Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate.
The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.
D000085:Acetates / Q000494:pharmacology; D000328:Adult; D000368:Aged; D001051:Apoenzymes; D001059:Apoproteins; D002021:Buffers; D002474:Cell-Free System; D002951:Citrates / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008875:Middle Aged; D011732:Pyridoxal Phosphate / Q000097:blood*; D013997:Time Factors; D014445:Tyrosine Decarboxylase
D000085:Acetates; D001051:Apoenzymes; D001059:Apoproteins; D002021:Buffers; D002951:Citrates; D011732:Pyridoxal Phosphate; D014445:Tyrosine Decarboxylase