Photoaffinity labeling of the acetylcholine transporter.

The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.     

Fluorometric labeling of tetrahydroprogesterones.

7-Diethylaminocourmarin-3-carbohydrazide was used to label the ketone group of the tetrahydroprogesterones to form fluorescent derivatives with high sensitivity. The four isomeric 3-hydroxypregnanes separated readily on high-performance, thin-layer chromatography after derivatization. This separation was not possible with the underivatized isomers. Standards and steroids from biologic mixtures were separated and showed similar characteristics. The methods used are described.     

Photoaffinity labeling of the angiotensin II receptor; pharmacology of the labeling peptides in the dark.

The biological activities of photoaffinity labeling analogs of angiotensin II (ATII) and their precursors were measured in rabbit aorta strips in the dark. Most of the analogs behave as reversible, specific agonists, one as a competitive inhibitor. The activities are discussed in line with the current view of structural requirements. The modifications consisted of substitutions on the aromatic nuclei of Tyr4 and Phe8 in [Sar1]ATII with (4'-NO2) Phe, (4'-NH2)Phe, (4'N3)Phe, (4'-N2 +)Phe, and (4'-NH2-3', 5'-I2)Phe. It is shown that the affinity of the ATII analogs modified in position 4 depends on the electronegativity and not on space-filling properties of the aromatic residue; rising electronegativity lowers the affinity, i.e. [sar1, (4'-NO2)Phe4]ATII has no more measurable activity. Substituting the aromatic side chain in position 8 of [Sar1]ATII gives well-binding analogs with intrinsic activities from 0 to 100% and activity seems to depend only on stereochemical requirements. Agonists and partial agonists bear rather small groups like -NH2, -N3, -NO2, and -N2 +. The only antagonist [Sar1, (4'-NH2-3',5'-I2)Phe8]ATII resembles the antagonist E1Sar1, Leu8]ATII in competitivity and binding.     

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.     

Enzymatic labeling of arbitrary proteins.

This paper describes an enzymatic labeling technique (ELT), using transglutaminases. On the basis of the ELT, isotopic nuclei are easily incorporated into the gamma-carboxyamide groups of glutamine residues in arbitrary proteins, without changing their chemical structures. We have also shown that, by using ELT, protein aggregation was easily checked for NMR studies and that it can be applicable for the screening of weakly bound ligands for proteins. Owing to the simple preparation of the isotope-labeled proteins, ELT should be useful for speeding up various structural and functional analyses of arbitrary proteins.     

External yeast beta-fructosidase. Affinity labeling of the active site.

Conduritol-B-epoxide, a compound structurally related to the substrates of external yeast beta-fructosidase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26), is an active-site directed inhibitor of this enzyme. The inactivation is irreversible and first-order with respect to time and inhibitor concentration. From the kinetic data obtained, it is concluded that one molecule of inhibitor reacts with one molecule of the enzyme causing inactivation. The inactivation is prevented by the presence of substrates. The pH-dependence of inactivation shows two dissociating groups in the enzyme with pKa values 3.05 and 6.8 being involved in the inactivation process. A carboxylate at the active site with pKa 3.05 is suggested to be the reactive group with conduritol-B-epoxide.     

Method for the genetic labeling of cryptic plasmids.

A recently developed method for detecting transposition was employed to genetically "label" conjugative plasmids such as F and Ent P307, which do not normally exhibit a readily identifiable phenotype.     

End labeling of enzymatically decapped mRNA.

A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.     

Specific chemical labeling of DNA fragments.

We describe a simple method for specific chemical labeling of DNA fragments at their 3'-termini. The procedure includes enzymatic addition of 4-thiouridine, followed by reaction in mild non-denaturing conditions with the highly reactive alpha-haloacetamido derivatives of several chemical labels. The attached reporter molecule can be removed by extended treatment with beta-mercaptoethanol. Among the potential applications of this labeling method is the study of specific protein-DNA interactions in solution.     

Radioactive labeling of alpha-bungarotoxin with tritium.

α-[³H] bungarotoxin (Bgt) was prepared by catalytic reduction of ¹²⁵I-labeled α-Bgt with tritium. Specific activities of 10–15 Ci/mmol were attained. The radioactive label was found in tyrosine. Tritiated α-Bgt appears to bind specifically to the cholinergic receptor of diaphragm and to a similar component of cerebral cortex. This specificity and the high specific radioactivity attained provide a useful tool for the study of acetylcholine receptor in brain and other tissues with low receptor concentration.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

New frontiers in gold labeling.

Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab' antibody fragments, making small and stable probes. Silver enhancement then makes these small gold particles easily visible by EM, LM, and directly by eye. Another advance is the combination of fluorescent and gold probes for correlative microscopy. Chemical crosslinking of gold particles to many biologically active molecules has made possible many novel probes, such as gold-lipids, gold-Ni-NTA, and gold-ATP.     

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.     

Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.

The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.     

Photoaffinity labeling of soluble auxin-binding proteins.

The photoaffinity labeling agent azido-IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analogue of the endogenous auxin indole-3-acetic acid, was used to search for auxin-binding proteins in the soluble fraction of Hyoscyamus muticus cells. Azido-IAA became covalently attached to three polypeptides with a high specific activity. The labeling was specific for IAA and not due to random tagging. Two polypeptides with molecular masses of 31 and 24 kDa in the 0-30% ammonium sulfate fraction were labeled after UV photolysis at 0 degree C but not at -196 degrees C, and appeared to have a high affinity indole-binding site(s) for which active, non-indole auxins were not good ligands. A third polypeptide with a molecular mass of 25 kDa present in the 50-60% ammonium sulfate fraction labeled exclusively at -196 degrees C and had a significant affinity for active auxins but not for inactive indoles. The azido-IAA labeling pattern, pI, competition results, and immunoprecipitation all indicate that the 31- and 24-kDa polypeptides are related to the basic form of endo-1,3-beta-glucanase (EC 3.2.1.39). Azido-IAA labeling polypeptides equivalent to the 31- and 24-kDa species were apparently also present in the cell wall. The low pH optimum for binding of azido-IAA to the 25-kDa polypeptide suggests the location of the active protein in a compartment such as the vacuole or a transport vesicle rather than in the cytosol.     

Fluorescence labeling of the human erythrocyte anion transport system.

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419--2427). The vesicles have a functional anion transprot system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescnece titrations show a maximum labeling stoichiometry of 1.3 +/- 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu2+ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion transport system. Sulfate transport in the labeled vesicles remains fully functional. We detected self-energy transfer between bound FMA molecules by fluorescence depolarization. With excitation at 450--50 nm P decreases from 0.4, when less than half of the proteins are labeled, to 0.1 at saturation. This depolarization is not observed with red edge excitation (510--530 nm). Addition of 0.1% sodium dodecyl sulfate (SDS) changes P to 0.32, regardless of the excitation wavelength or degree of saturation with FMA. These results indicate that the band 3 proteins are close enough to allow energy transfer between fluorophores(Ro = 37.4 A), which does not occur upon red edge excitation or when the proteins are separated by SDS. We conclude that the functional anion transport system exists as a dimer or higher oligomer of band 3 proteins in these membranes, confirming previous suggestions derived using other methods. Future applications are discussed.