|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta)
1 NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School
of Marine and Atmospheric Science, University of Miami, Miami, FL 33149,
USA
2 Joint Genome Institute, Genomic Diversity Laboratory, Walnut Creek, CA
94598, USA
3 Department of Biochemistry and Microbiology, University of Victoria,
Victoria, BC, Canada
* Author for correspondence (e-mail: pwalsh{at}rsmas.miami.edu)
Accepted 21 January 2003
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 mass-specific GSase activities rank in 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
post-translational 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.
Key words: glutamine synthetase, toadfish, ureotely, ornithine–urea cycle, ammonia detoxification, Opsanus beta, ammonia excretion, gill, ORF, UTR
Related articles in JEB:
This article has been cited by other articles:
|
|
 |
|
  P. A. Wright, S. L. Steele, A. Huitema, and N. J. Bernier Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia J. Exp. Biol., August 15, 2007; 210(16): 2905 - 2911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Buckley, A. Y. Gracey, and G. N. Somero The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis J. Exp. Biol., July 15, 2006; 209(14): 2660 - 2677. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Essex-Fraser, S. L. Steele, N. J. Bernier, B. W. Murray, E. D. Stevens, and P. A. Wright Expression of Four Glutamine Synthetase Genes in the Early Stages of Development of Rainbow Trout (Oncorhynchus mykiss) in Relationship to Nitrogen Excretion J. Biol. Chem., May 27, 2005; 280(21): 20268 - 20273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Barimo, S. L. Steele, P. A. Wright, and P. J. Walsh Dogmas and controversies in the handling of nitrogenous wastes: Ureotely and ammonia tolerance in early life stages of the gulf toadfish, Opsanus beta J. Exp. Biol., May 15, 2004; 207(12): 2011 - 2020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Murray, E. R. Busby, T. P. Mommsen, and P. A. Wright Evolution of glutamine synthetase in vertebrates: multiple glutamine synthetase genes expressed in rainbow trout (Oncorhynchus mykiss) J. Exp. Biol., May 1, 2003; 206(9): 1511 - 1521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Phillips FISH FIND A FROG IN THE FAMILY J. Exp. Biol., May 1, 2003; 206(9): 1429 - 1430. [Full Text] [PDF]
|
|
