Pyridoxal phosphate Volume

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. 

This chapter describes model studies of hydride transfer entirely with respect to nicotinamide coenzymes, flavin coenzymes and quinone coenzymes. Other coenzymes/cofactors may be alluded to but are not reviewed in detail. Some coenzymes involved either in hydride transfer or the transfer of other hydrogen species have been treated elsewhere in these volumes (thiamin diphosphate is treated by Hiibner et al., pyridoxal phosphate by Spies and Toney, folic acid by Benkovic... 

The distribution volume of intravenously administered pyridoxal phosphate is about twice the plasma volume (Lumeng et al, 914t). This could reflect binding to the walls of the vascular system or equilibration of protein bound pyridoxal phosphate with an interstitial pool. 

The more widespread route of catabolism could be via reaction 2. This is an extremely important reaction in higher plants as it is considered to be the primary source of CO2 in photorespiration (cf. Tolbert, this series, Vol. 2, Chapter 12 Keys, this volume. Chapter 9 Lorimer, Chapter 9, Vol. 8 this treatise). Reaction 2 can be carried out by particles from leaves of spinach (Kisaki et al., 1971) or tobacco (Bird et al., 1972a,b), and by pea cotyledon mitochondria (Clandinin and Cossins, 1975). There are some differences between the various systems in regard to the effect of oxygen and exogenous cofactors. The spinach and pea systems are stimulated by the addition of NAD, pyridoxal phosphate, and tetrahydrofolic acid. The tobacco leaf system does not require these exogenous cofactors however, electrons transported during the reaction can be linked to ATP synthesis with three ATP... 

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... 

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... 

Method. Solutions of amino acids in phosphate buffer (pH 9.3) are mixed with an equal volume of freshly prepared 0.4 M pyridoxal solution (adjusted to pH 9.3) and permitted to stand at 8 °C for 30 min. (The molar ratio of pyridoxal to amino acid should be >75 1.) At this point, 1 ml of sodium tetrahydroborate solution (100 mg/ml in 0.1 N sodium hydroxide) is added and the contents are gently shaken. Excess of sodium tetrahydroborate is destroyed by addition of sufficient hydrochloric acid (pH 1-2) prior to column chromatography. The pyridoxal derivatives are separated on a column (100 X 0.6 cm) of Aminex A-5 ion-exchange resin (Bio-Rad) at a mobile phase flow-rate of 33 ml/h. The eluting solvents consist of 0.2 N buffers at pH 3.40,4.44 and 4.86 and a 0.35 N buffer at pH 5.86 (all of the buffers are sodium citrate). The separation of a number of pyridoxyl-... 

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. 

Glutamic acid decarboxylase was assayed in a final volume of 100 / L containing 40 /tL of 200 mAf potassium phosphate buffer (pH 6.8), 10 / L of 5 mM L-glutamic acid, 5 / L of 0.2 mAf pyridoxal 5 -phosphate, 40 /tL of homogenate (2 mg protein/mL), and 1 fig of gabaquline (inhibitor of y-aminobutyric acid degradation). The reaction was stopped by the addition of 10 fiL of 100% trichloroacetic acid. After centrifugation, 5 fiL of standard 8-aminovaleric acid solution and 90 fiL of o-phthaldehyde solution (2 mg/mL 0.4 M borate buffer, pH 10.4) was added and the mixture was allowed to react for 3 to 5 minutes before injection of 20 fiL into the HPLC system. The reaction was linear for 20 minutes. 

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 10 mM glyoxylate, 80 mM L-alanine, 100 mM potassium phosphate buffer (pH 8.0), and 100 fiM pyridoxal-5 -phosphate in a final volume of 281 fiL. The reaction was initiated by adding alanine. After 30 minutes the reaction was stopped by mixing a 50 fiL aliquot of the incubated sample with 50 fiL of phenylhydrazine solution (1M in water) and 2.0 mL of water. After 15 minutes of incubation at room temperature, 50 fiL of the mixture was used for analysis by 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. 

Source Form Number of pyridoxal 5-phosphate groups Sedimentation coefiBcient, 20 (XlO") Diffusion coefficient, O20 (X 10 ) Partial specific volume, V Molecular weight p H 0 W w... 

The following procedure has been suggested. Amino acids and primary amines are dissolved in 0.5 M phosphate buffer, pH 9.3. This solution is mixed with the same volume of a freshly prepared 0.4 M pyridoxal solution in the same buffer (pyridoxal/amino acids = 5 1). 

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