Expression of Carbamoyl-phosphate Synthetase III mRNA during the Early Stages of Development and in Muscle of Adult Rainbow Trout (Oncorhynchus mykiss)

Muscle as the Primary Site of Urea Cycle Enzyme Activity in an Alkaline Lake-adapted Tilapia, Oreochromis alcalicus grahami

“…The high levels of all urea cycle enzymes in tilapia muscle reported here identify muscle as the likely major site for urea formation in this fish. This finding highlights the possible significance of recent reports of low levels of CPSase III and ornithine carbamoyltransferase activities in muscle of several other species of fish (12)(13)(14)(15). The presence in muscle of a urea cycle pathway that is normally expressed at high levels only under specific environmental (3)(4)(5)(7)(8)(9)(10)(11) or life cycle (12) circumstances may be a common characteristic of fish.…”

“…As shown in Fig. 1, a protein band with the same mobility as rat CPSase I can be readily detected in tilapia, but not in trout, which has very little CPSase III activity (12). The identity of this band as CPSase III was confirmed by showing that the sequence of the first 10 amino acid residues of the protein in this protein band was identical to the N-terminal sequence predicted for the mature protein after removal of the mitochondrial signal sequence as noted above, which also indicates that the enzyme is probably localized in the mitochondria.…”

“…Extracts for measuring units/gram of tissue of urea cycle enzymes in frozen samples (Table I) were prepared and assayed as described previously (7,12), except tissue samples were smaller (0.2 g), and the ATP-regenerating system was not used in the CPSase assay. Subcellular localization studies (Table II) of GSase, glutaminase, and arginase were conducted on-site using the isolation and assay methods previously described (7), except homogenates were centrifuged at 4°C at 82 ϫ g for 5 min to pellet the debris fraction and the resulting supernatant at 13,800 ϫ g for 5 min to pellet the mitochondrial fraction.…”

“…The sequence of the CPSase III cDNA was determined following previously described procedures for RNA isolation (from tilapia muscle), cDNA synthesis, analysis and isolation of PCR products, strategy for obtaining CPSase III-specific cDNA segments by PCR using consensus primers and sequence analysis of these segments, the strategy for sequencing the remainder of the CPSase III cDNA by primer walking, and sequence analysis (12,13). Numbering of amino acid residues of the deduced CPSase III amino acid sequence begins with the initial methionine residue of the entire translated product, i.e.…”

“…In liver of the two species of teleost fishes where expression of ureotelism has been well documented, CPSase III is localized exclusively in the mitochondrial matrix, as occurs in ureotelic terrestrial vertebrates, but in contrast to ureotelic terrestrial vertebrates, GSase is also localized entirely or partially in the mitochondria (7)(8)(9)(10)(11). CPSase III and ornithine carbamoyltransferase, which catalyzes the second step of the urea cycle, activities have recently been reported in muscle of several different species of teleostean fishes (12)(13)(14)(15), but the levels of CPSase III activity are extremely low (e.g. 0.5-2 nmol/min/g of muscle), and the physiological significance, if any, has not been established.…”