Search

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 approximately 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.

Female squirrelfish (Holocentridae) accumulate higher concentrations of hepatic zinc than any other known organism. In the liver cells, up to 70% of zinc is bound to metallothionein (MT), which is expressed at extremely high levels. These attributes are related to reproduction in ways that have not been fully characterized. In the present study, we have demonstrated that female-specific zinc and MT accumulation and distribution are strongly correlated to the onset of sexual maturity in Holocentrus adsenscionis. Sexual maturation not only resulted in increased concentrations of zinc in the liver and plasma, but also increased levels of hepatic MT mRNA. Furthermore, mature female squirrelfish exhibited greater proportions of MT protein in the nuclear liver cell fractions. To characterize the physiology further, we have examined the influence of the female sex hormone 17 beta-estradiol (E(2)). E(2) was not sufficient to elicit an increase in hepatic zinc concentrations or MT mRNA levels. E(2) administration did, however, result in increased levels of MT in the nuclear fraction as well as overall hepatic MT protein. E(2) also increased concentrations of zinc in the plasma. The changes in zinc concentration in the bloodstream followed the same time course as vitellogenin (VTG) transport from the liver. However, the high ratio of molar concentrations of zinc to VTG in the bloodstream suggest that VTG may not be the primary vehicle for hepato-ovarian zinc transport in squirrelfish.