Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.     

Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.     

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.     

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Binding studies with the allosteric effector phenylalanine.

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.     

A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.     

Reactive lysines of yeast glyceraldehyde 3-phosphate dehydrogenase. Attachment of a reporter group to a specific non-essential residue

The lysine-183 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, in contrast to the cysteine-149 residues, react independently with acylating and alkylating agents. Modification of all four residues is required to inactivate the enzyme in spite of the fact that this residue is apparently in the neighborhood of the cysteine-149 involved in half-of-the-sites activity. The modification of the lysine-183 residue, however, influences the half-of-the-sites effect since alkylation of the cysteine-149 residues of the enzyme whose lysine-183 residues are acetylated follows a linear pattern with each subunit acting independently. Four lysine residues outside the active site can be modified with fluorodinitrobenzene, causing 80% loss in enzyme activity. Once again each subunit acts independently. This same residue can also be modified by a fluorescein label which can serve as a reporter group for binding and conformational changes occurring at the active site. The results add support for the functional symmetry of the apo-enzyme and demonstrate how the co-operativity between subunits can be altered by amino acid modification.     

Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide.

3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.     

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.     

The reductive cleavage of myeloperoxidase in half, producing enzymically active hemi-myeloperoxidase

Reduction and alkylation of human myeloperoxidase under nondenaturing conditions results in the cleavage of this enzyme. Sedimentation equilibrium data is presented which shows that the molecular weight of the cleavage product (78,000 +/- 2,000) is half that of the native enzyme (153,000 +/- 4,000). We conclude that the cleavage product is the half-enzyme hemi-myeloperoxidase. Hemi-myeloperoxidase retains both heme groups and contains both subunit types (Mr = 57,500 and 14,000) in the same ratio as native myeloperoxidase. The two halves of native myeloperoxidase are apparently not dependent upon one another for peroxidatic activity, as the specific activity of the half-enzyme is the same as that of the native enzyme. Analytical ultracentrifugation studies show native myeloperoxidase has a sedimentation coefficient of 8.0 and an axial ratio of 5:1, while hemi-myeloperoxidase has a sedimentation coefficient of 4.3 and an axial ratio of 10:1. When [3H]iodoacetic acid was used to prepare hemi-myeloperoxidase, the label incorporated with a stoichiometry of 1.2 [3H]carboxymethyl groups per hemi-myeloperoxidase, with 90% of this label associated with the heavy subunit. From these observations we conclude that native myeloperoxidase contains two heavy-light protomers, which are joined along their long axes by a single disulfide bond between the heavy subunits. Selective reduction of this disulfide bond by the use of nondenaturing conditions results in the formation of hemi-myeloperoxidase, a catalytically active heavy-light protomer of native myeloperoxidase.     

Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures.

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.     

Estrogen receptor immunocytochemistry.

Estrogen receptor activity was preserved in fixed, paraffin-embedded tissue and demonstrated by binding of estrogen which, in turn, was detected immunocytochemically. Estrogen was added to rat endocervial epithelium to protect specifically receptors during fixation. The protective estrogen was apparently lost during embedding and had to be resupplied before staining. Estradiol-mediated immunocytochemical staining was inhibited by diethylstilbestrol and nafoxidine hydrochloride.     

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.     

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.     

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.     

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).     

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.     

Amino acid sequence at the active site of beta-glucosidase A from bitter almonds.

beta-Glucosidase A from bitter almonds was inhibited by the substrate analogue 6-bromo-3,4,5-trihydroxycyclo[2-3H]hex-1-ene oxide. Incorporation of 2 mol inhibitor/mol of dimeric enzyme resulted in total loss of activity. From tryptic digests of the labeled enzyme two radioactive peptides were isolated and their sequence determined (binding site of inhibitor underlined): peptide I, containing approx. 60% of the label: Ile-Thr-Glx-Glx-Gly-Val--Phe-Gly-Asp-Ser-Glx-(Ala, Asx2, Pro)-Lys and peptide II with approx. 30% of the label: Gly-Thr-Glx-Asp. The specifity of the reaction of beta-glucosidases (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with substrate-related epoxides indicates that the aspartic acid labeled in peptide I participates in the catalytic process of beta-glucoside hydrolysis. The labeling of a second site is interpreted in terms of two, mutually exclusive, binding modes of the inhibitor.     

Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten-antibody complex. The extent of covalent labelling was 0.5-0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3-5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29-34 and 95-114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.     

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: limitations to the interpretation of experiments characterizing the activity of individual isomers

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37 degrees C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37 degrees C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, alpha-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.