Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Abstract

Glutamine synthetase from bean nodules can be separated into two isoforms, GS,, and GSn2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, DyeMatrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GSn1 and GSn2 have been purified to homogeneity. Subunit structure analysis using twodimensional polyacrylamide gel electrophoresis revealed that GSnl was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GSn2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GSnl. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GSn2 was but one of the isozymes, and GSn1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric. GS2 (EC 6.3.1.2) catalyzes the formation ofglutamine from substrates glutamate, ammonia, and ATP. This enzyme and GOGAT (EC 1.4.1.14) coupled together form the GOGAT cycle, which is considered the major ammonia assimilation pathway in most higher plant tissues (17). In legume root nodules, ammonia produced by N2 fixation in the bacteroids is excreted into the nodule cytosol (9), where it is assimilated into glutamate by the GOGAT cycle (19).GS has been extensively studied because of its importance in plant nitrogen metabolism. It has been purified and characterized from root nodules of soybeans (15) that is found in roots. The physical and kinetic properties of GS., and GSn2 are similar (3). The holoenzyme of both isoforms have identical mol wt (380,000 D), and are both comprised of eight subunit polypeptides of 41,000 D. Lara et al. (13) reported that GS., and GSn2 are comprised of two different types of subunit polypeptides (,3 and y). The polypeptides have identical mol wt but different isoelectric points.In an organ such as the bean nodule, there are two different types of GS subunit polypeptides (13). Since the GS holoenzyme consists of eight polypeptides (3), it is not known whether the eight subunit polypeptides of the holoenzyme must be identical (i.e. homo-octamer), or they can be different (i.e. hetero-octamer). Earlier, we have shown that after GS,, and GS.2 had been separated by native PAGE and the gel had been stained with Coomassie blue, we could detect six to nine protein bands in the GS,, region and one protein band in GSn2 (23). W suggested that these proteins bands are individual isozymes of the GS isoforms. That is, the isoform GS., may be composed of six to nine hetero-and homo-octameric isozyme...

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells

“…The diversity in the structure of GSCyt from various species is now well documented. In bean root nodules, for instance, among the eight GS,,, isoforms identified, one is a homooctameric isozyme, whereas the others are made up by different ratios of two distinct polypeptide subunits (Cai and Wong, 1989). In Hevea, whether the two cytosolic polypeptides observed are different subunits of the same holoenzyme or two distinct isozymes is still to be determined.…”

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L.

“…GSni is a composite of eight isozymes comprised of and ,y subunits (4,15). Only the ,8-subunit is present in GSn2.…”

“…Subunit structure of GS,, and GSn2 was studied extensively by Cai and Wong (4). Both forms of GS are octamers.…”

Antipeptide Antibodies That Can Distinguish Specific Subunit Polypeptides of Glutamine Synthetase from Bean (Phaseolus vulgaris L.)

“…The subunit polypeptides are of two major types (designated (3 and y) (6,13). The (3 and y polypeptides have similar mol wts but different isoelectric points (3,13).…”