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PubMed2001

Affinity labels as tools for the identification of opioid receptor recognition sites.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Affinity Labels↗
PubMed1988

Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling.

Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor.

Affinity Labels↗
PubMed1990

An isozyme-selective affinity label for rat hepatic acetyltransferases.

Affinity chromatography of an ammonium sulfate precipitate obtained from rat hepatic cytosol resulted in the separation of two fractions of N-acetyltransferase (NAT) activity. NATI catalyzed the S-acetylcoenzyme A (AcCoA)-dependent acetylation of p-aminobenzoic acid (PABA); NAT II catalyzed the N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-dependent acetylation of 4-amino-azobenzene (AAB) (N,N-acetyltransferase), the AcCoA-dependent acetylation of procainamide (PA), and the N-arylhydroxamic acid N,O-acyltransferase (AHAT) activity that results in the conversion of N-OH-AAF and related hydroxamic acids to electrophilic reactants. 1-(Fluoren-2-yl)-2-propen-1-one (vinyl fluorenyl ketone, VFK) was shown to be a potent and irreversible inactivator of NAT II activities. A 200-fold higher concentration of VFK was required to inactivate NAT I activity than was required for inactivation of NAT II activities. Similar selectivity in the inactivation of the isozymes was observed when experiments were conducted with enzyme preparations that contained both NAT I and NAT II activities. The presence of substrates and products of the NAT II-catalyzed reactions such as AcCoA, 2-acetylaminofluorene (2-AAF), and N-acetyl-4-aminoazobenzene (N-Ac-AAB) protected NAT II from the inactivating effects of VFK, providing evidence that VFK is an active site directed inhibitor (affinity label) of NAT II. Studies with 1-(fluoren-2-yl)-2-propan-1-one (EFK), an analogue of VFK in which the alpha, beta-unsaturated vinyl ketone group of VFK has been replaced with an ethyl ketone group, demonstrated that the conjugated ketone of VFK is required for inactivation of enzyme activity. The results of these studies suggest that agents such as VFK should have utility as probes of acetyltransferase multiplicity and in the investigation of the active site topography of the enzymes.

Acetylation↗
PubMed1981

Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling.

Membranes from adult rabbit superior cervical ganglia, cross-linked to membrane-bound 125I-labeled nerve growth factor (NGF) by the photoreactive agent hydroxysuccinimidyl-p-azidobenzoate, were found to contain two labeled components with apparent Mr = 143,000 and Mr = 112,000. At high concentrations of the cross-linker, minor amounts of a Mr = 300,000 affinity labeled product were also observed. The affinity labeled species exhibit the characteristics expected of membrane receptors for NGF. The inhibition of specific 125I-NGF binding to membranes by increasing concentrations of unlabeled NGF parallels the inhibiton of the affinity labeling of these components. Insulin, insulin-like growth factor I, multiplication stimulating activity, and epidermal growth factor do not inhibit the affinity labeling reaction. Membrane preparations of various non-neuronal tissues do not show any detectable specific cross-linking to 125I-NGF. The affinity labeled species of superior cervical ganglia are proteins and contain intrapeptide disulfide bridges compacting their molecular structure. Peptide mapping experiments indicate a close structural relationship between the Mr = 143,000- and the Mr = 112,000-labeled proteins, suggesting a transformation of the former into the latter by limited proteolysis. The results suggest that the Mr = 143,000 affinity labeled protein represents a native NGF receptor component.

Affinity Labels↗
PubMed1978

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.

Adenosine↗
PubMedDate not supplied

[Interaction of RNA polymerase of Escherichia coli with substrate by affinity labeling and NMR with nuclear 31P].

The affinity labeling of E. coli RNA polymerase by periodate oxidized uridine triphosphate was carried out without and with the template (polydeoxynucleotides poly(dA) and poly(dT) ), and under the conditions of poly(dA) and poly(dT) transcription. The extent of RNA polymerase labeling was nearly the same in the presence of poly(dA) and poly(dT) (0.9 and 0.7). However, during the transcription of poly(dA) the extent of RNA polymerase labeling proved to be more than twice as high as that in the case of poly(dT) (0.75 and 0.3). The longitudinal relaxation times (T1) for phosphorus of ATP and dinucleotide pUpU in the complexes of RNA polymerase with the template (poly(dA) and poly(dT) in the presence of MnCl2 were determined. The distance from the phosphorus of enzyme-bound ATP and pUpU and the Mn2+ ion coordinated at the active center on RNA polymerase were calculated using the Solomon - Blombergen formula. According to the results of the affinity labeling of RNA polymerase and the NMR (nuclear magnetic resonance) experiments it can be inferred that the template influences the formation of the working conformation of the enzyme active center, specific with respect to the true substrate, in the stage of RNA elongation.

Affinity Labels↗
PubMed1996

Stereo-selective affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionic acid.

The role of configuration for the affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionate (BrImPpOH) has been studied. A saturation kinetics mechanism applies which includes formation of a reversible complex with the enzyme prior to alkylation of Cys-43. The pseudo first-order inactivation rate-constant, k2, and the dissociation constant for the reversible enzyme-affinity label complex. KEI, were determined at pH 7.4 and 23.5 degrees C. The stereo isomers of each affinity label exhibit different kinetic characteristics but, unlike with horse liver alcohol dehydrogenase, the discrimination between them is not absolute. For the different affinity labels, k2 varies with 2-chloro-3-(5-imidazolyl)methylpropionate (Me-ClImPpOH) > 2-chloro-3-(5-imidazolyl)propionate (ClImPpOH) > 2-chloro-3-(5-imidazolyl)propanol (ClImPOH), consistent with their order of inherent reactivity, and the specificity constant k2/KEI varies with (S)-Me-ClImPpOH > (S)-ClImPpOH > (S)-ClImPpOH > (R)-Me-ClImPpOH > (R)-ClImPpOH. Models of the affinity labels were built into the active site of the predicted subunit structure of the enzyme by using a computer-controlled display system. In each binary complex, the imidazole moiety of the affinity label was liganded to the catalytic zinc atom, and the angle Scys-C alpha-Cl was linear, in accordance with an SN2 mechanism. Both enantiomers of each label could form plausible complexes with the enzyme model, in agreement with the kinetic data. The enantiomeric selectivity, rather than absolute specificity, of the reaction appears due to the anion-binding site in sorbitol dehydrogenase being less developed than in horse liver alcohol dehydrogenase.

1-Propanol↗
PubMed1984

Affinity labeling of the androgen receptor in rat prostate cytosol with 17 beta-[(bromoacetyl)oxy]-5 alpha-androstan-3-one.

An androgen affinity label, 17 beta-[(bromoacetyl)-oxy]-5 alpha-androstan-3-one, has been synthesized in both radioactive and nonradioactive forms. The affinity label (170 Ci/mmol) was characterized and found to have a high degree of purity. Affinity labeling of the androgen receptor from rat ventral prostate was androgen specific and appeared to be directed at the steroid binding site of the protein. Covalent binding was achieved at 0 degrees C; however, heat treatment at 23 degrees C for 30 min enhanced covalent binding by 31%. The covalently bound steroid was resistant to extraction with organic solvents and precipitation with trichloracetate. The Stokes radius (4.2 nm) and sedimentation coefficient (4.5 S) were identical with those found for receptor bound noncovalently to dihydrotestosterone. Gel electrophoresis of the affinity-labeled receptor under denaturing conditions revealed a molecular weight of 86000. The same molecular weight was observed for the receptor from rat seminal vesicle. This affinity label will be useful in future studies on the structure and function of androgen receptors.

Affinity Labels↗
PubMed1998

Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase.

Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.

Affinity Labels↗
PubMed1988

Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal.

Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.

Adenine Nucleotides↗
PubMed1989

Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling.

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.

Affinity Labels↗
PubMed1991

Dexamethasone 21-(beta-isothiocyanatoethyl) thioether: a new affinity label for glucocorticoid receptors.

The C-21 methanesulfonate ester of the synthetic glucocorticoid dexamethasone (Dex) is an efficient electrophilic affinity label of glucocorticoid receptors and exhibits irreversible antiglucocorticoid activity. In an effort to obtain other affinity labeling steroids with differing biological activities, several new derivatives of Dex were prepared which contained a reactive electrophilic substituent at various distances from the C-21 position. All compounds displayed relatively low affinity for rat glucocorticoid receptors (less than or equal to 8% of that of Dex) in a cell-free competition assay. Nevertheless, one compound, dexamethasone 21-(beta-isothiocyanatoethyl) thioether (Dex-NCS), appeared to be an affinity label by virtue of its ability to block the cell-free exchange binding of [3H]Dex. [3H]Dex-NCS was thus synthesized and reacted with cell-free receptors to give, after analysis on denaturing SDS-polyacrylamide gels, only one specifically labeled species at 98 kDa, which is the molecular weight of authentic rat glucocorticoid receptor. These data directly establish Dex-NCS as a new affinity label for glucocorticoid receptors. Data on the reactivity of Dex-NCS and the stability of [3H]Dex-NCS-labeled receptors suggest that a cysteine SH group has been labeled.

Affinity Labels↗
PubMed2001

Discriminative disulfide-bonding affinity labeling of opioid receptor subtypes.

The affinity-labeling technique is an extremely important method in receptor biochemistry. The 3-nitro-2-pyridinesulfenyl (Npys) group, attached to a mercapto group, can react only with a free thiol group (the beta-mercapto group of cysteine residue) of the target receptor molecules, forming a disulfide bond. This disulfide bonding is mediated through the thiol-disulfide exchange reaction. Unlike other labeling methods, the approach utilizing such chemically activated thiol-containing ligands is able to reproduce an unlabeled protein by treatment with dithiothreitol, a reducing reagent. This provides several unique aspects for the studies elucidating the structure-function relationships between the peptide and the receptor. Based on the SNpys affinity technique, we have achieved the discriminative disulfide-bonding affinity labeling of the three different subtypes of opioid receptors: mu, delta and kappa. This article reviews our novel affinity techniques in the in vitro receptor biochemistry.

Amino Acid Sequence↗
PubMed1993

Using periodate-oxidized nucleotide as affinity label for the nucleotide site of proteins.

The active site of pigeon liver malic enzyme was labeled with a fluorescent affinity label, the periodate-oxidized aminopyridine adenine dinucleotide phosphate. The modified enzyme was subjected to proteolytic digestion with trypsin. The resulted peptides were then separated with reversed-phase high-performance liquid chromatography on Waters muBondapak C18 column. Two pure fluorescent peptides were obtained after three runs of the chromatography. The peptides were then subjected to automatic Edman degradation on a Beckman peptide sequencer and subsequently separated and identified with phenylthiohydantoin C18 column. No sequence was obtained. The possible reasons for the failure in sequencing the periodate-oxidized nucleotides labeled active site peptide and some possible pitfalls in using these reagents were discussed.

Adenine Nucleotides↗
PubMed1993

Steroidal affinity labels of the estrogen receptor. 1. 17 alpha-(Bromoacetoxy)alkyl/alkynylestradiols.

To develop steroidal affinity labels for the estrogen receptor, we prepared five electrophilic estradiol derivatives bearing the 17 alpha-propyl, 17 alpha-(1'-butynyl), or 17 alpha-(1'octynyl) chain, with either a terminal epoxy function (for the 17 alpha-propyl substituent) or a terminal bromoacetoxy function (for all three 17 alpha-substituent types). These compounds displayed low affinity for the lamb uterine estrogen receptor; with apparent relative affinity constants ranging from 0.02% to 0.24% that of estradiol. They were also rapidly transformed in cytosol, probably to the corresponding vicinal diols (epoxy compounds) or primary alcohols (bromoacetoxy compounds). Nevertheless, bromoacetates induced irreversible inactivation of the hormone-binding site but only with ligand-free binding sites. The effect of bromoacetates was prevented by treatment of the cytosol with the thiol-specific reagent methyl methanethiosulfonate. Inactivation of the receptor at 0 degrees C was rapid (< 1 h) and strongly dependent on both compound concentration and pH, with significant effects obtained at either > 150 nM (at pH 9) or pH > 7.5 (at 5 microM). Regardless of the conditions used, the order of efficiency for bromoacetates was always: 17 alpha-propyl derivative < 17 alpha-butynyl derivative < 17 alpha-octynyl derivative, with maximal inactivation of approximately 30% and approximately 70% of the hormone-binding sites obtained for the less active and more active compounds, respectively. Characteristics of the receptor inactivation suggest that (i) prepared bromoacetates are highly reactive affinity labels for the estrogen receptor, (ii) they react with similar (or even a single) nucleophilic amino acid residues located within or near the hormone-binding site of the receptor; these residues are probably the -SH of cysteines, and (iii) position 17 alpha of steroidal ligands is suitable for introducing electrophilic substituents to develop efficient affinity labels for the receptor.

Acetates↗
PubMed1982

Identification of thiol group at the estrogen-binding site on the cytoplasmic receptor from rabbit uterus by affinity labeling.

The interaction of estrogen with its receptor from rabbit uterus was studied using the affinity labeling technique. An affinity labeling reagent, 3-hydroxy-17 beta-(p-nitrophenyldithio)-1,3,5(10)-estratriene (Reagent A) was synthesized. The compound was designed to meet two requirements: (1) it binds specifically to the steroid-binding site of the receptor; (2) it has functional group capable of forming a covalent bond with an amino acid residue at or near the binding site. The first requirement was demonstrated by competitive binding assay and sedimentation analysis. If the binding affinity of 17 beta-estradiol is defined as 100, that of Reagent A is found to be 0.05. It was shown that the binding was specific for the estradiol receptor. The second requirement was examined by reaction of Reagent A with simple model compounds. 10 microM Reagent A gave rise to approx. 1.3 microM p-nitrothiophenol in the presence of a large excess of L-cysteine or reduced L-glutathione under the experimental conditions employed, indicating that a covalent bond was formed between Reagent A and the model compounds. Furthermore, I have presented evidence by using polyacrylamide gel electrophoresis that 3H-labeled Reagent A is linked covalently to the estrogen-binding site on the receptor of rabbit uterus. The experiment also fulfilled the first requirement. These results indicate that the thiol group present at the binding site is directly involved in the estradiol-receptor binding.

Affinity Labels↗
PubMed1989

A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.

Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.

Affinity Labels↗
PubMed1976

Affinity labelling of the estradiol-17 beta dehydrogenase from human placenta with substrate analogs.

Affinity labelling of the estradiol-17 beta dehydrogenase of human placenta has been performed using derivatives of estradiol-17 beta carrying alkylating groups in nine different positions on the steroid nucleus. The active-site-directed character of the inhibition is confirmed by the following observations: the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68 000 g of enzyme (dimer). The alkylation of a histidine residue which is fast and extensive when the alkylation side chain is on the C-3 carbon atom, is dramatically decreased when alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the beta face of the steroid nucleus. The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C-3 to C-16 of the steroid nucleus. The correlation of this result and that obtained using an alkylating analog of NAD (3-chloroacetyl-pyridine-adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme.

Acetamides↗