A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta)

Patrick J. Walsh, Gregory D. Mayer, Mónica Medina, Matthew L. Bernstein, John F. Barimo, Thomas P. Mommsen

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

We characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in tissues of the gulf toadfish Opsanus beta subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that massspecific GSase activities rankin the order of brain > liver > stomach ≈ kidney > intestine > gill > heart/spleen > muscle. When tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine, with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity increases significantly only in liver (fivefold) and muscle (twofold), tissues that previously showed significant expression of the other enzymes of urea synthesis. Western analyses of samples on SDS gels demonstrated that GSase-specific protein content reflected enzyme activity, and all tissues except muscle had a single, similarly sized GSase subunit of 49.4 kDa; muscle showed staining of two bands of 36.8 and 98.9 kDa, which may possibly result from another gene product or posttranslational modification. RT-PCR and RACE-PCR revealed the presence of a second GSase cDNA from gill tissue that shares only 73% nucleotide and amino acid sequence similarity with the GSase cDNA previously cloned from liver, and that lacks a mitochondrial leader-targeting sequence. RT-PCR and restriction digestion experiments demonstrated that mRNA from the original 'liver' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new 'gill' form shows expression primarily in the gill. Gill GSase activity shows apparently exclusive expression in the soluble compartment, while other tissues expressing the 'liver' form show both cytoplasmic and mitochondrial activities. Phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade containing the toadfish liver GSase and other described teleost GSase genes. The results are discussed in the context of a prior hypothesis on an ammonia-trapping mechanism in the gill of the toadfish.

Original languageEnglish (US)
Pages (from-to)1523-1533
Number of pages11
JournalJournal of Experimental Biology
Volume206
Issue number9
DOIs
StatePublished - May 1 2003

Fingerprint

Batrachoidiformes
Glutamate-Ammonia Ligase
glutamate-ammonia ligase
gills
Gene Expression
liver
muscle
gene
Liver
Batrachoididae
Muscles
genes
Intestines
muscles
Stomach
stomach
intestines
muscle tissues
Polymerase Chain Reaction
Genes

All Science Journal Classification (ASJC) codes

  • Ecology, Evolution, Behavior and Systematics
  • Physiology
  • Aquatic Science
  • Animal Science and Zoology
  • Molecular Biology
  • Insect Science

Cite this

Walsh, Patrick J. ; Mayer, Gregory D. ; Medina, Mónica ; Bernstein, Matthew L. ; Barimo, John F. ; Mommsen, Thomas P. / A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta). In: Journal of Experimental Biology. 2003 ; Vol. 206, No. 9. pp. 1523-1533.
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A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta). / Walsh, Patrick J.; Mayer, Gregory D.; Medina, Mónica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

In: Journal of Experimental Biology, Vol. 206, No. 9, 01.05.2003, p. 1523-1533.

Research output: Contribution to journalArticle

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T1 - A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta)

AU - Walsh, Patrick J.

AU - Mayer, Gregory D.

AU - Medina, Mónica

AU - Bernstein, Matthew L.

AU - Barimo, John F.

AU - Mommsen, Thomas P.

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N2 - We characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in tissues of the gulf toadfish Opsanus beta subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that massspecific GSase activities rankin the order of brain > liver > stomach ≈ kidney > intestine > gill > heart/spleen > muscle. When tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine, with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity increases significantly only in liver (fivefold) and muscle (twofold), tissues that previously showed significant expression of the other enzymes of urea synthesis. Western analyses of samples on SDS gels demonstrated that GSase-specific protein content reflected enzyme activity, and all tissues except muscle had a single, similarly sized GSase subunit of 49.4 kDa; muscle showed staining of two bands of 36.8 and 98.9 kDa, which may possibly result from another gene product or posttranslational modification. RT-PCR and RACE-PCR revealed the presence of a second GSase cDNA from gill tissue that shares only 73% nucleotide and amino acid sequence similarity with the GSase cDNA previously cloned from liver, and that lacks a mitochondrial leader-targeting sequence. RT-PCR and restriction digestion experiments demonstrated that mRNA from the original 'liver' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new 'gill' form shows expression primarily in the gill. Gill GSase activity shows apparently exclusive expression in the soluble compartment, while other tissues expressing the 'liver' form show both cytoplasmic and mitochondrial activities. Phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade containing the toadfish liver GSase and other described teleost GSase genes. The results are discussed in the context of a prior hypothesis on an ammonia-trapping mechanism in the gill of the toadfish.

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