Methods pyridoxal phosphate

The most widely used method of assessing vitamin Bg status is by the activation of erythrocyte aminotransferases by pyridoxal phosphate added in vitro, expressed as the activation coefficient. 

The leucocyte method estimates pyridoxal phosphate in isolated leucocytes it is based on a coenzyme-catalyzed tyrosine decarboxylase system from S. faecalis (B32). Enough data are not yet on hand to evaluate this method. The determination of circulating or available vitamin Ba should offer a more direct approach. 

This method does not seem to be applicable if R is aromatic, as would be necessary to make the arsonomethyl analogue of pyridoxal phosphate. Despite protection of groups capable of interference, the reaction proved impossible, and evidence of elimination of HBr with formation of R—CH=CH—COOH from R—CH2—CHBr—COOH in attempting step 6 was obtained (105). 

The current model for FeS cluster formation is based mainly on studies of S. cerevisiae, for which methods of genetic manipulation are well established (reviewed in Lill and Miihlenhoff 2005) (Fig. 6.3). The central pair of components of Fe cluster formation consist of mitochondrial IscS (Nfs), a pyridoxal phosphate-dependent desulfurase, and IscU (Isu), which serves as a scaffold for the formation of a transient FeS cluster. Sulfur released by IscS from cysteine is transferred to IscU and combined with iron to form a labile FeS... 

In conclusion, suicide enzyme inactivators offer a powerful method for the selective irreversible inhibition of enzymes. Although this review has concentrated on pyridoxal phosphate-dependent enzymes the approach is also valid for the irreversible inhibition of other types of enzymes (6 ) and may offer a means for the rational design of therapeutically-useful substances. 

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. 

As shown in Table 9.5, there are a number of indices of vitamin Be status available plasma concentrations of the vitamin, urinary excretion of 4-pyridoxic acid, activation of erythrocyte aminotransferases by pyridoxal phosphate added in vitro, and the ability to metabolize test doses of tryptophan and methionine. None is wholly satisfactory and where more than one index has been used in population studies, there is poor agreement between the different methods (Bender, 1989b Bates et al., 1999a). 

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-... 

Vitamin Bg status can be evaluated by direct measurement of plasma pyridoxine or pyridoxal phosphate by microbiological, enzymatic, radioimmunological, or chemical methods. Measurement of urinary xanthurenic acid or other intermediates of the kynurenine pathway (Chapter 17) are used to assess indirectly the adequacy of vitamin Bg for metabolic needs. 

Some results indicate that different attempts of FucA immobilization by covalent attachment provoked severe enzyme inactivation (Fessner and Walter 1997). FucA and DERA from E. coli and SHMT from Streptococcus thermophilus have been immobilized by multipoint covalent attachment to glyoxyl-agarose. Although this immobilization method had been very successful with many different enzymes (Guisdn et al. 1993), results obtained with these aldolases were dissimilar. For FucA, in spite of an immobilization yield of 80-90%, enzyme inactivation occurred during immobilization process and only 10-20% of activity was retained (Suau et al. 2005). On the other hand, SHMT immobilization yield was 100%, but the immobilized activity was lost during the sodium borohydride reduction step, probably due to the reduction of the Schiff base established between the cofactor (pyridoxal phosphate) and the aldolase. Finally, 100% of immobilization yield and 65% of retained activity in the immobilized derivative was achieved with DERA. 

In the course of testing our chromatographic method for measuring vitamin B6 on a variety of samples, we happened to include goat milk, which contains a very high concentration of pyridoxal phosphate (Coburn and Mahuren, 1983). We then noticed that bovine milk contained about the same total vitamin B-6 content but had a lower concentration of pyridoxal phosphate. Further study revealed a strong inverse relationship between the pyridoxal phosphate content of milk and the alkaline phosphatase... 

The immunochemical method was based on the precipitation of apoenzyme or holoenzyme With antibodies produced after immunization by holoenzyme. The amount of precipitated protein was higher in the case of holoenzyme than in the case of apoenzyme. These workers concluded that the binding of pyridoxal phosphate had induced changes in the secondary and tertiary structures of aspartate aminotransferase. 

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. 

The most widely used method of assessing vitamin B status is by the activation of erythrocyte transaminases by pyridoxal phosphate added in vitro, expressed as the activation coefficient (section 11.6.4.1). The ability to metabolize a test dose of tryptophan (section 11.9.5.1) or methionine (section 11.9.5.2) has also been used. 

A Hamfelt. A simplified method for determination of pyridoxal phosphate in biological samples. Uppsala J Med Sci 91 105-109, 1986. 

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