Volume pyridoxal phosphate enzymes

The standard incubation mixture contained 0.2 Af phosphate buffer (pH 7.5), 0.02 m Af pyridoxal phosphate, 0.1 mAf patgyline, 0.2 mAf L-dihydroxyphe-nylalanine, 0.1 mAf 5-hydroxytryptophan, and enzyme in a total volume of 200 fiL. After incubation at 37°C for 30 minutes, the reactions were terminated by addition of 800 fiL of chilled 0.1 Af perchloric acid containing 0.1 mAf sodium metabisulfite and 0.2 mAf EDTA. After centrifugation, 10 fiL aliquots were used for HPLC analysis. 

We have emphasized before that only some pointers to the solution of problems in steady state kinetics will be given in this volume. To conclude this summary some aspects of enzyme reactions involving two substrates will be discussed. First, two conventions must be mentioned. In many reactions involving HjO, H", or OH the concentrations of water and its ions are not considered stoichiometrically. The ionic concentrations are taken into account in terms of rapid equilibria (see sections 3.4 and 6.4). The distinction between substrates, coenzymes and prosthetic groups may not always be a sharp one. We shall treat coenzymes (NAD", NADH, ATP, etc.) as a second substrate. The term arises from the fact that, unlike other substrates, coenzymes are continuously recycled. The difference between coenzymes and prosthetic groups (biotin, riboflavin, pyridoxal phosphate, etc.) is that the latter are more or less firmly attached to the active site of the enzyme the lifetime of the complex is very long compared... 

Ornithine Decarboxylase Assays. The double-chamber assay system of Moskal and Basu (59) was used to measure enzyme activity in the form of L C] carbon dioxIHe evolution. The assay conditions of O Brien and Diamond (60) were used and consisted of the following components (in micromoles, unless otherwise stated) in a total volume of 100 jul sodium phosphate buffer, pH 7.2, 5.0 EDTA, 1.0 dithiothreitol, 5.0 pyridoxal-5 -monophosphate, 0.2 L-ornithine (specific activity 0.5 x 10° cpm/-jumole), 0.1 and protein, 0.1-0.5 mg. Incubations were carried out at 37°C for 60 min, and the reactions were terminated by the addition of 200 jul of 2M sodium citrate followed by a post-incubation period of 3 hours at 37°C to insure maximal release of radiolabeled carbon dioxide. 

The reaction mixture contained 5 mAf L-glutamate and 0.5 to 10 mU of enzyme in 100 mAf sodium phosphate (pH 7.2) containing 0.1 mAf pyridoxal 5 -phosphate and 1 mAf S-2-aminoethylisothiouronium bromide. The reaction was started by adding 5 to 10 pL of enzyme solution to give a final volume of 50 pL. Aliquots of 10 pL were removed at 0,10, and 20 minutes and mixed with 10 pL of prechilled 0.2 Af perchloric acid. After centrifugation, 10 pL of the supernate was mixed with 190 pL of the HPLC mobile phase HPLC... 

The reaction mixture contained in a final volume of 1 mL 20 mAf sodium phosphate buffer (pH 5.0), 2.5 mAf dithiothreitol, 0.1 mAf pyridoxal 5 -phosphate, 0.4 /xCi L-[2,3-3H]ornithine, 100 mAf ornithine hydrochloride, and 0.3 mL of either E. coli ornithine decarboxylase or tissue homogenate. The E. coli enzyme was assayed at pH 5.0 and 37°C the pH values of reaction mixtures were adjusted to pH 7.3 before assaying homogenates of mammalian tissues. The reaction was stopped by injection onto the column. 

---