Properties of an Aminotransferase of Pea (Pisum sativum L.)

Abstract

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purfied from shoot tips of pea (Pisum sativum L. cv. Transaminases (aminotransferases, EC 2.6. 1), particularly those of plants, are quite incompletely described in regard to their amino acid and keto acid specificities. Some partially purified preparations are highly specific for particular amino acids (1, 2, 5, 30). Other preparations reportedly transaminate a variety of amino acids (7,8,12,15,25,29). Many workers have concentrated on transamination of aromatic amino acids. Two transaminase fractions have been isolated from rat brain, one of which showed highest activity toward tyrosine and phenylalanine and the other of which showed highest activity toward tryptophan and 5-hydroxytryptophan (4, 6, 26). Partially purified tryptophan transaminase from the bacterium Clostridium sporogenes catalyzed the transamination of tyrosine and phenylalanine, as well as tryptophan (23). A transaminase fraction from mung bean (Phaseolus aureus) also transaminated tryptophan, phenylalanine, and tyrosine, as well as several aliphatic amino acids (7,8).The purpose of these investigations was to purify partially and ascertain some properties of a transaminase fraction from pea shoots and to attempt to corroborate the evidence reported by Moore (20) and kept under a 16-hr photoperiod at 20 + 1 C with a light intensity of 900 to 1000 ft-c and an 8-hr dark period at 16 + 1 C. After 10 to 14 days, 50 to 70 g of shoot tips were removed immediately above the fifth node and placed in liquid nitrogen for overnight storage prior to use.Preparation of Enzyme Extract. Each sample of shoot tips was homogenized in a chilled mortar and pestle with 2 ml of 0.5 M borate buffer (pH 8.5), containing 150 ltM chloramphenicol and 1 mm dithiothreitol, per g fresh weight of tissue. The homogenate then was centrifuged at 40,000g for 10 min at 4 C, and the resulting supernatant was the crude enzyme extract. The extract was then brought to 25% (v/v) acetone with the addition of ice-cold acetone. After setting in an ice bath for 10 min, the 25% (v/v) acetone-crude extract was centrifuged at 10,000g for 10 min. The supernatant was then brought to 60% (v/v) acetone, allowed to set for 10 min in an ice bath and then was centrifuged at 10,000g for 10 min. The resultant supernatant was discarded, and the pellet was resuspended in 0.5 M borate buffer (pH 8.5) in a total of one-sixth of the original volume of crude extract. This suspension was then centrifuged at 40,000g for 10 min, and the resulting goldyellow colored supernatant was used as the acetone-precipitated enzyme fraction. In some studies, the acetone-precipitated enzyme was added to a column of hydroxylapatite (1.6 x 17 cm) which was previously equilibrated with 0.02 M KH,PO4-Na2HPO, buffer at pH 8.5. The column was then washed with 70 ml of 0.05 M sodium-potassium phosphate buffer (pH 8.5) and 50 ml of 0.08 M sodium-potassium phosphate buffer (pH 8.5). The enzyme was then eluted with 0.15 M sodium-potassium phosphate buffer...

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

“…However, the enzyme activity was lost upon attempted further purification. The characteristics found for this enzyme match the findings for tryptophan aminotransferase in other plants (10,12,19 this enzyme is clearly present in all species examined and it can be concluded that in these species of Nicotiana, tryptophan aminotransferase, indoleacetaldehyde oxidase, and indoleacetaldehyde reductase are gene products ofpotentially crucial importance in the metabolic pathway leading from tryptophan to IAA. Conversely, it appears from the results obtained here that tryptophan decarboxylase and tryptamine are not significantly involved in this pathway.…”

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

“…The in vitro enzyme assay using recombinant TAA1 was performed according to the previously described borate buffer assay (Matheron and Moore, ; Tao et al ., ) with minor modifications. The reaction mixture was prepared in 500 μl of 500 m m borate buffer (pH 8.5) containing 300 μ m l ‐Trp, 1 m m sodium pyruvate, 10 μ m pyridoxal phosphate (PLP), 1 μg of purified TAA1 recombinant protein and the test compound at 30 μ m .…”

Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM

“…The tryptophan aminotransferase activity was determined by the previously described method (Matheron and Moore 1973; Khandaswami and Vaidyanathan 1973) with some modifications. The final assay concentration contained 0·5 ml of enzyme extract and 2·5 ml of 0·5 mol borate buffer (pH 8·5) containing 0·1 μmol of pyridoxal phosphate, 40 μmol of l ‐tryptophan and 20 μmol of α‐ketoglutarate.…”