Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine beta-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5'-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5'-phosphate fell by >95%, and saturation of cystathionine beta-synthase apoenzyme with pyridoxal 5'-phosphate decreased from a predepletion value of 70% to <10%. When such depleted cells were grown in media containing pyridoxal concentrations ranging from 0 to 1,000 ng/ml, cellular pyridoxal 5'-phosphate reached a maximum of 30 ng/mg cell protein at a medium pyridoxal concentration of 100 ng/ml. Maximal saturation of aposynthase with coenzyme in control cells was reached at a medium pyridoxal concentration of 10 ng/ml. In contrast, maximal saturation of residual aposynthase in cells from an in vivo responsive patient was achieved at a medium pyridoxal concentration of 25-50 ng/ml, whereas that from cells from an in vivo unresponsive patient was reached at 100 ng/ml. Estimates of the affinity of control and mutant cystathionine beta-synthase for pyridoxal 5'-phosphate in cell extracts supported the differences observed in intact cells. The apparent K(m) of cystathionine beta-synthase for pyridoxal 5'-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the K(m) for pyridoxal 5'-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine beta-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5'-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5'-phosphate.

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Affinity of cystathionine beta-synthase for pyridoxal 5'-phosphate in cultured cells. A mechanism for pyridoxine-responsive homocystinuria.

The pyridoxal phosphate contents of several enzymes have been compared both by fluorometry and by absorption spectrophotometry of the phenylhydrazone, and give results in good agreement. The fluorometric method is as rapid and simple as the spectrophotometric method and in addition is about as sensitive as the best biological assay methods. The characteristic excitation and emission spectra also permit qualitative identification of the coenzyme.

Fluorometric determination of pyridoxal phosphate in enzymes.

Previous work supporting the vitamin A dependency of adrenal function in rats neglected to take into account a secondary effect of the deficiency, a decrease in hepatic ascorbic acid biosynthesis. Vitamin A-depleted rats maintained on a diet free of ascorbate had a decrease in the activity of adrenal 3 beta-hydroxysteroid dehydrogenase, and extensive adrenocortical degeneration. The use of an ascorbate supplement prevented the symptoms. The results suggest that previous evidence for direct involvement of vitamin A in steroidogenesis may have been due to the production of a secondary deficiency, a chronic scorbutic condition.

https://science.sciencemag.org/content/sci/191/4226/472.full.pdf

Vitamin A: not required for adrenal steroidogenesis in rats

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label

Depletion of cultured human fibroblasts of pyridoxal 5′-phospate: Effect on activities of aspartate aminotransferase, alanine aminotransferase, and cystathionine β-synthase

Fluorometric determination of pyridoxal phosphate in microgram samples of tissue

A microfluorometric adaptation of the spectrophotometric method for determination of isocitric dehydrogenase activity was developed for use with μg samples of tissue, e.g., microtome sections. The method was based on measurement of NADPH formed when NADP oxidizes isocitrate. A study was carried out of the quantitative histological distribution of the enzyme in the rat adrenal, and the influence of single subcutaneous injections of ACTH insaline solution (25 mg/kg), as well as of injections (10 mg/rat) twice daily for 5 days of ACTH in gel. These treatments had little effect on the activity or its localization, which was maximal in the reticularis and the fascicular-reticular border zone. The relationship of the isocitric dehydrogenase activity to formation of NADPH for steroid biosynthesis in the adrenal was considered. (Endocrinology 83: 596, 1968)

Quantitative histological distribution of isocitric dehydrogenase in the rat adrenal with effects of adrenocorticotropin, and determination of the enzyme in microgram samples of tissue.

Kenneth M. Grigor

Papers

Fluorometric determination of pyridoxal phosphate in microgram samples of tissue

Quantitative histological distribution of isocitric dehydrogenase in the rat adrenal with effects of adrenocorticotropin, and determination of the enzyme in microgram samples of tissue.