An improved method for the determination of pyridoxal-5-phosphate using tyrosine decarboxylase is described.Pyridoxal-5-phosphate (PLP) is the coenzyme form of vitamin B6 for a multiplicity of biosynthetic and catabolic enzyme systems. The methods generally used for the determination of PLP in biological samples are either enzymatic or chemical procedures. The method described here is basically the same as other enzymatic procedures (Bhagavan et al., 1976;Chabner and Livingston, 1970;Reinken, 1972), using radioactive tyrosine and the apoenzyme, tyrosine decarboxylase from Streptococcusfaecalis. However, instead of collecting ]4CO2, the other product of the enzymatic reaction, [3H]tyramine, was determined after separation from the substrate, [3H]tyrosine, by a mini DEAE-cellulose column.The assay mixture for PLP measurement contained 0.1 mol/l sodium acetate buffer, pH 5.5, the enzyme, tyrosine decarboxylase (approximately 20mU), PLP (10-300pg) and [aH]tyrosine (0.5~tCi/0.25pmol) in a total volume of 0.3 ml. The reaction mixture was preincubated in the absence of tyrosine for 20 min to allow the formation of the holo enzyme. The reaction was then initiated by addition of tyrosine, incubated for 15 min at 37°C, and terminated by heating the mixture for 3 min at 95 °C. After cooling the mixture in an ice bath, 100 lal of the supernatant was loaded on to a mini DEAEcellulose column (0.9 x 1.0cm) prepared with a 2.5 ml disposable syringe. The preparation of the OH form of
The developmental patterns of four lysosomal enzymes have been investigated in liver, kidney, lung, heart, spleen, muscle and brain tissues of human fetuses at various gestational ages. The largest increment in the activity of all four enzymes, namely acid α-glucosidase, α-galactosidase, β-galactosidase and acid phosphatase has been observed in kidney with a 6- to 12-fold increase between the second and third trimester of gestation. The activity of all liver and spleen enzymes also increased considerably during these periods. In muscle, however, only α-glucosidase and acid phosphatase showed an increase in the activity, and in lung, acid phosphatase and β-galactosidase. Most of brain and heart enzymes, except acid phosphatase, did not change significantly during the observation period. The activities of these lysosomal enzymes were also measured in tissues of a normal adult individual, and aspects of the neonatal and postnatal development of these enzymes were discussed.
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