Tuberculous myositis in a patient with ankylosing spondylitis treated with adalimumab.

We present a rare case of tuberculous myositis in a 36-year-old man with long-standing ankylosing spondylitis treated with adalimumab. We review the association between antitumor necrosis factor therapy and tuberculous myositis. Our case illustrates that the index of suspicion of tuberculosis in these patients, even with atypical clinical features, must be very high and emphasizes that this rare infection may occur even with negative tuberculosis screening before therapy was started.

32P-labeled opioid peptides with high affinity for the delta-opioid receptor.

We describe a mild and convenient labeling method for obtaining opioid radioligands which exhibit high specific activity together with a high affinity for the delta-opioid receptor. We chemically synthesized and tested the affinity of enkephalin- and deltorphin-like peptides that contain a phosphorylation site at their C-terminus. The peptide YdAGFLTPRRASLGC (peptide B), labeled to 700 Ci/mmol in the presence of cAMP-dependent protein kinase and [gamma-32P]ATP, bound to the receptor with high affinity (Kd = 3.62 +/- 0.29 nM). This peptide was also chemically coupled to bovine serum albumin and provided a multivalent opioid protein (B-BSA) with interesting properties: compared with peptide B, B-BSA was a better substrate for the kinase (100% 32P incorporation, sp act > or = 7000 Ci/mmol when labeled) and a better ligand for the receptor (Kd = 0.20 +/- 0.02 nM). The concept of peptide extension by a short phosphorylatable sequence should be more generally applicable to other small peptidic hormones or neurotransmitters and provide useful probes for biochemical studies and expression cloning of membrane receptors.

The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization.

A random primed expression cDNA library was constructed from the RNA of NG 108-15 cells. Pools of plasmid DNA were transfected into COS cells, which were screened for their ability to bind 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr, a tritiated agonist for the delta-opioid receptor. A cDNA was isolated that encodes a 371-amino acid-residue protein presenting all the structural characteristics of receptors that interact with guanine nucleotide-binding proteins. Noticeable features are (i) the high hydrophobicity of the encoded protein, (ii) its low sequence similarity to both catecholamine receptors and peptide-binding receptors, although it presents the typical aspartate residue involved in catecholamine binding of the first group and the characteristic short third cytoplasmic loop of the second group. When expressed in COS cells, the receptor exhibits pharmacological properties similar to those of the native receptor: high-affinity binding sites for 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr (Kd = 1.4 nM), stereospecific binding sites for the - enantiomers of levorphanol and naloxone, and the selectivity profile of a delta receptor, as determined by competition experiments with a set of mu-, delta-, and kappa-opioid ligands.

p-Butyroxybenzenediazonium fluoroborate, substrate of acetylcholinesterase and butyrylcholinesterase, discriminates between the two enzymes by a specific affinity labelling.

p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6.10(-5) M and 1.3. 10(-4)M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within two active sites.

Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer.

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.

Photosuicide inactivation of acetylcholinesterase by nitrosamine derivatives.

Methyl(acetoxymethyl)nitrosamine and methyl-(butyroxymethyl)nitrosamine are respectively substrate (KM - 10(-2) M) and competitive inhibitor (Ki = 2 x 10(-3) M) of electric eel acetylcholinesterase (EC 3.1.1.7). Irradiation of an incubation mixture of this enzyme with either nitrosamine leads to an irreversible loss of enzyme activity. The inactivation rates are dependent on photolysis wavelength, light intensity, and inhibitor concentration. Experiments where acetylcholinesterase was radioactively labeled by [14C]-methyl(acetoxymethyl)nitrosamine show that the incorporation of 1 mol of radioactive label per active site is sufficient to cause complete enzyme inactivation irrespective of the reaction conditions used. Methyl(acetoxymethyl)nitrosamine shows no affinity for horse serum butyrylcholinesterase (EC 3.1.1.8) while methyl(butyroxymethyl)nitrosamine is a competitive inhibitor (Ki = 2 x 10(-3) M), but no irreversible inhibition is induced by the action of light. We propose that a suicide type of inhibition [Bloch, K. (1969) Acc. Chem. Res. 2, 193-198] is responsible for the inactivation of acetylcholinesterase, based on photoactivation of nitrosamines only when associated with an acidic hydrogen of the active site.

Specific photoaffinity labeling induced by energy transfer: application to irreversible inhibition of acetylcholinesterase.

p-Dimethylaminobenzene diazonium fluoroborate belongs to a class of potential photoaffinity labeling reagents which, by irradiation, produces a highly reactive electrophilic species. In addition, it can be photodecomposed by photoexcited tryptophan derivatives (e.g., N-acetyltryptophanamide and tryptophan residues belonging to acetylcholinesterase) by an energy transfer reaction. This substance is a competitive inhibitor of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and is able to inactivate the enzyme either by photoaffinity labeling after irradiation at 410 nm or by an energy transfer reaction after irradiation at 295 nm. The efficiency of this method is demonstrated by an increase of the rate of enzyme inactivation as well as by a decrease of nonselective labeling with a radioactive inhibitor p-[methyl-3H]-dimethylaminobenzene diazonium fluoroborate.

NAD(P)+ analogues: tools for the investigation of the active site of oestradiol 17beta-dehydrogenase from human placenta.

Oestradiol-17beta:NAD+ 17-oxidoreductase from human placenta can accept coenzyme analogues of NAD+ and NADP+ where the amide group is replaced by methyl ketone, nitrile or thioamide. The inhibition with analogues of NAD+ has been studied. The presence of a substituent at C-3 of the pyridinium ring is necessary for the binding. The inhibition by C-4 methylated analogues is very poor, and the effect of a methyl group at C-5 depends on the substituent at C-3. The 1,4,5,6-tetrahydronicotinamide adenine dinucleotide is a competitive inhibitor. Nicotinamide 8-bromoadenine dinucleotide and nicotinamide 8-thioadenine dinucleotide are efficient hydrogen acceptors.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.