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1.
N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].  相似文献   

2.
Muscle tropomyosin was modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) at several different pH values. NBD-chloride reacts specifically with SH residue at neutral pH but it reacts with both SH residue and amino residues at alkaline pH. The polymerizability of tropomyosin at low ionic strength and the binding property of tropomyosin to F-actin were not affected by the modification of SH residues but they were lost rapidly by the modification of amino groups, in accordance with the previous report [Johnson, P. & Smillie, L.B. (1977) Biochemistry 16, 2264-2269]. By the addition of heavy meromyosin, labeled tropomyosin which could not bind to F-actin recovered the binding ability to F-actin and it could regulate the superprecipitation of actomyosin in the presence of troponin. Further modification of amino groups (labeling ratios more than 5) led to loss of the regulating ability completely.  相似文献   

3.
Simultaneous formulation binding and structure modification of the binding site leads to binding process that can be analyzed within the framework of the non-linear theory of dynamic systems. Such an approach allows us to obtain several properties of the binding center: plurality of stationary (stable and unstable) states at binding, recognition of bistable and hysteretic binding modes. It is also shown that adsorption centre deformation leads to a S-shaped adsorption curve.  相似文献   

4.
It was discovered that illumination of the complex formed by the solubilized acetylcholine receptor from Torpedo marmorata and Lys25-p-azidobenzoyl derivative of neurotoxin II results in the appearance on the receptor of up to 4 additional binding sites. Acetylcholine and neurotoxin II, but not the long-chain neurotoxins bind specifically to these sites. The additional binding sites could be also detected after illuminating the receptor complex with other photoactivable derivatives, provided the latter were displaced from one of the two main binding sites by hexa(trifluoroacetyl)neurotoxin II. A similar, but less pronounced effect, was observed on binding Lys25 (Ac) derivative of neurotoxin II. The formation of the additional binding sites was found to depend on the activity of the receptor preparations as well as on the mutual influence of the two main toxin-binding sites.  相似文献   

5.
4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group.  相似文献   

6.
The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.  相似文献   

7.
It is generally held with respect to heterotrimeric guanine nucleotide binding protein-coupled receptors that binding of ligand stabilizes a conformation of receptor that activates adenylyl cyclase. It is not formally appreciated if, in the case of G-protein-coupled receptors with large extracellular domains (ECDs), ECDs directly participate in the activation process. The large ECD of the glycoprotein hormone receptors (GPHRs) is 350 amino acids in length, composed of seven leucine-rich repeat domains, and necessary and sufficient for high affinity binding of the glycoprotein hormones. Peptide challenge experiments to identify regions in the follicle-stimulating hormone (FSH) receptor (FSHR) ECD that could bind its cognate ligand identified only a single synthetic peptide corresponding to residues 221-252, which replicated a leucine-rich repeat domain of the FSHR ECD and which had intrinsic activity. This peptide inhibited human FSH binding to the human FSHR (hFSHR) and also inhibited human FSH-induced signal transduction in Y-1 cells expressing recombinant hFSHR. The hFSHR-(221-252) domain was not accessible to anti-peptide antibody probes, suggesting that this domain resides at an interface between the hFSHR ECD and transmembrane domains. CD spectroscopy of the peptide in dodecyl phosphocholine micelles showed an increase in the ordered structure of the peptide. CD and NMR spectroscopies of the peptide in trifluoroethanol confirmed that hFSHR-(221-252) has the propensity to form ordered secondary structure. Importantly and consistent with the foregoing results, dodecyl phosphocholine induced a significant increase in the ordered secondary structure of the purified hFSHR ECD as well. These data provide biophysical evidence of the influence of environment on GPHR ECD subdomain secondary structure and identify a specific activation domain that can autologously modify GPHR activity.  相似文献   

8.
Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (Ki = 15 nM) interacted reversibly with the binding site for folate (Kd = 0.8 nM). Cells treated with activated [3H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.  相似文献   

9.
Immunoadsorbents were modified with monomethoxy-polyethylene glycol (PEG; average molecular weights of 5000 (PEG-5000) and 1900 (PEG-1900)) activated with cyanuric acid (activated PEG) by four different methods. In the two methods, anti-BSA antibodies were modified with activated PEG with and without protection of antigen binding sites with BSA and then were coupled to CNBr-activated Sepharose 4B. In the other two methods, Immunoadsorbents, which were prepared by coupling anti-BSA antibodies to CNBr-activated Sepharose 4B, were modified with activated PEG with and without the protection. The effects of PEG modification by these four methods on the binding ratio (the ratio of the numbers of moles of antigen adsorbed to the numbers of moles of binding sites of antibody coupled), the antigen binding property and the resistance to proteolytic digestion of immunoadsorbents were studied. The decrease in the binding ratio by the modification with activated PEG was small enough to use modified immunoadsorbents for industrial purification processes. The resistance to proteolytic digestion of immunoadsorbents was improved by modification with activated PEG. The modification without protection of antigen binding sites gave higher resistance to proteolytic digestion than that with protection, while the former caused larger decrease in the binding ratio of modification. The immunoadsorbents modified with activated PEG-5000 showed higher resistance to proteolytic digestion than those modified with activated PEG-1900.  相似文献   

10.
P A Whitson  K S Matthews 《Biochemistry》1987,26(20):6502-6507
The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino acid analysis or incorporation of 14C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approximately 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.  相似文献   

11.
The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.  相似文献   

12.
Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.  相似文献   

13.
Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.  相似文献   

14.
We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.  相似文献   

15.
Fluorescein 5'-isothiocyanate (FITC) was used to modify lysine residues of the strychnine-sensitive glycine receptor. Pretreatment of rat spinal cord synaptic plasma membranes with FITC specifically affected the ability of glycine to displace [3H]strychnine binding. Glycine completely prevented the effect of FITC modification, suggesting the existence of lysine group(s) either at or in the vicinity of the agonist binding site. Labeling of purified glycine receptor with FITC indicates that such lysine residue(s) are located in the 48,000 daltons polypeptide. Chemical cleavage of the FITC-labeled 48-kilodalton subunit with N-chlorosuccinimide reveals two major labeled fragments of Mr 13.9 kilodalton and 8.5-kilodalton, respectively, the labeling of each being protected by glycine.  相似文献   

16.
Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.  相似文献   

17.
V N Hingorani  L F Chang  Y K Ho 《Biochemistry》1989,28(18):7424-7432
The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points.

Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Man1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Man1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.  相似文献   


19.
Polyomaviruses are small, nonenveloped DNA tumor viruses with restricted host ranges. Virus binding to cell surface receptors is one determinant of viral tropism. Although murine polyomavirus is among the best characterized viruses, little is known about the sialic acid-containing receptor and its interaction with viral particles. By using nonradioactive virus binding assays as recently described for the B-lymphotropic papovavirus, murine polyomavirus particles were found to bind in a saturable and noncooperative manner to 25,000 receptors per 3T6 mouse fibroblast. The virus-receptor interaction at 4 degrees C was of high affinity (Kd = 1.8 x 10(-11) M), very fast (k1 = 1.7 x 10(7) M(-1) s(-1)), and stable (half-life = 38 min). Elongation of the N-acyl side chain of sialic acid by biosynthetic modulation with synthetic precursor analogs has been shown for other polyomaviruses to influence both sialic acid-dependent binding and infection (O. T. Keppler, P. Stehling, M. Herrmann, H. Kayser, D. Grunow, W. Reutter, and M. Pawlita, J. Biol. Chem. 270:1308-1314, 1995). In 3T6 cells in which about one-third of the sialic acids were modified, infection and binding of polyomavirus particles were significantly reduced. The number of receptors per cell was decreased to 18,000, with the remaining receptors displaying the same affinity as in untreated cells. Molecular modeling studies based on the three-dimensional structure of a mouse polyomavirus-sialyllactose complex recently solved by T. Stehle and coworkers (T. Stehle, Y. W. Yan, T. L. Benjamin, and S. C. Harrison, Nature 369:160-163, 1994) were performed. They suggest that the elongation of the N-acyl side chain by a single methylene group leads to steric hinderence, with the peptide backbone of a loop walling the tip of the shallow sialic acid binding groove. This collision appears to be incompatible with functional binding. The data are taken as a basis to discuss possible features of the organization and topology of the cellular receptor for mouse polyomavirus.  相似文献   

20.
The dialdehyde of oxidized 1,N6-etheno-ATP and adenosine triphosphopyridoxal were used as probes of the catalytic site of 4-aminobutyrate aminotransferase. Both compounds react with lysine residues critically connected with aminotransferase activity. The binding of 1 mol of oxidized 1,N6-etheno-ATP per mol of enzyme or the binding of 1 mol of adenosine triphosphopyridoxal abrogates catalytic activity. The presence of substrate alpha-ketoglutarate (4 mM) prevents inactivation of the aminotransferase by either one of the ATP analogs. Reduction of the enzyme modified with oxidized 1,N6-etheno-ATP yields a chromophore which displays a maximum of emission at 415 nm and a fluorescent lifetime of 21.6 ns. The degree of exposure of the ethenoadenine ring to collisional encounters with the strong quencher KI was determined at pH 7.0. The ethenoadenine ring of the bound ligand is partially shielded from collisional encounters with the quencher. Steady-state emission anisotropy measurements of the bound ligand reveal that oxidized 1,N6-etheno-ATP is not rigidly attached to the protein matrix. It is postulated that the catalytic domain of 4-aminobutyrate aminotransferase is accessible to bulky reagents of greater length than the substrates 4-aminobutyrate and alpha-ketoglutarate.  相似文献   

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