Pyridoxal phosphate assay

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

Table 6.2.3 Enzyme assay to assess AADC activity set-up and incubations. CEN Centrifuge, INC incubation, PCA perchloric acid, PLP pyridoxal phosphate...

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. 

The source of enzyme was a homogenate of rat brain prepared by sonication in 0.01 M 3-N-morpholinopropanesulfonic acid (pH 7.4). The homogenate was diluted 1 1 with buffer containing 4 mM pyridoxal phosphate, 0.6 M tetraethylamine, and 2 mAf 2-aminoethyl isothiomonium. The final suspension was treated with 0.4% Triton X-100 (final concentration), and the supernate obtained by centrifugation was used for assays. 

In contrast, when ATP is omitted and pyridoxal phosphate is added for the decarboxylase assay, the formation of GABA can be detected (Fig. 10.3). The activities, were prepared by homogenizing bovine brain. After centrifugation at lOOOg to remove debris, the supernatant solution was used directly for experiments. Bovine retinas were also prepared in the same manner. 

Some pyridoxal phosphate-dependent enzymes are normally fuUy saturated with cofactor and show the same activity on assay in vitro whether additional pyridoxal phosphate is present in the incubation medium or not. Examples of this class of enzymes include liver cysteine sulfinate decarboxylase (which is involved in the synthesis of taurine from cysteine Section 14.5.1) and the brain and liver glutamate and aspartate aminotransferases. 

A question of stability. Pyridoxal phosphate (PLP) is a coenzyme for the enzyme ornithine aminotransferase. The enzyme was purified from cells grovm in PLP-deficient media as well as from cells grown in media that contained pyridoxal phosphate. The stability of the enzyme was then measured by incubating the enzyme at 37°C and assaying for the amount of enzyme activity remaining. The following results were obtained. 

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-... 

Pyridoxal phosphate is a cofactor for both of the aspartate and alanine aminotransferases (AST ALT) and is often supplied as a separate component of reagent assay kits. The inclusion of pyridoxal phosphate can increase the measured activities of both aminotransferases activity by a small percentage (Stokol and Erb 1998), but this also can alter statistical differences observed between treatment groups in rat studies (Evans and Whitehorn 1995). 

Chabner, B., and Livingston, D., A simple enzymic assay for pyridoxal phosphate. Anal. Biochem. 34, 413--423 (1970). 

Multiple forms of cysteine synthase have been demonstrated in a number of plants. Two peaks of cysteine synthase were obtained by ammonium sulfate fractionation of acetone powders of spinach (Giovanelli and Mudd, 1968). A highly purified cysteine synthase from rape leaves was resolved into two fractions by polyacrylamide gel electrophoresis. The two had similar sizes and subunit compositions but one contained pyridoxal phosphate, whereas the other contained a derivative of pyridoxal phosphate (Masada et al., 1975). The electrophoretic mobility of the latter enzyme could be converted to that of the former by incubation with pyridoxal phosphate. Two forms of cysteine synthase have been extracted from both Phaseolus vulgaris and P. polyanthus (Table II) (BertagnoIIi and Wedding, 1977). It is unlikely that the multiple forms of the Phaseolus enzyme are due to modification of the pyridoxal phosphate prosthetic group, since pyridoxal phosphate was included in all media used for purification and assay of these forms. 

Direct fluorimetry of pyridoxal phosphate and pyridoxal is unreliable due to interfering fluorescent compounds, and thus sample cleanup prior to the assay is required. Oxidation of pyridoxal phosphate to pyridoxic acid phosphate by alkaline cyanide, or reaction with bisulfite or semicarbazide can enhance the fluorescence and hence the sensitivity of the assay. 

This enzyme is a pyridoxal phosphate protein which can also catalyse reactions with hydroxyphenylalanine and 3-hydroxyphenylserine. It catalyses the formation of dopamine which is the precursor of noradrenaline. It may be assayed radiochemically utilising the evolution of C labelled CO2. 

This enzyme is a pyridoxal phosphate protein, which catalyses the formation of 7-aminobutyric acid, a possible chemical transmitter in the CNS. It can be used in an indicator reaction for the radiochemical assay of transaminases. It is assayed radiochemically using Clabelled glutamate and COj evolution [391]. 

Bacterial Amino Acid Decarboxylases. Tyrosine decarboxylase was used extensively in studies on the nature of the amino acid decarboxylases. This activity is found in several microorganisms and the enzyme was purified over 100-fold from S. faecalis. It is easily resolved, and the apoenzyme has been used to assay pyridoxal phosphate (PALPO). Final concentrations of PALPO between 10 M and 10 M are conveniently measured by the rate of CO2 liberation from tyrosine. Phenylalanine is decarboxylated by this or a similar enzyme. Tyrosine decarboxylase and all of the other amino acid decarboxylases described in this section act only on the L-isomers of their respective substrates. 

Histidine decarboxylase from E. coli has a pH optimum near 4.0, while a similar enzyme assayed in Clostridium welchii has a pH optimum near 2.5. Extracts of acetone- or dioxane-treated C. welchii, however, have maximal rates of histidine decarboxylation at pH 4.5. The reason for the difference in pH optima of intact cells and extracts is conventionally attributed to pH effects on cell permeability, but other factors may be responsible. It is noteworthy in this connection that the Km of histidine is the same for both types of preparation. For years no cofactor could be found for this enzyme, but recently it was found that both pyridoxal phosphate and Fe+++ or Al are required. ... 

This quick assay is performed as follows (Verma et al., 1978) Retinoids and TPA are each dissolved in acetone and are applied separately to the shaved dorsal skin of mice in a volume of 0.2 ml. The mice are pretreated with the appropriate dose of retinoid 1 h before application of 17 nmol of TPA. The mice are sacrificed 4.5 h after dosing with TPA, which is the time of maximum ODC induction in the skin. The epidermis from individual mice is separated by brief heat treatment. The pooled epidermal preparations from three or four mice are homogenized in neutral phosphate buffer containing 0.1 mM pyridoxal phosphate and 0.1 n M EDTA and centrifuged briefly at high speed to obtain a clear extract. The ODC activity is determined on this extract by measuring the release... 

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. 

BE Haskell, EE Snell. An improved apotryptonase assay for pyridoxal phosphate. Anal Biochem 45 567-576, 1972. 

As with ADC, some interferences have been observed when ODC activity is measured, especially in crude or semicrude plant extracts (Birecka et al., 1985a). Some ways to prevent toe oxidative decarboxylation of ornithine by plant extracts in toe presence of pyridoxal phosphate have been reported (Smith and Marshall, 1988a- c). Ammonium sulfate precipitation of toe enzyme preparation and the presence in the enzyme assay of dithiothreitol and ethylenedia-minetetraacetic acid (EDTA) are important steps to be used to eliminate this nonenzymatic decarboxylation of ornithine. 

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). 

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