Pyridoxal-5 -phosphate assay methods

Tryptophanase catalyzes the conversion of tryptophan to indole and acetic acid. Pyridoxal phosphate is a required cofactor. The HPLC method developed to assay this activity involves the separation of the tryptophan from the indole. 

Decarboxylation of amino acids is a typical feature of the bacterial decomposition of proteins. Both phenylethylamine and tyramine were isolated from putrid meat by Barger and Walpole (30), who considered it extremely probable that they were derived from phenylalanine and tyrosine, respectively. No cell-free preparation of phenylalanine decarboxylase appears to have been reported, but decarboxylation by a crude Streptococcus faecalis preparation provides a valuable method of phenylalanine assay (887). Bacterial tyrosine decarboxylase has been studied in detail (495), especially by Gale and co-workers (summarized in 284). It requires pyridoxal phosphate as coenzyme (26, 326, 327) and, unlike mammalian tyrosine decarboxylase, also attacks dihydroxyphenylalanine. Decarboxylation normally only occurs in acid media and is considered primarily to be a protective mechanism tending to restore the pH to neu-... 

The best method for the assay of pyridoxal phosphate is the use of tyrosine decarboxylase as described by Gunsalus, Bellamy, and Umbreit. The enzyme is prepared from a dried powder of cells of S. faecalis R. which has been grown deficient in vitamin Be by growth In a vitamin-Be-free alanine-rich medium. Thus, the decarboxylase is obtained almost completely resolved. This is a convenient preparation, since such a powder is stable for long periods and since the resolution of transaminases, decarboxylases, and tryptophanases isolated from tissues is a rather difficult task. The assay is performed manometrically by measuring the rate of CO2 liberation from tyrosine by the dried powder in the presence of pyridoxal phosphate. The rate of CO2 evolution is a function of the concentration of pyridoxal phosphate. 

This scant information about aminopropyltransferases is due in part to the difficulty of measuring their activities, since one of the precursors, dSAM, is unstable and not easily available. Although its synthesis has been improved, an extinction coefficient has not been published (Samejima et al., 1978). We have developed a method for the evaluation of spermidine synthase in oat leaves (Tiburcio et al., 1986a) by a coupled reaction without using dSAM. We add SAM, [ ] putrescine, and pyridoxal phosphate to the assay mixture. Incorporation of the label into spermidine can be detected after 45 min of incubation at 37 C. Labeled spermidine is separated from labeled putrescine or spermine by elution with HCl in Dowex 50 W-H" " columns (Tiburcio et al., 1986a). A similar procedure can be used for the evaluation of spermine synthase (A. F. Tiburcio et al., unpublished observations). 

Wada and Snell (150) have developed a method for the assay of pyridoxine and pyridoxamine phosphate, based on enzymatic oxidation to pyridoxal which is allowed to react with phenylhydrazine. 

Potassium cyanide catalyzed oxidation of PLP with subsequent quantification of the highly fluorescent oxidation product, 4-pyridoxic acid-5 -phosphate, has been used to determine PLP levels in human plasma, brain tissue and cell cultures (74,126,127). The sensitivity of this method has been reported to be even better than methods based on PLP semicarbazone formation (127). PL is not determined by these methods this vitamer is oxidized to 4-pyridoxic acid lactone, which shows little fluorescence at pH 2.5 to 3.8 of the mobile phases used (Table 2). Hess and Vuilleumier (128) overcame this problem by alkaline delactonization of pyridoxic acid lactone to yield the highly fluorescent 4-PA. The determination of PL as 4-PA suffers from the disadvantage that each sample has to be analyzed twice. In the first analysis, cyanide oxidation is omitted and the basal 4-PA concentration is measured. In the second analysis, PL is converted to 4-PA as described. The concentration of PL is determined from the difference between the two assays for 4-PA. 

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