Logo of plntphysLink to Publisher's site
Plant Physiol. Aug 1996; 111(4): 1169–1175.
PMCID: PMC160993

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Abstract

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.

Full Text

The Full Text of this article is available as a PDF (2.2M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Amy NK, Garrett RH. Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana. Plant Physiol. 1974 Oct;54(4):629–637. [PMC free article] [PubMed]
  • Beudeker RF, Tabita FR. Characterization of glutamine synthetase isoforms from chlorella. Plant Physiol. 1985 Apr;77(4):791–794. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Brown JR, Masuchi Y, Robb FT, Doolittle WF. Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. J Mol Evol. 1994 Jun;38(6):566–576. [PubMed]
  • Caizzi R, Bozzetti MP, Caggese C, Ritossa F. Homologous nuclear genes encode cytoplasmic and mitochondrial glutamine synthetase in Drosophila melanogaster. J Mol Biol. 1990 Mar 5;212(1):17–26. [PubMed]
  • Darrow RA, Knotts RR. Two forms of glutamine synthetase in free-living root-nodule bacteria. Biochem Biophys Res Commun. 1977 Sep 23;78(2):554–559. [PubMed]
  • Gao Y, Smith GJ, Alberte RS. Nitrate Reductase from the Marine Diatom Skeletonema costatum (Biochemical and Immunological Characterization). Plant Physiol. 1993 Dec;103(4):1437–1445. [PMC free article] [PubMed]
  • Garcia-Fernandez JM, Lopez-Ruiz A, Toribio F, Roldan JM, Diez J. Occurrence of Only One Form of Glutamine Synthetase in the Green Alga Monoraphidium braunii. Plant Physiol. 1994 Feb;104(2):425–430. [PMC free article] [PubMed]
  • Gassmann M, Thömmes P, Weiser T, Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. [PubMed]
  • Goodman HJ, Woods DR. Cloning and nucleotide sequence of the Butyrivibrio fibrisolvens gene encoding a type III glutamine synthetase. J Gen Microbiol. 1993 Jul;139(7):1487–1493. [PubMed]
  • Hirel B, Gadal P. Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES. Plant Physiol. 1980 Oct;66(4):619–623. [PMC free article] [PubMed]
  • Magasanik B. Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilization genes. Trends Biochem Sci. 1988 Dec;13(12):475–479. [PubMed]
  • McNally SF, Hirel B, Gadal P, Mann AF, Stewart GR. Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf. Plant Physiol. 1983 May;72(1):22–25. [PMC free article] [PubMed]
  • Mitchell AP, Magasanik B. Purification and properties of glutamine synthetase from Saccharomyces cerevisiae. J Biol Chem. 1983 Jan 10;258(1):119–124. [PubMed]
  • Nozaki Y, Schechter NM, Reynolds JA, Tanford C. Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry. 1976 Aug 24;15(17):3884–3890. [PubMed]
  • Orr J, Haselkorn R. Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120. J Biol Chem. 1981 Dec 25;256(24):13099–13104. [PubMed]
  • Orr J, Haselkorn R. Regulation of glutamine synthetase activity and synthesis in free-living and symbiotic Anabaena spp. J Bacteriol. 1982 Nov;152(2):626–635. [PMC free article] [PubMed]
  • Ramalho CB, Hastings JW, Colepicolo P. Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels. Plant Physiol. 1995 Jan;107(1):225–231. [PMC free article] [PubMed]
  • Rasulov AS, Evstigneeva ZG, Kretovich VL, Stel'mashchuk V Ia, Samsonidze TG, Kiselev NA. Ochistka, svoistva i chetvertichnaia struktura glutaminsintetazy Khlorelly. Biokhimiia. 1977 Feb;42(2):350–358. [PubMed]
  • Reyes JC, Florencio FJ. A mutant lacking the glutamine synthetase gene (glnA) is impaired in the regulation of the nitrate assimilation system in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol. 1994 Dec;176(24):7516–7523. [PMC free article] [PubMed]
  • Sivasankar S, Oaks A. Regulation of Nitrate Reductase during Early Seedling Growth (A Role for Asparagine and Glutamine). Plant Physiol. 1995 Apr;107(4):1225–1231. [PMC free article] [PubMed]
  • Southern JA, Parker JR, Woods DR. Expression and purification of glutamine synthetase cloned from Bacteroides fragilis. J Gen Microbiol. 1986 Oct;132(10):2827–2835. [PubMed]
  • Sumar N, Casselton PJ, McNally SF, Stewart GR. Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri. Plant Physiol. 1984 Feb;74(2):204–207. [PMC free article] [PubMed]
  • Tréguer P, Nelson DM, Van Bennekom AJ, Demaster DJ, Leynaert A, Quéguiner B. The silica balance in the world ocean: a reestimate. Science. 1995 Apr 21;268(5209):375–379. [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...