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The preparation and characterization of mono-iodinated photoreactive analogs of insulin
Photoreactive derivatives of insulin (B29-(p-azidobenzoyl-insulin) iodinated primarily in either the B26 or A14 tyrosine of insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The binding affinities and photoaffinity labeling characteristics of these derivatives were studied in isolated rat adipocytes. Under nonreducing conditions, three forms of the insulin receptor were labeled equally by the B26-derivative, the A14-derivative, and the mixture of the iodinated derivatives. When dithiothreitol was used to reduce the radiolabeled receptors, the radioactivity associated with the binding subunit was much less than that in the intact receptor and the magnitude of the decrease was proportional to the amount of iodine in the A chain of the photoderivatives. Use of the photoreactive derivative iodinated primarily in the B26 position resulted in greater labeling of insulin receptor subunits since most of the radioactivity (80%) remained associated with the receptor upon reduction.
Photoreactive derivatives of insulin (B29-(p-azidobenzoyl-insulin) iodinated primarily in either the B26 or A14 tyrosine of insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The binding affinities and photoaffinity labeling characteristics of these derivatives were studied in isolated rat adipocytes. Under nonreducing conditions, three forms of the insulin receptor were labeled equally by the B26-derivative, the A14-derivative, and the mixture of the iodinated derivatives. When dithiothreitol was used to reduce the radiolabeled receptors, the radioactivity associated with the binding subunit was much less than that in the intact receptor and the magnitude of the decrease was proportional to the amount of iodine in the A chain of the photoderivatives. Use of the photoreactive derivative iodinated primarily in the B26 position resulted in greater labeling of insulin receptor subunits since most of the radioactivity (80%) remained associated with the receptor upon reduction.