Independent bindings of Mn2+ and Mg2+ to the active site of B. cereus glutamine synthetase

Glutamine synthetase purified from Bacillus cereus IFO 3131 was modified by iodoacetamide and the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Only Mg2+-dependent activity was inactivated by iodoacetamide, whereas only Mn2+-dependent activity was inactivated by FSBA. When iodoacetamide-treated enzyme was reacted with FSBA, Mn2+-dependent activity was also inactivated. Mg2+ plus Mn2+-dependent activity was inactivated in any case. The results suggested that the binding sites of Mn2+ and Mg2+ are separate from each other in the active site of B. cereus glutamine synthetase and that bindings of Mg2+ and Mn2+ to each site are required for normal activity in vivo.     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation.

Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.     

Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Temperature-sensitive mutant of Bacillus subtilis glutamine synthetase obtained by random mutation

Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg2(+)-dependent activity and Mg2+ plus Mn2(+)-dependent activity of the mutant were less than 5% of those of the wild-type at 37 degrees C, and these activities decreased above 15 degrees C, whereas the Mn2(+)-dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the Km values for NH3 or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg2+, although addition of glutamate, ATP, and Mn2+ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg2+.     

Pea leaf glutamine synthetase: regulatory properties

Of a variety of purine and pyrimidine nucleotides tested, only ADP and 5'AMP significantly inhibited the Mg(2+)-dependent activity of pea leaf glutamine synthetase. They were less effective inhibitors where Mn(2+) replaced Mg(2+). They were competitive inhibitors with respect to ATP, with inhibition constant (Ki) values of 1.2 and 1.8 mm, respectively. The energy charge significantly affects the activity of glutamine synthetase, especially with Mg(2+). Of a variety of amino acids tested, l-histidine and l-ornithine were the most inhibitory, but significant inhibition was seen only where Mn(2+) was present. Both amino acids appeared to compete with l-glutamate, and the Ki values were 1.9 mm for l-histidine (pH 6.2) and 7.8 mm for l-ornithine (pH 6.2). l-Alanine, glycine, and l-serine caused slight inhibition (Mn(2+)-dependent activity) and were not competitive with ATP or l-glutamate.Carbamyl phosphate was an effective inhibitor only when Mn(2+) was present, and did not compete with substrates. Inorganic phosphate and pyrophosphate caused significant inhibition of the Mg(2+)-dependent activity.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is modified and inhibited by p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The modification appears to occur at the NAD(H)-binding site when TH alone or TH in the presence of NADPH is incubated with FSBA. However, when this site is protected by NADH, then FSBA inhibits TH more slowly and modifies a different, though specific, site. This second site could be the NADP(H)-binding site. Using [3H]FSBA in the presence of NADPH, the NAD(H)-binding site was modified, and a single tryptic peptide carrying the label was isolated and sequenced. The amino acid sequence of this peptide was Glu-Ser-Gly-Glu-Gly-Gln-Gly-Gly-Tyr*-Ala-Lys. The modified residue was Tyr. The labeled peptide isolated after incubating TH with [3H]FSBA in the presence of NADH could not be completely purified. However, amino acid analysis and partial sequencing made it possible to identify this segment on the amino acid sequence of bovine TH as derived from its cDNA by Yamaguchi et al. (private communication).     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

The modification of sulfhydryl groups of glutamine synthetase from Bacillus stearothermophilus with 5, 5'-dithiobis(2-nitrobenzoic acid).

The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.     

Purification and characterization of a repressor for the Bacillus cereus glnRA operon.

We report the overexpression, purification, and properties of the regulatory protein, GlnR, for glutamine synthetase synthesis of Bacillus cereus. The protein was found to be a dimer with a molecular weight of approximately 30,000, and its subunit molecular weight was 15,000 in agreement with that (15,025) of deduced amino acid sequence of GlnR. The purified GlnR protein bound specifically to the promoter region of the glnRA operon of B. cereus and Bacillus subtilis. The binding of the GlnR protein to the DNA fragment was enhanced by the presence of glutamine synthetase, the product of glnA, of B. cereus or B. subtilis, although the affinity of the GlnR protein for DNA was not affected in the presence of glutamate, glutamine, Mg2+, Mn2+, or ammonia. These results indicate the existence of an interaction between GlnR and glutamine synthetase, and support the hypothesis that the regulation of glnA expression requires both GlnR protein and glutamine synthetase in Bacillus.     

Subunit interaction elicited by partial inactivation with L-methionine sulfoximine and ATP differently affects the biosynthetic and gamma-glutamyltransferase reactions catalyzed by yeast glutamine synthetase

Yeast glutamine synthetase can be irreversibly inactivated in the presence of L-methionine sulfoximine, ATP, and a divalent cation Mn2+ or Mg2+. Kinetic studies with partially inactivated enzymes show that inactivation of a given subunit in the octameric glutamine synthetase affects the activities of its neighboring subunit such that the rate of the inactivation as well as the gamma-glutamyltransferase activity of the noninactivated subunits decreases while their biosynthetic activity is enhanced. This outcome of subunit interaction is the same irrespective of whether Mn2+ or Mg2+ is used to fulfill the divalent cation requirement of glutamine synthetase for the inactivation reaction and the gamma-glutamyltransferase reaction. Although only Vmax is affected in the gamma-glutamyltransferase assay, both Km (glutamate) and Vmax are changed in the biosynthetic assay.     

Regulation of glutamine synthetase activity by phosphorylation instead of by adenylylation

o-Phosphotyrosyl glutamine synthetase (P-GS) was isolated from highly adenylated glutamine synthetase (AMP-GS) purified from Mycobacterium phlei, by treatment with micrococcal nuclease. The physical characteristics of P-GS were quite similar to those of AMP-GS except for the UV-absorption spectrum. In either Mg2+- or Mn2+-dependent biosynthetic reactions, the kinetic properties, such as optimum pH, Vmax, and apparent Km for each of three substrates of P-GS, were found to be in good agreement with those of AMP-GS. The biosynthetic activity of P-GS was markedly increased after treatment with alkaline phosphatase similarly as in the deadenylylation of AMP-GS by snake venom phosphodiesterase treatment. These results revealed that repression of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue, without recourse to adenylylation.     

The role of specific cations in regulation of cyanobacterial glutamine synthetase

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.