Conformational change at the active center of serine-proteases upon their binding to alpha 2-macroglobulin

The inhibition rates and spectral characteristics of 2 probes specific for the active-site serine residue of proteases were examined for evidence of conformational change of the proteases upon their binding to alpha 2-macroglobulin (alpha 2M). Elastase, chymotrypsin, trypsin, and plasmin were reacted with (7-nitrobenz-2-oxa-1,3-diazole) aminoethyl- and aminopentyl methylphosphonofluoridate. The inhibition rate constants depend on the chain length of the aminoalkyl moiety of the probe and range from 10(5) to 10(4) M-1 min-1 for elastase and chymotrypsin. They are significantly modified when the proteases are stoichiometrically bound to alpha 2M. The absorption maximum of the chromophore appears in the range of 460-470 nm and 475-480 nm for the aminoethyl- and aminopentyl- conjugates, respectively. The fluorescence emission is maximal around 530 nm with a low quantum yield of about 3%. These spectral characteristics are altered in different ways by the covalent or non-covalent binding mode of the protease to alpha 2M. Finally, the CD spectrum of the NBD aminoethyl and aminopentyl elastase and chymotrypsin conjugates exhibits intense optical activity in the absorbing band of the NBD-moiety. These chiral properties are greatly altered upon binding of the protease to alpha 2M. All these results strongly suggest a conformational change in the protease at its active center upon its binding to alpha 2M; this conformational change could be taken into account to explain the alteration of the catalytic properties of the alpha 2M-bound proteases.     

Reaction of chicken egg white lysozyme with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. II. Sites of modification.

1. Procedure for the isolation of peptides from proteins bearing the chemically labile aromatic ether, O-tyrosyl-4-nitrobenz-2-oxa-1,3-diazole group, is described. 2. The tyrosyl residue reactive towards 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in chicken egg white lysozyme (Aboderin, A. A., Boedefeld, E. and Luisi, P. L., (1973) Biochim. Biophys. Acta 328. 20-30) is tyrosine-23. The amino group in the protein whose reaction with the reagent is dependent on the prior reaction of tyrosine-23 is the epsilon-amino group of lysine-33.     

Involvement of tyrosyl residues in the structure-function relationships of D-beta-hydroxybutyrate dehydrogenase: a phospholipid-requiring enzyme

The involvement of tyrosyl residues in the function of D-beta-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme, has been investigated by using several tyrosyl modifying reagents, i.e., N-acetylimidazole, a hydrophilic reagent, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane, two hydrophobic reagents. Modification of the tyrosyl residues highly inactivates the derived enzyme: Treatment of the enzyme with 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole leads to an absorbance at 380 nm and to an incorporation of about 1 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole per polypeptide chain for complete inactivation. Inactivation by N-acetylimidazole induces a decrease in absorbance at 280 nm which can be reversed by hydroxylamine treatment. On the other hand, the ligands of the active site, such as methylmalonate, a pseudosubstrate, and NAD+ (or NADH), do not protect the enzyme against inactivation. In contrast, the presence of phospholipids strongly protects the enzyme against hydrophobic reagents. Finally, previous modification of the enzyme with N-acetylimidazole does not affect the incorporation of 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole while modification with tetranitromethane does. These results indicate the existence of two classes of tyrosyl residues which are essential for enzymatic activity, and demonstrate their location outside of the active site. One of these residues appears to be located close to the enzyme-phospholipid interacting sites. These essential residues may also be essential for maintenance of the correct active conformation.     

A new chromogenic substrate for gamma-glutamyl transpeptidase.

A benzfurazan derivative of glutathione l-γ-glutamyl-(S-4-nitrobenz-2-oxa-1,3-diazole)-l-cysteinylglycine (GS-NBD) with an absorption maximum at 419 nm is readily acted upon by γ-glutamyl transpeptidase to yield the S-benzfurazan derivative of cysteinylglycine. An internal S→N shift occurs immediately to yield the N-benzfurazan derivative, which in turn reacts with the sulfhydryl reagent 4,4′-dithiodipyridine to produce the mixed disulfide with an intense absorption at 461 nm. The maximum difference in molar extinction coefficient is 13,200 and occurs at 470 nm. This general device should be applicable to the assay of many other peptidases.     

Synthesis and properties of a fluorescent derivative of phosphatidylcholine

1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidyl choline, (NBD-PC) was prepared by alkylation of ?-amino caproic acid with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1), followed by esterification of lysophosphatidylcholine. The compound was purified by silicic acid chromatography, and exhibited a single spot on thin layer chromatography using acidic, basic and neutral solvent systems, when visualized by UV, molybdate spray, primuline, or charring. The UV-visible absorption spectrum, and the uncorrected fluorescence excitation spectrum of NBD-PC in absolute ethanol showed maxima at approximately 340 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescence intensity and emission maximum wavelength of NBD-PC are strongly dependent on solvent dielectric constant, and the relative fluorescent intensity of NBD-PC in absolute ethanol is directly proportional to its concentration from 1 ng/ml to approximately 3 μg/ml. Incorporation of NBD-PC into membranes of human lymphocytes cultures in the presence or absence of phytohemagglutinin (PHA) resulted in a marked increase in the relative fluorescent intensity of the bound fluorochrome, and a 15 nm blue shift in its emission maximum wavelength. Fluorescence titration data indicate that the unstimulated lymphocytes bound 912 pmoles NBD-PC/mg protein with an association constant of 3.45 × 10⁷ M, while the PHA stimulated cells bound 1200 pmoles NBD-PC/mg protein with an association constant of 2.82 × 10⁷ M. The temperature dependence of the fluorescent intensity of NBD-PC incorporated in control, and PHA stimulated lymphocytes showed discontinuities at 15 and 24 °C respectively. Fluorescence polarization of NBD-PC incorporated in the membranes of stimulated lymphocytes was greater than the polarization of the fluorochrome in non-stimulated cells, suggesting that the plasma membranes of PHA stimulated lymphocytes contain regions of higher microviscosity.     

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for lambda max of the fully oxygenated haemocyanin were 531.5 nm (pH less than 7.8) and 530.0 nm (pH greater than 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH less than 7.2) and 535.2 nm (pH greater than 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891-1895]. Only four amino acids of the four-hexamer were labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.     

A new route to trihydroxamate-containing artificial siderophores and synthesis of a new fluorescent probe

A fluorescent labelled artificial siderophore 1 was synthesized by coupling a 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative to the terminal amino group of a new trihydroxamate-containing amine 2, a ferrichrome-type siderophore that was obtained from tris(hydroxymethyl)aminomethane. Compound 1 was shown to be a suitable tool for experiments on siderophore transport and uptake processes in various organisms cells and particularly in Candida albicans cells.     

The active protein-conducting channel of Escherichia coli contains an apolar patch

Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by translocase, a complex of a protein-conducting channel, SecYEG, and a peripheral motor domain, SecA. SecYEG has been proposed to constitute an aqueous path for proteins to pass the membrane in an unfolded state. To probe the solvation state of the active channel, the polarity sensitive fluorophore N-((2-(iodoacetoxy)ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-1,3-diazole was introduced at specific positions in the C-terminal region of the secretory protein proOmpA. Fluorescence measurements with defined proOmpA-DHFR translocation intermediates indicate mostly a water-exposed environment with a hydrophobic region in the center of the channel.     

Cysteine-25 of adenylate kinase reacts with dithiothreitol to form an adduct upon aging of the enzyme.

Adenylate kinase (AK) ages in solution in the presence of DL-dithiothreitol (DTT) with a gradual activity decrease. Upon dilution with 4 M guanidine hydrochloride denatured native and aged AK, both recover to the same activity as the fresh enzyme. Mass spectroscopy and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole chloride modification kinetics studies identify that the residue cysteine-25 of the enzyme reacts with DTT to form an adduct. The formation of the unusual bridging DTT adduct of AK appears to be the result of a stable DTT-protein complex. The K(M) for AMP, ADP and MgATP of the DTT-modified enzyme does not differ significantly from that of the intact enzyme, whereas the secondary and tertiary structures of the enzyme change obviously. These results indicate that cysteine-25 may not be involved directly in substrate binding, but may play an important role in maintaining secondary and tertiary structures of native AK, as well as the conformation interconversion in the catalytic cycle.     

A fluorescent chiral chemosensor for the recognition of the two enantiomers of chiral carboxylates

A new 7-nitrobenz-2-oxa-1,3-diazole (NBD)-based fluorescent chiral chemosensor (NBD-1) was prepared and applied to the recognition of the two enantiomers of the tetrabutylammonium salts of N-t-Boc-α-amino acids and chiral carboxylic acids including naproxen. In particular, the chiral recognition by the new fluorescent chiral chemosensor for the two enantiomers of N-t-Boc-threonine (tetrabutylammonium salt) was quite excellent, the Stern-Volmer constant ratio (K(D)/K(L)) for the two enantiomers being as high as 4.89.     

Fluorescent derivatives of bile salts. I. Synthesis and properties of NBD-amino derivatives of bile salts.

In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature.     

Substrate specificity and colorimetric assay for recombinant TrzN derived from Arthrobacter aurescens TC1

The TrzN protein, which is involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1, was cloned and expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.     

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.     

Fluorescence studies of chicken liver fatty acid synthase. Segmental flexibility and distance measurements

The 4'-phosphopantetheine of chicken liver fatty acid synthase was specifically labeled with the fluorescent substrate analog coenzyme A 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoate at low salt concentrations. A serine at the active site of the thioesterase was specifically labeled with the fluorescent compounds 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminopentylmethylphosphono fluoridate and/or pyrenebutyl methylphosphonofluoridate. Dynamic anisotropy measurements indicate the thioesterase has considerable segmental flexibility, whereas the fluorescent labeled 4'-phosphopantetheine does not display detectable local or segmental flexibility. Fluorescence resonance energy transfer measurements indicate that the distance between the fluorescent label at the end of the 4'-phosphopantetheine and NADPH bound to the beta-ketoacyl reductase or enoyl reductase site on the same polypeptide chain is essentially the same, approximately 38 A. The two types of reductases were distinguished by specifically blocking enoyl reductase with pyridoxal 5'-phosphate. No significant energy transfer occurs between sites on different polypeptide chains so that the distances must be greater than 55 A. The distance between the serine on the thioesterase and the 4'-phosphopantetheine on the same polypeptide is 48 A; again no interpolypeptide chain energy transfer was observed. The distance between the serines of the two thioesterases within a fatty acid synthase molecule is greater than 56 A. The monomeric enzyme obtained at 1 degree C does not have beta-ketoacyl synthase and reductase activities. Also fluorescent titrations indicate NADPH is not bound to beta-ketoacyl reductase in monomeric enzyme. The addition of potassium phosphate to the monomers at 1 degree C rapidly dimerizes the enzyme and restores the beta-ketoacyl reductase activity. The beta-ketoacyl synthase activity is slowly restored when the dimer is raised to room temperature. The results obtained suggest that relatively large conformational changes may be part of the catalytic cycle.     

Alkaline structural transition of 4-nitrobenz-2-oxa-1,3-diazolyl-Lysozyme. Kinetic and spectroscopic investigations.

When lysozyme is reacted with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-CL), A 1:1 covalent product is produced, in which the NBD group arylates the phenolic hydroxyl group of Tyr-23 (Aboderin, A. A., and Boedefeld, E. (1976) Biochim. Biophys. Acta 420, 177). Changing the pH from neutral to alkaline conditions results in a large spectral shift of the absorption band associated with the NBD chromophore (Aboderin, A. A., Boedefeld, E., and Luisi, P. L. (1973) Biochim. Biophys. Acta 328, 30). In the present work it is shown that this spectral change is due to the formation of a sigma complex in which a hydroxyl ion is added to the aromatic nucleus of the nitrobenzoxadiazole system. Circular dichroic studies suggest that the NBD group is held in a conformationally rigid state in the protein. The kinetics of the spectral change accompanying the formation of the sigma complex has been investigated with a rapid mixing stopped flow spectrophotometer both in the modified enzyme and in the low molecular weight model compounds N-acetyl-(O-NBD)-L-tyrosinamide and glycyl-(O-NBD)-L-tyrosine. In the pH range from 10.1 to 12.7, the time course of the reaction is first order in the case of the modified enzyme (k = 4.8 s-1) and bimolecular and much slower (under pseudo-first order conditions) in the low molecular weight compounds. It is suggested that in the enzyme the reaction proceeds much faster because of the hydrophobic environment around the reacting groups. It is further suggested that the unimolecularity in the enzyme is due to a rate-determining isomerization step, probably connected with a local rearrangement of the protein conformation following the ionization of Tyr-20.     

7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin: Synthesis of a fluorescent actin probe

Phallacidin was labeled with the fluorophore, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, after a series of modifications. The complete purification and synthesis of intermediates are described. A simple laboratory procedure for production of the fluorescent toxin is outlined. The fluorescent phallacidin was used to stain plant and animal cellular actin. Because of the low molecular weight, high actin specificity and visible excitation profile of the conjugated toxin, fluorescent phallacidin, has proven useful in the fluorescence microscopic study of actin in vivo and in vitro.     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

Reaction of tyrosyl-modifying reagents with the ligand- and DNA-binding domains of the rabbit liver glucocorticoid receptor

We have studied the effects of p-nitrobenzenesulfonyl fluoride, 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and tetranitromethane on the glucocorticoid receptor from rabbit liver. Our results show that all tyrosine modifying reagents inhibit the binding of [3H]dexamethasone to the receptor. Equilibrium binding experiments revealed that only 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid is a competitive inhibitor while the other chemical probes decrease the concentration of binding sites. Transformation of glucocorticoid-receptor complexes was markedly reduced when heat treatment was performed in the presence of tyrosyl-directed reagents. Taken together, these results indicate for the first time that critical tyrosyl moieties may be involved in both hormone binding and transformation of the glucocorticoid receptor.     

Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter

Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+-dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p-chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N-ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter.