Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio

Abstract

The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, a and ,B, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of a-and ,B-polypeptides and the isoenzymic pattem was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of jB-polypeptide and of the more cathodal isoenzymes, whereas a-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the a-and the catabolic reaction with the /i-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions.NAD(H)-GDH isoenzymes from a perennial plant species, grapevine ( Vitis vinifera L.). The amination reaction was fully activated by about 100 /M Ca2", whereas the deamination reaction was not affected by the addition of Ca2". We (1 1) also raised rabbit antiserum against leaf NAD(H)-GDH isoenzyme 1. The antiserum recognized the seven isoenzymes and precipitated all the enzyme activity from leaf, shoot, and root tissues. Protein blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kD, which was considered to correspond to the NAD(H)-GDH subunit.In this paper, we present evidence from more detailed electrophoretic studies suggesting that the seven grapevine GDH isoenzymes consist of two polypeptides with similar antigenic properties but with different molecular mass and charge; they occur in an ordered ratio and NADH-GDH/ NAD-GDH activity is regulated by the differential quantitative participation of the two polypeptides in the seven isoenzymes. GDH is a mitochondrial enzyme (15) and has been purified to homogeneity from a few plant species (6, 18,21). Recently, we (10) purified to homogeneity the major of the seven 'Abbreviation: GDH, glutamate dehydrogenase. MATERIALS AND METHODS 104Grapevine ( Vitis vinifera L. cv Sultanina) virus-free plants were grown in vitro and micropropagated by the one-node technique in Roubelakis medium (23). Leaf, shoot, and root tissues were used for enzymatic studies. In addition, green leaf, shoot, and root segments of0.5 to 1.0 cm were positioned for callogenesis on a modified Murashige and Skoog medium (17) supplemented with plant growth regulators at 2/5 ,M 6-benzoami...

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

“…Transfer of electrophoretically resolved proteins onto nitrocellulose filters (0.2-mm pore size; Schleicher and Schuell) was performed according to Loulakakis and Roubelakis-Angelakis (1990b). GDH isoenzymes were localized in nondenaturating polyacrylamide gels using the in situ staining technique as analytically described by Loulakakis and Roubelakis-Angelakis (1991).…”

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

“…The seven NAD(H)-GDH isoenzymes in Arabidopsis are composed of two subunits that combine in different ratios in a manner similar to those of grape (Loulakakis and Roubelakis-Angelakis, 1991) and avocado (Loulakakis et al, 1994) NAD(H)-GDH isoenzymes; however, the subunit makeup of the anodal and cathodal isoenzymes in Arabidopsis are the reverse of those of grape and avocado. In Arabidopsis the slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of a subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of /3 subunits.…”

“…Data from feeding studies using grape calli also suggest that NAD(H)-GDH may play a role in nitrogen metabolism. Native PAGE analyses of protein extracts from grape calli treated with ammonia or Gln contained NAD(H)-GDH isoenzymes of high electrophoretic mobility (Loulakakis and Roubelakis-Angelakis, 1991). Two-dimensional gel electrophoresis of these samples revealed that they were composed of a single type of subunit designated a.…”

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

“…In higher plants, GDH functions as a homohexamer or heterohexamer consisting of two subunits, a and b, that differ minimally in terms of their mass and charge. The a and b polypeptides combine in different ratios to form seven possible NADH-GDH isoenzymes in A. thaliana, N. plumbaginifolia or V. vinifera (Fontaine et al 2006;Loulakakis and Roubelakis-Angelakis 1991;Masclaux-Daubresse et al 2002). Only one isoform was found to be present in triticale, whereas L. luteus contains fourteen isoforms of this enzyme (Kwinta et al 2001;Ratajczak et al 1986).…”