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PubMed1996

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit.

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).

Affinity Labels↗
PubMed1978

Characterization of azidobenzamidines as photoaffinity labels for trypsin.

Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversible in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-l-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation. Maximum inactivation of trypsin is achieved at pH 6.2 using either azidobenzamidine derivative. Evaluation of the pH dependence of photoaffinity labeling may provide a sensitive tool for probing conformational changes in inhibitor binding sites. These studies provide a basis for the use of azidobenzamidines as photoaffinity analogs of lysine and arginine side chains.

Affinity Labels↗
PubMed1991

Sites of phosphorylation and photoaffinity labeling of the 1,25-dihydroxyvitamin D3 receptor.

The 1,25-dihydroxyvitamin D3 receptor is a member of the steroid/thyroid hormone receptor gene family and is thought to act by regulating transcription of specific genes. In this report, we have used peptide mapping of porcine 1,25-dihydroxyvitamin D3 receptor to localize the sites of phosphorylation, photoaffinity labeling, and monoclonal antibody binding. Receptor was immunoprecipitated from [32P]orthophosphate-labeled pig kidney LLC-PK1 cells grown in the absence and presence of 1,25-dihydroxyvitamin D3. Phosphorylation of receptor was induced by 1,25-dihydroxyvitamin D3. The phosphorylated receptor was digested with Staph A V8 protease within Cleveland gels and the 32P label was found entirely in a 23-kDa fragment. Similarly, receptor that was photoaffinity labeled with 1,25-dihydroxy-[26,27-3H]vitamin D3 was subjected to peptide mapping by Cleveland gels. The primary site of photoaffinity label incorporation was in the same 23-kDa peptide. This peptide was localized to a region in the center of the receptor protein, spanning part of the previously designated hinge region and roughly one-half the proposed steroid binding domain. Because phosphorylation did not occur in the DNA binding domain, it may not be involved in the binding of receptor to DNA. The localization of phosphorylation sites to this 23-kDa peptide may suggest that phosphorylation is involved in steroid binding and/or activation of the receptor.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex.

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.

Adenosine Triphosphate↗
PubMed1992

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: characterization by radioligand binding and photoaffinity labelling.

A1 adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[3H]dipropyl-xanthine ([3H]DPCPX) gave a Kd value of 0.7 nM and a Bmax value of 82 +/- 13 fmol/mg protein. For the highly A1-selective agonist 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 +/- 29 fmol/mg protein was estimated. Competition of agonists for [3H]DPCPX binding gave a pharmacological profile with R-N6-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N6-125I-p-hydroxyphenylisopropyladenosine ([125I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A1 receptors labelled in brain membranes. Therefore, coated vesicles contain A1 adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A1 receptors in these vesicles are not a processed receptor form. These results confirm that A1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A1 receptor systems in coated vesicles and in plasma membranes are identical.

Affinity Labels↗
PubMed1995

Direct identification of a polyamine binding domain on the regulatory subunit of the protein kinase casein kinase 2 by photoaffinity labeling.

Phosphorylation of many protein substrates by the protein kinase casein kinase 2 (CK2) is stimulated severalfold in the presence of polyamines such as spermine. Previous experiments have shown that CK2 is a polyamine binding protein and that the regulatory beta subunit is required for this binding activity. To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiated photoactivable analog of spermine, [3H]sperminediazonium. The photoaffinity labeled beta subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide T72EQAAEM78 and the minor one was a 22-amino acid peptide comprising residues Ile98 to Met119. Thr72 and His108 were identified as the labeled amino acids of the Thr72-Met78 and Ile98-Met119 peptides, respectively. In the same manner, we succeeded in determining the residue Leu220 as an alpha subunit residue covalently bound to the probe. The photoaffinity labeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we propose a provisional structural model. These observations suggest a possible mechanism for CK2 activation by polyamines at the molecular level.

Affinity Labels↗
PubMed1984

Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8'-azidoadenosine 3',5'-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2',3'-cyclic nucleotide 3'-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1993

Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose.

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins. Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.

Amino Acid Sequence↗
PubMed1989

1-beta-D-arabinofuranosyl-5-(E)-(4-azidostyryl)-uracil 5'-triphosphate: synthesis and utilization as a photoaffinity labeling reagent for DNA polymerase alpha from cherry salmon, Oncorhynchus masou.

For developing a photoaffinity labeling reagent for DNA polymerase alpha, we synthesized 1-beta-D-arabinofuranosyl-5-(4-azidostyryl)-uracil 5'-triphosphate [5-(E)-(4-azidostyryl)-araUTP] bearing a photoreactive aryl azido group. This compound was easily decomposed by photolysis by light above 300 nm. This analogue strongly inhibited DNA polymerase alpha purified from cherry salmon, Oncorhynchus masou, testes; the mode of inhibition was mixed when the enzyme reaction was carried out under room light, and was competitive to dTTP in the dark. From the results of photoaffinity labeling experiments using an analogue containing [gamma-32P], it appeared that this analogue could bind to dTTP binding site of DNA polymerase alpha. Thus, this compound should be very useful for analysis of nucleotide binding sites of this enzyme.

Affinity Labels↗
PubMed1994

Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.

The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin.

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.

Affinity Labels↗
PubMed1984

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction.

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.

Adenine Nucleotides↗
PubMed1987

Photoaffinity labeling with [3H]RU 28362: a powerful tool for the study of rat brain glucocorticoid receptors.

In order to study the receptor system for adrenocortical steroids in rat brain the synthetic glucocorticoid RU 28362 (11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl) androsta-1,4,6-trien-3-one) has been used for photoaffinity labeling. Competition and dissociation studies revealed a single class of binding sites for RU 28362 in rat brain cytosol. Photoaffinity labeling was performed by u.v.-irradiation for 2 min with a coupling efficiency of about 25%. The high efficiency permitted investigation of crude cytosolic preparations under denaturating conditions. Sodium dodecyl sulfate (SDS) and high resolution two-dimensional gel electrophoresis confirmed the high specificity of the photoaffinity labeling. The molecular weight (93 kD) as well as the isoelectric point (5.6) evaluated by these methods corresponded well to data reported for the classical glucocorticoid receptor in rat liver.

Affinity Labels↗
PubMed1993

Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

The membrane-associated 45- and 55-kDa forms of phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) from Saccharomyces cerevisiae are inhibited by ADP by a competitive mechanism with respect to ATP. We initiated studies toward defining the ATP and ADP sites on the PI 4-kinases using azidonucleotide photoaffinity labeling probes. The photoprobe 8-azido-ATP fulfilled the criteria of a specific photoaffinity label for the 45- and 55-kDa PI 4-kinases. 8-Azido-ATP was a substrate and a competitive inhibitor of the PI 4-kinases with Ki values similar to the Km for ATP. 8-Azido-ATP photoinactivated the enzymes and was photoincorporated into the enzymes in a dose-dependent manner at concentrations similar to the Ki values for the photoprobe. ATP, the true substrate, provided specific protection against photoinactivation and photoincorporation of the PI 4-kinases with 8-azido-ATP, whereas GTP, a nonspecific nucleotide, provided no protection against photoinactivation and photoincorporation. Photoaffinity labeling of the PI 4-kinases with 8-azido-ATP was specifically prevented with ADP. The photoprobe 8-azido-ADP also fulfilled the criteria needed to validate its use as a specific photoprobe for the PI 4-kinases. Photoinactivation of the PI 4-kinases with 8-azido-ADP was prevented specifically with ATP. Taken together, these data supported the conclusion that the ATP and ADP sites on the membrane-associated 45- and 55-kDa PI 4-kinases from S. cerevisiae were the same.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Photoaffinity labeling of peripheral benzodiazepine receptors in R-3327 Dunning prostatic tumors.

Photoaffinity labeling of peripheral benzodiazepine receptors (PBZr) in mitochondrial and microsomal fractions from AT-1, H, and G Dunning R-3327 tumor sublines was performed using the photoaffinity ligand 3H-PK 14105. Subsequent sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed specific labeling of a 17 kDa protein in the microsomal fractions from these tumors. In the mitochondrial fractions, however, two bands with the apparent molecular mass of 17 and 18 kDa in AT-1 and H tumors were identified, whereas only the 17 kDa band was present in G tumors. Using another ligand, 3H-flunitrazepam, to photoaffinity label PBZr, proteins with molecular masses of 17, 30, 42, and 48 kDa were identified. These data suggest the possibility of different subclasses of PBZrs in Dunning prostatic tumors.

Affinity Labels↗
PubMed1993

Improved method for autoradiographic localization of beta-adrenoceptors using photoaffinity labelling.

Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of beta-adrenoceptors with "dipping" autoradiography and a modified trichrome stain. Incubation of 16 microns cryosections of rat lung tissue with the iodinated, photoaffinity labeling, non-selective, beta-adrenergic agonist [125I]-cyanopindololazide II ([125I]-CYPA II) (100 pmol/l) in the absence or presence of 1 mumol (+/-)-propranolol revealed strong, specific beta-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photoemulsion by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of beta-adrenoceptors with [125I]-CYPA II allows the use of "dipping" autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiographic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.

Affinity Labels↗
PubMed1988

Photoaffinity labeling adenosine A1 receptors with an antagonist 125I-labeled aryl azide derivative of 8-phenylxanthine.

We have derivatized a series of 125I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes. Photoincorporation into both polypeptides was competitively inhibited by adenosine analogues with a potency order typical of adenosine A1 receptors, but the 24,000-dalton polypeptide bound both agonists and antagonists with lower affinity than the 34,000-dalton polypeptide. Specific photolabeling of receptors in brain membranes of rat, guinea pig, dog, and cow did not show any variation in the 34,000-dalton adenosine receptor binding subunit. The adenosine agonist photoaffinity label [125I]N6-(4-azido-3-iodobenzyl)adenosine also specifically photolabeled the 34,000-dalton polypeptide, but photoincorporation of the agonist was less efficient than the antagonist and, unlike the antagonist, was greatly reduced by guanosine 5'-(beta,gamma-imidotriphosphate). The results indicate that the antagonist photoaffinity label may be more useful than agonists particularly for labeling uncoupled receptors.

Affinity Labels↗
PubMed1995

Purification and photoaffinity labeling of herpes simplex virus type-1 thymidine kinase.

The molecular basis for the treatment of human herpesviruses with nucleoside drugs is the phosphorylation of these drugs by the viral-encoded thymidine kinases. In order to better understand the structural and enzymatic mechanisms by which herpesviral thymidine kinases recognize their substrates, photoaffinity labeling with [alpha-32P]5-azido-2'-deoxyuridine-5'-monophosphate and [ gamma-32P]8-azidoadenosine-5'-triphosphate was used to characterize the thymidine, thymidylate, and ATP active sites of the herpes simplex virus-1 (HSV-1) thymidine kinase. For this study, HSV-1 thymidine kinase and a site-specific mutant enzyme (C336Y, known to confer acyclovir resistance) were expressed in bacteria and purified by a rapid, two-step protocol. The specificity of photoaffinity labeling of these HSV-1 thymidine kinases was demonstrated by the ability of site-directed substrates such as thymidine, thymidylate, acyclovir, 5-bromovinyl-2'-deoxyuridine, and ATP to inhibit photoinsertion. Differences in inhibition patterns of photoaffinity labeling correlated with kinetic differences between the wild-type and C336Y HSV-1 thymidine kinases. Cumulative results suggest that the acyclovir-resistant cysteine 336 mutation primarily affects the ATP binding site; yet it also leads to alteration in the binding affinity of nucleoside drugs in the thymidine site. In this study, azidonucleotide photoaffinity analogs are shown to be effective tools for studying the active-site environment of HSV-1 thymidine kinase and related site-specific mutants.

Adenosine Triphosphate↗