Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias).

C A Casey,P M Anderson

doi:10.1016/s0021-9258(18)34352-7

Abstract

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).

Highlights

0.6 M as a mechanism of osmoregulationin a saltwater related to a role in urea synthesis from glutamine
The metabolic pathway for urea synthesis in elasmobranchs is apparently analogous to the pathw(ai.ye
Ammonia generated inside the mitochondria is detoxified by conversion to citrulline, which exits the mitochondria and ultimatelygives rise to urea, which is excreted. This intramitochondrial detoxication is accomplished as a result of the specificlocation of ammoniaandacetylglutamate-dependentcarbamoyl-phosphatesynthetase and ornithine carbamoyltransferase in the mitochondrial matrix; the other threeenzymes of the urea cycle, argininosuccinate synthetase, argininosuccinate lyase, and arginase, are located in the cytosol

Summary

Cycle Enzymes in SpiDnyogfish

The washed pellets were resuspended in 5 ml of fractionation activity of the mitochondrial enzymes of interest was assayed as buffer for use in subsequent experiments. The presence of nuclei and mitochondria in the respective subcellular fractions was verified by electron microscopy (Fig. l),by staining with fluorescent dyes specific for DNA or for viable mitochondria, and by the presence of the classical mitochondrial marker enzymes, glutamate dehydrogenase and cytochrome oxidase (Table I). The results of both electron (Fig. 1) and fluorescent microscopy showed that nuclei were virtually absent from the mitochondrial fraction, but, as obthis enzyme, as described by Webb and Brown [8].

Glutamine Synthetase and Urea Cycle Enzymes in Spiny Dogfish

Findings

Argininosuccinate lyase