An NADP-dependent Glutamate Dehydrogenase in Chloroplasts from the Marine Green Alga <i>Caulerpa simpliciuscula</i>

Gayler, Kenwyn R.; Morgan, W. R.

doi:10.1104/pp.58.3.283

Cited by 42 publications

(21 citation statements)

References 21 publications

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“…Although the P. laminosum enzyme was purified more than 4,400-fold, the preparation was not homogeneous when analyzed by polyacrylamide gel electrophoresis, indicating that this protein was present in a very low concentration even in ammonium-grown cells. However, the purified enzymes from other sources showed lower specific activities (between 3.5 and 8.5 U/mg of protein) (11,17) and were less highly purified (78-and 6-fold, respectively). Our enzyme recovery compares to that of the enzyme from the fungus Cenococcum graniforme (25) but is much lower than that of Chlorella spp.…”

Section: Discussionmentioning

confidence: 92%

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

et al. 1988

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The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-flxing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30°C it showed a specific activity of 32.9 gimol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (K,m = 1.25 mM) and NADPH (Km = 64 ,uM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [SJ0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60°C (Ea = 5.1 kcal/ mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

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Section: Discussionmentioning

confidence: 92%

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

et al. 1988

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“…This may apply in the case of the alga Caulerpa sirnpliciuscula (Gayler and Morgan, 1976;McKenzie et al. 1979).…”

Section: Discussionmentioning

confidence: 99%

“…Cells were har-(1976) and Turpin and Harrison (1978) further support vested during the exponential phase of growth except this view. Thus while GDH activity might be of prime in the case of Isochrysis galbana in which senescent importance in nitrogen assimilation in organisms such cells were assayed because they exhibited greater as Caulerpa simpliciuscula (Gayler and Morgan, 1976; levels of GS activity. McKenzie et al, 1979) and in Stichococcus bacillaris (Everest and Syrett, in press), it appears to play only a secondary role in marine phytoplankton.…”

Section: Methodsmentioning

confidence: 99%

Detection of glutamine synthetase activity in marine phytoplankton: optimization of the biosynthetic assay

Bressler¹,

Ahmed²

1984

Mar. Ecol. Prog. Ser.

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A comprehensive study of the key ammonium-assimilating enzyme, glutarnine synthetase (GS), was made in a variety of marine phytoplankton. GS activity was detected in 13 out of the 15 species examined comprising 6 algal classes. Both the biosynthetic GS reaction as well as the transferase reaction were measured with phytoplankton extracts for comparative purposes. In evaluating the differences between the 2 assays it was found that the biosynthetic assay was more sensitive than the transferase assay in terms of final product formed/assay. The transferase assay yielded a more stable final product but the reaction is not specific for GS (Meister, 1974). A series of kinetic studies on the GS enzyme were carried out on a number of marine species. These studies included the determination of the pH optimum, linearity of the reaction with time, apparent energies of activation (E,) and apparent Y, values for the substrates: NH,', glutamate and ATP. In addition, the optimum requirement for the divalent cation, Mgf + was ascertained. The most significant finding of these studies was the broad distribution of GS activity in marine phytoplankton. The apparent K, values for ammonium were in the micromolar range. This is in marked contrast to earlier observations of the requirement of glutamate dehydrogenase (GDH), an alternate ammonium-assimilating enzyme, which has been shown to have a K, for ammonium ions in the millimolar range. These data support the view that GS is the primary ammonium-assimilating enzyme in phytoplankton found in areas of intermittent nitrogen supply.

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“…It had been previously shown that C. simplicwscula chloroplasts contained an NADP-dependent glutamate dehydrogenase with a low Km for ammonia (5). This unusual glutamate dehydrogenase has now been shown to be augmented by similar levels of the conventional glutamine synthetase and GOGAT.…”

Section: Discussionmentioning

confidence: 89%

Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

McKenzie¹,

Ch'Ng²,

Gayler³

1979

Plant Physiol.

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The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways.Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 jumoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):a-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 pmoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low Km for ammonia which has been described previously in these chloroplasts provides the second potential pathway.Chloroplasts isolated from siphonous green algae have been shown recently to synthesize a different, and often wider, array of photosynthetic products from ''CO2 than higher plant chloroplasts (7,19). In this they resemble the chloroplasts isolated from Acetabularia where a similar range of products is synthesized by the isolated organelle (17). In this paper the studies of the metabolic competence of chloroplasts from siphonous green algae are extended to include the pathways of assimilation of ammonium into amino acids.In chloroplasts of higher plants ammonium is incorporated into amino acids via the two enzymic steps, glutamine synthetase (EC 6.3.1.2) followed by GOGAT2 (Jo). It has been suggested that the situation in chloroplasts isolated from the marine alga Caulerpa simpliciuscula is different, since they have been found to contain high levels of an NADP-dependent glutamate dehydrogenase, with an abnormally low apparent Km for ammonia of 0.4 to 0.7 mM (5). This enzyme could provide an alternative means of ammonium incorporation into amino acids in these algae.GOGAT activity has been demonstrated both in photosynthetic organisms which are more advanced in evolution than C. simpliciuscula and also in those which are more primitive (10, 11). We would expect that GOGAT activity should also be present in C. simpliciuscula unless this species represents a radical departure in evolution from other members of the plant kingdom. Here, we show that the chloroplasts of this siphonous green alga contain the enzymic capacities for both pathways of ammonium assimilation into amino acids. MATERIALS AND METHODSPlant Material. C. simpliciuscula plants were gathered at a depth of 1 to 2 m from the sea at Williamstown, Victoria and held in an illuminated tank of aerated and recirculating seawater on a 12-h light/12-h dark cycle at 16 C.Chloroplast Isolation. The chloroplast fraction was prepared from fronds of C. simpliciuscula by the method described prev...

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An NADP-dependent Glutamate Dehydrogenase in Chloroplasts from the Marine Green Alga Caulerpa simpliciuscula

Cited by 42 publications

References 21 publications

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Detection of glutamine synthetase activity in marine phytoplankton: optimization of the biosynthetic assay

Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

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