The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

Tbe enzymic basis for the flow of .ltrgeu from arginie to e during the first 3 days of gIlnaon has bed In extracts from cotton (Goss_ypm hnm) cotyledons. Eviene that a synthetase reguates n accuma t (for tsrt to the axis) Is Further, eviene that the bWuk of the passed from one generatio to the next In dicots through a cycle Invdvi the fIn seq ee r arg storae protein -arginine -* asparaIe s discussed.Data on the pathway of ammonia assimilation in nodules of leguminous roots and green leaves have been accumulating during the past several years, two review articles appearing recently (19,20). In green leaves, ammonia from nitrate assimilation serves as a nitrogen source for amino acid synthesis, whereas in root nodules, ammonia produced by nitrogen fixation serves as a nitrogen source. In both cases, much ofthe carbon skeletons for amino acid synthesis presumably comes as a result of photosynthesis. The situation is likely to be different in dicot seeds, such as legumes and cotton, that do not depend on external nitrogen sources. The storage proteins and starch or lipid satisfy their nutritional needs at least in the early days of germination. In cotton, amino acids and carbon skeletons released by the hydrolysis of the stored material appear to go through active interconversions as indicated by the lack of correlation between the amino acid composition of the storage proteins (13) and that of the free amino acid content of germinating cotyledons (4). The predominant nitrogen storage unit in the cotton storage protein is arginine which contains at least 25% of the total stored nitrogen (13), whereas most of the nitrogen transported from cotyledons to the growing axis is by way of asparagine (4). It seems that a major pathway of nitrogen metabolism in germinating cotyledons lies in the transfer of nitrogen from ARG' to ASN. Furthermore, we have observed that cotyledons germinated in actinomycin D (Act D) do not accumulate ASN to the extent that untreated cotyledons do, but rather accumulate massive amounts of glutamine (4). This suggests that nitrogen for the build-up of ASN may pass through glutamine and that the enzyme(s) responsible for synthesis of ASN at the expense of glutamine may be derived from mRNA newly synthesized in germination.Against this background we have examined the levels during germination ofactivities in crude extracts ofcotyledons ofenzymes 'Abbreviations: Act D: actinomycin D; GDH: glutamate dehydrogenase; GS: glutamine synthetase; GOGAT: glutamate synthase; AS: asparagine synthetase; ASN: asparagine; ARG: arginine.likely to be involved in the transfer of nitrogen from ARG to ASN, and have examined the effects of Act D and cycloheximide on some of these activity levels.Arginase (EC 3.5.3.1; L-argimne amidinohydrolase; reaction 1) and urease (EC 3.5.1.5; urea amidohydrolase; reaction 2) activities were assayed to establish the possible pathway for the release of ammonia from arginine of the storage protein. purchased from Morrow Bros., Farmington, Georgia, were germinated in the dar...

Section: Resultsmentioning

confidence: 57%

Developmental Biochemistry of Cotton Seed Embryogenesis and Germination

Dilworth¹,

Dure²

1978

“…However, some GDH activity was also detected in the mitochondrial fraction. The presence of GDH in higher plant mitochondria has been well documented by several workers (4,9,17,26,27,29), and the fact that a fraction of GDH in SB3 cells could be mitochondrial cannot be ruled out at present. However, the results presented here suggest that a major fraction of GDH is of plastidial origin.…”

Section: Discussionmentioning

confidence: 89%

A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

Bhadula

Shargool²

1991

The subcellular distribution of L-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and 35S-methionine. Using antibodies raised against purified GDH, we identified a 35S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.

“…The mitochondrion may be expected to feature prominently as a location for some of the enzyme whose total activities were monitored in this study (5,8,13,15).…”

Section: Discussionmentioning

confidence: 94%

“…8). Although the action of GDH to form glutamate in plant cells has been discounted recently by some authors (13), the development of GDH activities in cotyledon cells is itself evidence of some function, and amination is the thermodynamically favored direction at physiological pH (5,25,28 assimilating NH4' in a number of microorganisms (e.g. ref.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

Murray¹,

Kennedy²

1980

MATERIALS AND METHODSIn the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: a-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.Changes in the activities of alanine: a-ketoglutarate aminotransferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.13) have also been measured, in cotyledons as well as seedcoats. On a fresh weight basis, the highest activities of asparaginase and both aminotransferases developed in the seedcoats, whereas the highest activities of the remaining enzymes developed in the cotyledons.The data indicate that the amide groups of imported asparagine and glutamine are metabolized differently, largely by asparaginase and glutamate synthase, respectively. The NH4' released by the action of asparaginase is evidently reassimilated in cotyledon cells by the joint action of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. The data emphasize the central importance of a-ketoglutarate-glutamate cycling in the redistribution of amino groups associated with the net synthesis of amino acids and reserve proteins. PLANT MATERIAL AND PREPARATION OF EXTRACTSPlants of tall (cv. Telephone) and dwarf (cv. Melbourne Market) varieties of garden pea, P. sativum L., were grown, respectively, in the open garden or in pots of garden soil. Flowers were tagged at full blossom and pods then were removed at intervals throughout development, which occurred in spring (October, November) for cv. Melbourne Market and from November to mid-December (1979) for cv. Telephone.The procedures for extraction of separated seedcoat and cotyledon samples were as described (17, 18), with the sample size increased to four or five seeds. The extraction medium consisted of 25 mm K+-phosphate (pH 7.5), 0.5 mm 2-mercaptoethanol and 0.03% (v/v) Triton X-100. After centrifugation (17), duplicate aliquots were removed for treatment with ethanol, for the determination of protein content (ethanol-insoluble) and a-amino group content (ethanol-soluble) as previously described (16, 17).Measured portions of each clarified extract (1.0-1.5 ml) were passed through columns of Sephadex G-25 (0.9 x 25 cm) equilibrated with extraction medium to obtain enzyme solutions free of NH4, and amino acids. Complete removal of ethanol-soluble, ninhydrin-positive metabolites from the excluded fractions was confirmed by analysis (17).A number of recent studies have attempted to elucidate the nutritive and regulatory influences that the living seedcoats exert upon the embryo during its development (4,(17)(18)(19). Lacking the phenolic compounds that hinder the extraction of proteins from the seedcoats of many other species, developing pea seeds (Pisum sativum L.) offer an ideal system in wh...