Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Affinity Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources
PubMed1990

Laminin interactions with ductal pancreatic adenocarcinoma cells: identification of laminin- and collagen-binding proteins.

Laminin promotes the modulation of human pancreatic adenocarcinoma cells from a proliferative to a resting phenotype. This process includes restoration of cell polarity, increase of protein biosynthesis, and increase of glycoprotein secretion. The growth correlates with the amount of laminin coated on the culture dish. Adenocarcinoma cells do not synthesize collagen type I, fibronectin or laminin. Prolonged propagation of the cells on laminin substratum enhances the expression of laminin-binding sites on the cell surface. Laminin binds to cell plasma membrane vesicles with a KB of about 2.2 x 10(10). By affinity chromatography of [35S]methionine-labeled, detergent-extracted, cells on immobilized laminin, a Mr 82,000 polypeptide could be enriched. By simultaneous chromatography on immobilized collagen type I, a Mr 34,000 polypeptide was retained. In laminin overlay experiments on transblotted plasma membrane proteins, Mr 82,000 and 70,000 polypeptides were labeled. Affinity chromatography on laminin-Sepharose of tumor cell membranes from cells grown in nude mice tumors retained Mr 100,000, 82,000, 70,000 and 55,000 polypeptides bound to the column. Endoglycosidase F treatment of these proteins reduced the number of higher Mr proteins, leaving a Mr 70,000 polypeptide, together with smaller peptides. Using the enriched binding protein fraction as antigens, monoclonal antibodies (mAbs) were prepared in mice. Two of the mAbs were further analysed; they recognized simultaneously the complete pattern of high and low Mr polypeptides identified to date by the other methods. Antibody 2A1-H7 was capable of inhibiting attachment and spreading of the cells on laminin and collagen I, but not on tissue-culture plastic. These data may indicate a molecular heterogeneity, partly based on diverse glycosylation, responsible for the variations in the molecular weight of the binding proteins. The adaptation processes of the tumor cells during growth on the extracellular matrix may indicate a regulatory function on tumor growth and metastasis in vivo.

Adenocarcinoma↗
PubMed1991

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.

Adenosine↗
PubMed1988

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations.

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.

Affinity Labels↗
PubMed2006

One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity.

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Binding Sites↗
PubMed2000

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe.

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.

Amino Acid Sequence↗
PubMed1984

Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen.

We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 +/- 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pI of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10(-6) M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.

Affinity Labels↗
PubMed1988

Purification of nucleotide-linked peptide.

Affinity labeling of nucleotide-binding enzymes/proteins with 32P-labeled nucleotides is a powerful technique to identify nucleotide-binding proteins as well as to radiolabel the specific binding site. We have used this approach for labeling a nucleotide-binding domain in DNA polymerase and have isolated peptides bearing the linked nucleotides. The method used for separating tryptic peptides on hydrophobic matrices with an acetonitrile gradient in 0.1% trifluoroacetic acid as eluent results in loss of radioactivity, presumably through dissociation of the cross-linked nucleotide. This can be averted by the use of a non-acidic medium in the peptide purification protocol. We have devised a relatively simple procedure to concentrate the nucleotide-linked peptides by chromatography on DEAE-Sephadex A25. Most neutral and basic peptides as well as free nucleotides are removed by eluting the DEAE-Sephadex column with 0.2 M ammonium bicarbonate. The nucleotide-linked peptide is then eluted with 0.6 M ammonium bicarbonate. Radioactivity in the collected fractions is conveniently determined by scintillation counting. Labeled peptide in the 0.6 M ammonium bicarbonate eluate can be purified on a C4 reversed-phase column with an acetonitrile gradient in phosphate buffer (pH 6.8). By this procedure, 32P-labeled nucleotide linked with protein/peptide can be quantitatively purified with minimum loss.

Affinity Labels↗
PubMed1992

The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.

Affinity Labels↗
PubMed1984

Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli.

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) inactivates dodecameric glutamine synthetase from Escherichia coli with concomitant labeling of one site/subunit (Foster, W.B., Griffith, M.J., and Kingdon, H.S. (1981) J. Biol. Chem. 246, 882-886). Cyanogen bromide cleavage of the FSBA-inactivated enzyme in 70% HCOOH produced a large peptide (Mr congruent to 4600) in approximately 75% yield containing N epsilon-4-carboxybenzenesulfonyl (CBS) lysine. The CBS-peptide was purified by high performance liquid chromatography on the basis of its relatively high UV absorbance at 246 nm (delta epsilon congruent to 19,000 M-1 cm-1). The appearance of the labeled peptide coincided with the loss of a peptide (lacking tyrosine and tryptophan) from the unmodified, active enzyme; the CBS-peptide also could be distinguished from cysteine-containing peptides labeled with thionitrobenzoate and from the adenylylated peptide. The identical peptide was labeled when the unadenylylated enzyme was inactivated with FSBA in the absence or presence of Mn2+ or Mg2+ (+/- L-glutamate) or when the enzyme was adenylylated. Conformational differences among these enzyme forms therefore are not detected by affinity labeling of ATP sites. The FSBA-labeled peptide spans residues 9-48 from the N-terminal end of the subunit polypeptide chain, and sequence analysis by automated Edman degradation revealed that CBS-lysine was at position 47. Thus, lysyl residue 47 appears to be near the subunit gamma-phosphate-binding site for ATP in the native glutamine synthetase structure. The primary structure of the CBS-labeled peptide is presented.

Adenosine↗
PubMed1989

GTP interacts through its ribose and phosphate moieties with different subunits of the eukaryotic initiation factor eIF-2.

We have previously shown that a GTP derivative bearing p-azidoaniline at the gamma-phosphate group specifically labels the gamma-subunit of eukaryotic initiation factor eIF-2. In the present study a new GTP derivative carrying the photoreactive group at the ribose moiety of GTP was applied for affinity labeling of eIF-2 in different initiation complexes. Using this GTP analogue the beta-subunit of eIF-2 was found to be specifically labeled in all complexes investigated. It is concluded that GTP interacts with both the beta- and gamma-subunit of eIF-2: the guanosine moiety is in contact with the beta-subunit and the gamma-phosphate group with the gamma-subunit.

Affinity Labels↗
PubMed1988

Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors.

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

Affinity Labels↗
PubMed1992

Photoaffinity substrates for P-glycoprotein.

A variety of compounds can inhibit the function of P-glycoprotein (Pgp) by binding to it and preventing the efflux of anticancer drug substrates. While the molecular architecture of the drug binding site(s) in Pgp is not known, it is clear that modulators in general appear to conform to some general physical-chemical rules. In this paper, we discuss the basic concepts of drug recognition by Pgp as currently understood. We also examine the compounds used to photoaffinity label this protein and discuss their utility in identifying drug binding sites. Finally, we show that a photoaffinity analog of daunorubicin, [3H]azidobenzoyl-daunorubicin ([3H]AB-DNR), is a good affinity labeling reagent for Pgp. A finding of interest is that vinblastine and verapamil compete more effectively than daunorubicin for [3H]AB-DNR binding to Pgp, suggesting that vinblastine and verapamil have similar structural features not shared by daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent Ka for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21 microM, a value similar to that for guanosine 5'-[beta,gamma-imido]triphosphate. m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-32P]m-AcAGTP, followed by immunoprecipitation of G-protein Gq, identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of alpha-subunits of heterotrimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.

Adenylyl Cyclases↗
PubMed1993

Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic alpha-bungarotoxin binding sites with a derivative of alpha-bungarotoxin.

Neuronal/nicotinic alpha-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.

Affinity Labels↗
PubMed1997

Bacterial expression of human vitamin D-binding protein (Gc2) in functional form.

In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, in Escherichia coli. The recombinant protein (reDBP) was expressed as a fusion partner of glutathione S-transferase in order to facilitate proper folding of the reDBP; E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure.

Actins↗
PubMed1994

Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate.

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.

Acetylcholinesterase↗
PubMed1989

Two different forms of endothelin receptors in rat lung.

We provided evidence for the presence of two distinct types of rat endothelin (ET) receptors having different molecular masses by affinity labelling of rat lung membranes with 125I-ET-1, ET-2 and ET-3 and SDS-PAGE followed by autoradiography. One type, which is 44 kDa has a higher affinity for ET-1 and -2 than ET-3, whereas the other type, which is 32 kDa, preferably interacts with ET-3 rather than ET-1 and -2.

Affinity Labels↗
PubMed1996

Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue.

P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300-380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 --> Lys-81 and Asp-82 --> Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue.

Affinity Labels↗