A New Diazirine for Protein Modification by Flash Photolysis: Comparison With an Azide

INTRODUCTLON 2, MATERIALS AND METHODSVoltage regulated L-type calcium channels are widely distributed in many excitable cells of tissues such as skeletal, cardiac, and smooth muscle or brain. These channels have high affinity binding sites for a variety of drugs, e.g. 1,4-dihydropyridines, phenylalkylamines, bcnzothiazepines, and diphenylbutylpiperidincs ctc, (see [I] for review). The 1,4-dihydropyridines (DHP) are among the most useful ligands to identify the Ltype calcium channels even in broken cell preparations, DHP binds to the a-1 subunit of the L-type channels [2]. Since the primary structures of cr-1 subunits from various tissues (skeletal, cardiac, and smooth muscle) are known [3-51, it is a nmjor goal to identify the DHP binding site(s) within the primary amino acid sequences, Photoaffinity labeling plays an essential role to achieve this goal. Previously we have reported several reagents [&?'I and toxin derivatives for specifically photolabeling the sodium channels [G-10]. Recently we have synthesized diazipine (Fig. l), a novel photoreactive derivative of DHP possessing a phenyldiazirine as a carbene precursor [ll]. In this paper we describe the properties of diazipine, in terms of binding and photolabeling of the a-1 subunit in purified preparation of caldium channels from rabbit skeletal muscles. By comparison with azidopine, the currently used photoreactive DHP [Ia] characteristics of diazipine in binding affinity, photoincorporation efficiency, and photoproduct stability will also be described. Bitrdirtg experinren tsInto the WGA-purified calcium channels (4~41: of protein) in 50 mM TrisXl (pH 7,4) containing 2 mM CaC12, IOOrM cisdiltiazcm, and 0.1% digitonin 'binding buffer', ("Hlcliazipine was added in various concentrations (l-80 nM) and final volumes were 5004, The mixture was incubated at 25% for I h in the dark, Two 100 cl aliquots of the incubates were loaded on a Scphadex G-50 (fine) column (2 ml) which was pre-equilibrated with the binding buffcr and gel-filtrated by ccntrifugation as described [l3]. Radioactivity of the filtrate was measured by scintillation counting, Binding in the presence of IO PM (&)PN200-1 IO was also carried out to measure non-specific binding, Duplicate runs were performed for each data point.For

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Chromogenic diazirine: A new spectrophotometric approach for photoaffinity labeling

Hatanaka

Yoshida

Nakayama

et al. 1989

Bioorganic Chemistry

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A New Diazirine for Protein Modification by Flash Photolysis: Comparison With an Azide

Cited by 3 publications

References 18 publications

Synthesis and Properties of Diazipine, a Novel Photoaffinity Probe for Calcium Channel-Linked Dihydropyridine Receptors

Synthesis and Properties of Diazipine, a Novel Photoaffinity Probe for Calcium Channel-Linked Dihydropyridine Receptors

Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels

Chromogenic diazirine: A new spectrophotometric approach for photoaffinity labeling

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