841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent Km being 145–177 pm. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent KD = 446 ± 77 pM) and the binding capacity was also significantly enhanced (1663 ± 208/cell) without any change in the apparent Km for glycerol release (187 ± 7.1 pm). 相似文献
5.
Paul Majkut Iris Clau?nitzer Helmut Merk Christian Freund Christian P. R. Hackenberger Michael Gerrits 《PloS one》2013,8(12)
The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2’s were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level. 相似文献
6.
The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNAThr with an enlarged 8-nt anticodon loop (). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNAHis. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNAHis. A loss of the first nucleotide (G−1) in tRNAHis converts it to a substrate for MST1 with a Km value (0.7 μM) comparable to that of (0.3 μM), and addition of G−1 to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment. 相似文献
7.
8.
《Life sciences》1995,57(15):1401-1410
PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a 10000-fold selectivity versus human D4 receptors (Ki, 7000 nM) using [3H]spiperone as the radioligand in CHO-K1-cells. Studies with [3H]PD 128907, showed saturable, high affinity binding to human D3 receptors expressed in CHO-K1 cells (CHO-K1-D3) with an equilibrium dissociation constant (Kd) of 0.99 nM and a binding density (Bmax) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D2 receptors (CHO-K1-D2). The rank order of potency for inhibition of [3H]PD 128907 binding with reference DA agents was consistent with reported values for D3 receptors. These results indicate that [3H]PD 128907 is a new, highly selective D3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D3 receptors. 相似文献
9.
Specific binding proteins for 1,25-dihydroxyvitamin D3 were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[3H]vitamin D3 with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (Kd = 4.2 × 10?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D3, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and vitamin D3 did not effectively compete with 1,25-dihydroxyvitamin D3 for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D3. In vitro subcellular distribution of 1,25-dihydroxy[3H]vitamin D3 demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[3H]vitamin D3 was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[3H]vitamin D3 was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D3 in this tissue. 相似文献
10.
Tanja Kosenko Mia Golder Geoffrey Leblond Willy Weng Thomas A. Lagace 《The Journal of biological chemistry》2013,288(12):8279-8288
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a KD ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation. 相似文献
11.
Are Kristiansen Kjell M. Vårum Hans Grasdalen 《Biochimica et Biophysica Acta (BBA)/General Subjects》1998,1425(1):137-150
This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (FA) induced nearly identical changes in the 1H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (KaveD) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. KaveD decreased with increasing FA-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to KaveD of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed. 相似文献
12.
Background
Two meningococcal serogroup B vaccines contain Factor H binding protein (FHbp). Binding of Factor H (FH) to FHbp was thought to be specific for human or chimpanzee FH. However, in a previous study an amino acid polymorphism in rhesus macaque FH domain 6, tyrosine at position 352 (Y352) was associated with high binding to FHbp, whereas histidine at position 352 (H352) was associated with low binding.Methods and Results
Here we report that a second FH polymorphism at position 360 also affects macaque FH binding. Of 43 macaques, 11 had high FH binding and 32 had low binding. As in our previous study, all 11 animals with high binding had Y352, and 24 with low binding had H352. However the remaining eight with low FH binding had Y352, which was predicted to yield high binding. All eight had S360 instead of P360. Thus, three allelic variants at positions 352 and 360 affect macaque FH binding to FHbp: HP (low), YS (low), and YP (high). We measured binding affinity of each FH sequence type to FHbp by surface plasmon resonance. Two animals with high binding types (YS/YP and HP/YP) had dissociation constants (K D) of 10.4 and 18.2 nM, respectively, which were similar to human FH (19.8 nM). Two macaques with low binding (HP/HP and HP/YS) had K D values approximately five-fold higher (100.3 and 99.5 nM, respectively). A third macaque with low binding (YS/YS) had a K D value too high to be measured.Conclusions
Macaques have at least three allelic variants encoding FH with different affinities for FHbp (five genotypic combinations of these variants). Since in previous studies binding of FH to FHbp vaccines decreased protective antibody responses, our data will aid in selection of macaques with FH binding that is similar to humans for further investigation of FHbp vaccine immunogenicity. 相似文献13.
Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (KD ~ 4 µM) than to flavin adenine dinucleotide (KD ~ 12 µM) or to riboflavin (KD ~ 13 µM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs. 相似文献
14.
Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [3H]spiroperidol. The specific binding is saturable with two components (KD1 = 1.9 nM, KD2 = 36.2 nM). Determination of the KD by kinetic studies measuring rate constants for association and dissociation provided KD values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [3H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [3H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response. 相似文献
15.
Joseph D. Robinson 《Archives of biochemistry and biophysics》1973,156(1):232-243
Inactivation of the (Na+ + K+)-dependent ATPase by 50 μm BeCl2 occurred during brief incubations in the presence of both Mg2+ and K+. Inactivation followed, initially, a first-order time course, with rate constants sensitive to the concentration of K+ (other components held constant). From these data dissociation constants can be calculated for K+ binding to sites controlling inactivation. Comparisons of relative affinities for K+ analogs (T1+ and NH4+), and of sensitivity to reagents altering K+ activation (phlorizin and dimethylsulfoxide) indicate that the same K+ sites operate for both Be2+ inactivation and enzyme activation. With 3 mm MgCl2 the dissociation constant, KD, for K+ was 1.4 mm, but decreased 20-fold on addition of both Na+ and CTP. Alone, Na+ increased the apparent KD for K+, either by direct competition or indirectly from its own site, with a KD of 7 mm. The data suggest a model for K+ transport with K+ sites on the outer membrane surface that increase in affinity after formation of the phosphorylated enzyme intermediate, sufficiently to bind K+ in a high Na+ environment. Translocation may occur by an “oscillating pore” mechanism discharging K+ at the inner surface, while leaving demonstrable sites of moderate affinity at the outer end of the pore (which preclude attempts to document low-affinity discharge sites). 相似文献
16.
Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms. Improved X-ray scattering and analytical ultracentrifugation methodology for wild-type FH permitted a clarification of these oligomeric properties. Data at lower concentrations revealed a dependence of the X-ray radius of gyration values on concentration that corresponded to the weak self-association of FH. Global sedimentation equilibrium fits indicated that a monomer-dimer equilibrium best described the data up to 1.3 mg/ml with a fitted dissociation constant KD of 28 μM and that higher oligomers formed at increased concentrations. The KD showed that about 85-95% of serum FH will be monomeric in the absence of other factors. Size-distribution analyses in sedimentation velocity experiments showed that monomeric FH was the major species but that as many as six oligomeric forms co-existed with it. The data were explained in terms of two weak dimerisation sites recently identified in the SCR-6/8 and SCR-16/20 fragments of FH with similar KD values. These observations indicate a mechanism for the progressive self-association of FH and may be relevant for complement regulation and the formation of drusen deposits that are associated with age-related macular degeneration. 相似文献
17.
Unspecific binding of a protamine, namely fluorescein-labelled clupeine Z, to double-stranded calf thymus DNA was studied using fluorescence titration methods and chemical relaxation techniques. Both equilibrium and kinetic data have been analysed using general theoretical approaches discussed in the accompanying paper. The results agree well with the predictions made on the basis of a standard co-operative binding model.Basic parameters evaluated are the co-operative binding constant (K), the coefficient measuring co-operative interaction between nearest neighbours (q), the number of nucleotides occupied by one protamine molecule (n) and the rate constant of dissociation at the ends of bound ligand sequences (KD). Values obtained at 20 °C, pH 7.5 and 0.4 m-NaCl were K = 5.8 × 107m?1, q = 1700, n = 20 and KD = 0.29 s?1. They have been found to be sensitive to the concentration of added salt (NaCl). This effect apparently reflects the essentially electrostatic nature of the binding process. The results can be satisfactorily described in terms of competitive binding of sodium ions. 相似文献
18.
《Journal of receptor and signal transduction research》2013,33(1-4):355-375
AbstractAs we have shown earlier (-)125lodocyanopindolol (125ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (Bhiaff) and low affinity (Bloaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with 125ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (KD=1.5±0.2 pmol/l) of Bhiaff binding sites and 5210±510 [sites/cell] (KD=420±80 pmol/l) of Bloaff. During EPI infusion we observed biphasic modulation of the Bhiaff and an inverse modulation of the Bloaff. After 40 min of EPI Bhiaff increased to 1970±280 [sites/cell] (KD=4.2±0.8 pmol/l), whereas Bloaff decreased to 2720±280 [sites/cell] (KD=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI Bhiaff changed to 1310±240 [sites/cell] (KD=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (KD=190±100 pmol/l) Bloaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for 125ICYP on MNL during EPI infusion. 相似文献
19.
《BBA》1986,849(1):121-130
The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (KD(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The KD(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The KD(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with KD = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 104 M−1s−1. (6) The rate constant for 1-naphthoyl-ATP binding to TF1 is 6.6 · 103 M−1 · s−1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (kA−1 = 3 · 10−3s−1) and a slower second phase (kA−2 < 0.2 · 10−3s−1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex. 相似文献
20.
Gary B. Henderson Barbara Grzelakowska-Sztabert Edward M. Zevely F.M. Huennekens 《Archives of biochemistry and biophysics》1980,202(1):144-149
A binding component with a high affinity for 5-methyltetrahydrofolate (KD = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 104 sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the KD values for 5-methyltetrahydrofolate and methotrexate binding and the Kt values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate. 相似文献