5'-Deoxypyridoxal interaction with dexamethasone receptor: a new probe for the structure and function of steroid receptors

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Abstract
5''-Deoxypyridoxal, a vitamin B-6 analog, increased the rate of dissociation of [3H]dexamethasone from [human cervical carcinoma] HeLa S3 cytoplasmic glucocorticoid receptor complexes in vitro. This effect was achieved at millimolar concentrations of 5''-deoxypyridoxal, suggesting a low-affinity interaction of 5''-deoxypyridoxal with receptor. Loss of [3H]dexamethasone-receptor binding in the presence of 5''-deoxypyridoxal was pH dependent, and a plot of Kd vs. pH fit a simple sigmoidal titration curve with an inflection point at pH 7.8, suggesting that deprotonation of a single functional group on 5''-deoxypyridoxal increases Kd. Loss of [3H]dexamethasone binding in the presence or absence of unlabeled steroid also increased with pH, but no inflection point occurred over the range of pH tested. A titration of 5''-deoxypyridoxal indicated a pK of 7.94 for the pyridinium proteon, suggesting deprotonation of the pyridinium nitrogen may account for the pH dependence of Kd of dexamethasone from receptor. 5''-Deoxypyridoxal also caused a decrease in nuclear [3H]dexamethasone-receptor binding when incubated with whole HeLa S3 cells at 37.degree. C. Furthermore, 5''-deoxypyridoxal was effective in reducing nuclear binding of dexamethasone when added either simultaneously with [3H]dexamethasone or after achievement of equilibrium of steroid with receptor. The reduction in nuclear [3H]dexamethasone binding is highly specific for 5''-deoxypyridoxal. Several analogs of this compound, including 5''-deoxypyridoxamine, were ineffective. In addition, this test was reversible following removal of extracellular 5''-deoxypyridoxal. Under these conditions, 5''-deoxypyridoxal was competitive with dexamethasone for binding to nuclear receptor, with inhibition constant = 8.1 .times. 10-6 M. Scatchard plot analysis of dexamethasone-receptor binding in the presence or absence of 5''-deoxypyridoxal was consistent with an apparent reduced affinity of [3H]dexamethasone for receptor, which again suggests competitive interaction or allosteric interaction mediated dissociation. Glucocorticoids are known to stimulate alkaline phosphatase activity within HeLa S3 cells. In whole cell incubations, 5''-deoxypyridoxal was effective in reducing the dexamethasone-induced increase in alkaline phosphatase activity by 60% under conditions in which cell viability and cell growth were not affected.