A Simple Method for the Preparation of D-α-Aminoadipic Acid

Abstract

High quantity (1 g and more) of racemically and chromatograph-ically pure D-α-aminoadipic acid was Drepared by selective metabolism of the L-isomer of the commercially available DL-α-aminoadi-pate by Pseudomonas putida. The overall yield of this preparation averaged 40%. The final product has a [a]²⁵ _D value of ?25°. This procedure can be useful in the synthesis of high purity D-α-amino-adipate, a compound shown recently to be a useful tool in the study of neurotransmission mechanism mediating synaptic excitation.     

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.     

Membrane phase state and the rearrangement of hematopoietic cell surface receptors.

Transformed murine hematopoietic cells of several lineages bound the fluorescent membrane probe merocyanine 540, whereas their normal counterparts did not. Similar selective binding was reproduced in artificial liposomes which bound this probe above their phase transition temperature, but not below it. The regions of the membrane to which merocyanine 540 binds along with the receptors for the lectin concanavalin A, but not the receptors for the lectin wheat germ agglutinin, were rearranged during the course of induced differentiation of erythroleukemia cells. Based on these findings, we propose a model of hematopoietic cell surface differentiation in which proteins such as concanavalin A receptors, which are destined for removal from the plasma membrane, are specifically associated with disordered, liquid-like lipid domains which can be visualized with merocyanine 540. For the specific case of erythroid differentiation, these domains and their associated proteins are collected at the region of the membrane where nuclear extrusion occurs and are eliminated from the reticulocyte plasma membrane by the enucleation event.     

Interactions of substrate and non-substrate effectors with p-hydroxybenzoate hydroxylase from pseudomonas fluorescens

3,4-Dihydroxybenzoate (3,4-DOHB), 2,4-dihydroxybenzoate (2,4-DOHB), and benzoate facilitate the interaction of p-hydroxybenzoate hydroxylase with TPNH. The two dihydroxybenzoate effectors form 1:1 complexes with the enzyme, inducing large spectral perturbations and fluorescence quenching. The dissociation constants for 2,4-DOHB and 3,4-DOHB are 0.15 and 0.50 mM respectively. During the reaction of enzyme with TPNH and oxygen, all the 2,4-DOHB, <5% of the benzoate, and none of the 3,4-DOHB is hydroxylated.