subcellular location of glutamine synthetase and urea cycle

THE .JOURNAL OF BIOLOGICAL CHEMISTRY

Prcnted m U S.A. Vol. 257, No. 14, Issue of July 25. pp. 8449-8453, 1982

Subcellular Location of Glutamine Synthetase and Urea Cycle Enzymes in Liver of Spiny Dogfish (SquaZus acanthias)*

(Received for publication, December 14, 1981, and in revised form, February 5, 1982)

Carol A. Casey and Paul M. Anderson From the Department of Biochemistry, School of Medicine, University of Minnesota, Duluth, Duluth, Minnesota 55812

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 man- tkias. 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 K,,, for arginine is 1.2 m, and the enzyme has an M, = 100,000).

Marine elasmobranchs (sharks, skates, and rays) retain urea in their tissues and body fluids at concentrations as high as 0.6 M as a mechanism of osmoregulation in a salt water environment (1,Z). The metabolic pathway for urea synthesis in elasmobranchs is apparently analogous to the pathway (i.e. the urea cycle) in mammalian, ureotelic species (3, 4). The rate of incorporation of [14C]bicarbonate into urea in uiuo, or in tissue slices, and the tissue activities of the five urea cycle enzymes, all of which have been shown to be present, are sufficient to account for the necessary rate of daily urea production (4-6). In contrast to mammalian species, however, little is known about the properties, regulation, and metabolic interrelationships of the urea cycle in elasmobranchs, or of the enzymes involved. Since the function is related to osmoregulation and maintenance of a high urea concentration instead of (or in addition to) control of the rate of removal of nitrogen waste as urea, it might be expected that the urea cycle in elasmobranchs would have unique properties different from those which have been observed for the urea cycle in ureotelic species.

Recent studies in our laboratory have established that high levels of a glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase are present in liver of marine elas-

* This work was supported by Grant PCM-78 24130 from the National Science Foundation. A preliminary report of a portion of this work was presented at the 179th Annual Meeting of the American Chemical Society, Division of Biological Chemistry, August 1981 (Abstr. 134). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

mobranchs (6). The properties of this enzyme from the marine elasmobranch, Squalus acanthias, are similar to those of the classical ammonia- and acetylglutamate-dependent carbamoyl-phosphate synthetase which catalyzes the fist reaction of the urea cycle in mammalian, ureotelic species, except that glutamine is the nitrogen-donating substrate instead of ammonia (7). Webb and Brown recently reported that the levels of glutamine synthetase are also unusually high in liver of urea-retaining elasmobranchs (8, 9). On the basis of these observations, it has been suggested that the function of the unique glutamine-dependent carbamoyl-phosphate synthetase in elasmobranchs is to catalyze the first reaction of the urea cycle (7-9). If this is correct, then carbamoyl-phosphate and, consequently, one of the nitrogen atoms of urea, would be derived directly from glutamine rather than from ammonia, as occurs in mammalian, ureotelic species (7).

The present study was undertaken for the purpose of estab- lishing whether or not the subcellular distribution of urea cycle and related enzymes in a representative elasmobranch, S. acanthias (spiny dogfish), is the same as that for ureotelic species. The results are consistent with the view that the occurrence of high levels of both glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase in these ureoosmotic species is uniquely related to a role in urea synthesis from glutamine.

EXPERIMENTAL PROCEDURES

These studies were carried out at Friday Harbor Laboratories, University of Washington. Spiny dogfish were captured in the waters near San Juan Island by trawling at low speed. The animals were held until needed in a large circular holding tank provided with a constant supply of fresh seawater. [14C]Bicarbonate and t-[guanido- I4C]arginine were obtained from Research Products, International Corp.

Subcellular Fractionation-Freshly excised liver (3 g) from spiny dogfish was minced and suspended in 30 ml of fractionation buffer (0.25 M sucrose, 1 mM EDTA, 0.3 M urea, 0.15 M trimethylamine oxide, 0.15 M KCI, 20 mM Hepesl buffer, pH 7.5). The suspension was homogenized using a motor-driven Potter Elvehjem homogenizer with a loose fitting Teflon pestle; homogenization was accomplished using four strokes at 325 rpm. The homogenate was poured through six layers of coarse cheesecloth and centrifuged at 40 X g for 10 min to remove unbroken cells and other debris. The supernatant, subse- quently referred to as the extract, was removed by siphoning from the small amount of pellet at the bottom of the tube and the relatively large fat pad at the top of the tube. The extract was centrifuged at 250 X g for 10 min to sediment the nuclei. The supernatant was carefully removed from the resulting pellet (the nuclear fraction) by siphoning and was centrifuged at 15,000 X g for 10 min to give a well defined and fwm pellet (the mitochondrial fraction). The supernatant from this centrifugation is referred to as the soluble fraction. The nuclear and mitochondrial pellets were each washed once by gently resuspending the pellets in 10 ml of fractionation medium, followed by centrifugation at 250 X g and 15,000 X g, respectively, for 10 min.

’ The abbreviation used is: Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid.

8449

8450 Glutamine Synthetase and Urea Cycle Enzymes in Spiny Dogfish

The washed pellets were then resuspended in 5 ml of fractionation buffer for use in subsequent experiments. All steps of the subcellular fractionation scheme were carried out at 4 "C.

Electron and Fluorescent Microscopy-Preparation of samples from the nuclear and mitochondrial fractions for electron microscopy was performed as follows. The nuclei and/or mitochondria in an aliquot of the suspended particulate fractions were fixed by suspension in 3% glutaraldehyde in fractionation buffer and postfixed by suspension in 1% osmium tetroxide in fractionation buffer. The fixed pellets were then dehydrated by sequential ethanol washes and embedded in Epon-Araldite. Thin sections (70 nm) were cut, stained with 2% uranyl acetate for 2 min followed by 0.2% lead citrate for 2 min, and examined with a transmission electron microscope.

The presence of nuclei and/or mitochondria in the nuclear and mitochondrial fractions was also verified by use of two fluorescent dyes, Compound Hoechst 33258, a fluorescent stain specific for DNA (lo), and rhodamine 123, a fluorescent vital dye specific for viable mitochondria (11). A drop of the respective dye (5 pg/ml of fractionation buffer) was added directly to a drop of the suspended particulate fraction on a microscope slide in the dark. The stained subcellular particles were examined by epifluorescent illumination at 485 nm (fluorescein excitation) on a Zeiss photomicroscope.

Enzyme and Protein Assays-Prior to enzyme assay, the protein in the subcellular fractions was equilibrated with 0.05 M potassium phosphate buffer, pH 7.5, by passing 2-ml aliquots of the fractions through small columns (1 X 12 cm) of Sephadex G-25 equilibrated with the phosphate buffer at 4 "C; nuclear and mitochondrial proteins in the nuclear and mitochondrial fractions, respectively, were solubi- lized before this equilibration by brief sonication. All assays were carried out in duplicate at 26 "C. Spectrophotometric measurements were made with a Beckman DBGT spectrophotometer equipped with a Beckman 10-inch recorder. A unit of enzyme activity is defined as 1 pmol of product formed/min. Protein was measured by the dye- binding technique described by Bradford (12), using human y-globulin as the standard protein.

Glutamate dehydrogenase activity was determined by measuring the rate of oxidation of NADH at 340 nm in 0.05 M potassium phosphate buffer, pH 7.5, as described by Olson and Anfinsen (13). Cytochrome oxidase activity was determined by measuring the rate of oxidation of reduced cytochrome c a t 550 nm in 0.01 M potassium phosphate buffer, pH 7.0, as described by Wharton and Tzagoloff (14) using an extinction coefficient of 19.6 X lo3 M-' (15); cytochrome c was reduced prior to use by reaction with dithionite. Lactate dehydrogenase activity was determined by measuring the rate of oxidation of NADH at 340 nm, as described by Bergmeyer et al. (16). Glutamine synthetase activity was assayed by measuring the rate of formation of y-glutamyl hydroxamate in the transferase reaction catalyzed by this enzyme, as described by Webb and Brown (8). Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase activity was determined by measuring the rate of ['4C]carbamylphosphate formation from ['4C]bicarbonate, as described by Anderson (7). Or- nithine carbamoyltransferase activity was determined by measuring the micromoles of citrulline formed after 10 min by the method of Yashphe (17); the reaction mixtures contained ornithine (10 mM), lithium carbamyl phosphate (10 mM), potassium phosphate buffer (40 mM, pH 7.5), and an appropriate amount of each subcellular fraction in a final volume of 1 ml. Argininosuccinate synthetase was assayed by measuring the disappearance of citrulline, as described by Wixom et al. (18). The reaction mixtures contained ATP (5 mM), MgCL (6 m ~ ) , aspartate (5 m), citrulline (5 mM), Hepes buffer (50 mM, pH 7.5), and an appropriate amount of each subcellular fraction. A small aliquot (25 pl) was removed at 30-min intervals up to 2 h and added to 2 ml of potassium phosphate buffer (0.02 M, pH 7.8) containing 6 units of urease. After 10 min at 26 "C, the micromoles of citrulline were determined as described by Yashphe (17). Argininosuccinate lyase was assayed as described by Weiss and Davis (19). Arginase activity was determined by measuring the rate of formation of [''C] urea from ~-[guanido-'~C]arginine as described by Ruegg and Russell (20). Catalase was assayed as described by Luck (21).

Density Gradient Equilibrium Centrifugation of Mitochondria- An 0.5-ml aliquot (containing 2 mg of protein) of the suspended mitochondrial fraction in fractionation buffer was placed on top of a 4.7-ml 30-60% sorbitol gradient containing Hepes buffer (0.01 M, pH 7.6), KC1 (0.01 M), and EDTA (1 mM). The sample was centrifuged at 35,000 rpm in a Beckman SW 39L rotor for 120 min at 4 "C. The gradient was collected in 0.3-ml fractions to which were added 0.3 ml of a solution containing Hepes buffer (0.05 M, pH 7.6), KC1 (0.05 M), and Triton X-100 (0.5%) to solubilize the mitochondrial enzymes. The

activity of the mitochondrial enzymes of interest was assayed as described above.

Characterization of Arginase-Extracts containing arginase were prepared from pellets of the washed mitochondria1 fraction which had been stored at -70 "C. The packed mitochondria (0.25 cc) were suspended in 4.75 ml of 0.05 M potassium phosphate buffer, pH 8.0, at 4 "C and subjected to sonication for a short period of time. The protein concentration of the extract was about 4 mg/ml. Aliquots of the extract prepared in this way were used for the following experiments.

The K, for arginine was determined from the linear double recip- rocal plots of velocity versus concentration of [I4C]arginine. Arginase activity was determined as described above, using 1 pg of protein; under these conditions, less than 2% of the substrate was converted to product in each reaction mixture.

The sedimentation coefficient of arginase was determined by sucrose density gradient centrifugation, as described by Martin and Ames (22) and as discussed by O'Brien et al. (23). The linear sucrose gradients (5-20% sucrose in 0.1 M glycine buffer, pH 9.7) were prepared in a total volume of 4.9 ml. Centrifugation was carried out at 45,000 rpm (100,000 X g) in a Beckman SW 50.1 rotor for 8 h at 4 "C. The sample added to the top of the gradient consisted of 0.1 ml of the arginase-containing extract to which had been added an appropriate quantity of lactate dehydrogenase ( ~ 2 0 , ~ = 7.0) and catalase (SZO,~ = 11.3) which served as the standard reference proteins. After centrifugation, the gradients were collected in fractions of 0.18 ml, and the location of the three proteins was established by appropriate enzyme activity measurements.

Estimation of the molecular size of arginase by gel filtration chromatography was carried out by the method of Andrews (24) as previously described (7) , using Sephadex G-200 in a column (2.5 X 97 cm) equilibrated with 0.1 M potassium phosphate buffer, pH 7.6. The 2-ml sample added to the column contained 0.4 mg of protein from the arginase-containing extract and reference proteins as previously described (7).

RESULTS AND DISCUSSION

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 observed in other studies employing similar fractionation schemes (25-27), the nuclear fraction was contaminated with mitochondria. About 20-25% of the mitochondrial marker enzymes were consistently found in the nuclear fraction (Ta- ble I). Very little activity of either of the two mitochondrial enzymes was present in the soluble fraction, while virtually all lactate dehydrogenase activity, as would be expected, was present in the soluble fraction. We have observed that the isolated mitochondria are capable of synthesizing ['4C]citrulline from [14C]bicarbonate in the presence of succinate, glutamate, and ornithine at a rate equivalent to the maximum rate which could be achieved as calculated on the basis of the units of glutamine-dependent carbamoyl-phosphate synthetase present in the mitochondria.' This observation, together with the fact that the isolated mitochondria exhibited sub- stantial fluorescence in the presence of tbe fluorescent dye rhodamine 123, provides evidence that this subcellular fractionation scheme yields intact and viable mitochondria.

The results in Table I show that glutamine synthetase, the two enzymes catalyzing the first two steps of the urea cycle (carbamoyl-phosphate synthetase and ornithine carbamoyltransferase, respectively), and the enzyme which catalyzes the last step of the urea cycle (arginase) are mitochondrial enzymes. The distribution between the nuclear and mitochondrial fractions for all four of these enzymes is the same as that

* C. Casey and P. M. Anderson, unpublished experiments.

Glutamine Synthetase and Urea Cycle Enzymes in Spiny Dogfish 845 1

obtained with the mitochondrial enzyme markers. Additional evidence for the association of these four enzymes with mitochondria was obtained by subjecting an aliquot of the mitochondrial fraction to equilibrium density gradient centrifuga-

. tion on a 30-60% sorbitol gradient in a medium of relatively high ionic strength (Fig. 2) (19, 28). The distribution of these

4 enzymes after centrifugation was identical with the distribu- . , tion of the mitochondrial marker enzymes, all of which banded

at a density of 1.2, indicating the association of all six enzymes ' . with a common organelle. There was no evidence of significant

dissociation of any of these enzymes from the mitochondria . ~ during centrifugation. Arginase activity could not be solubi-

lized by repeated washing of the mitochondria with 0.15 M KC1, 0.15 M KC1 plus 2 mM CaC12, or 0.15 M KC1 plus 2 mM MgC12, suggesting that the association of arginase with the

€7 mitochondria is probably not an artifact due to nonspecific adsorption of arginase to mitochondria, which has been ob-

' served at low ionic strength in other species (29-31). The other two urea cycle enzymes, argininosuccinate synthetase and argininosuccinate lyase, are located in the soluble fraction; these enzyme activities were not present in the small pellet (presumably the microsomal fraction) obtained after centrif-

-: ' ugation of the soluble fraction at 105,000 X g for 1 h. The tissue activities of the urea cycle enzymes are lower than those

. . normally found in mammalian ureotelic liver, but the relative .. . - -9 . 1

h ' a-

FIG. 1. Representative electron micrographs of the nuclear (A) and mitochondrial (B) fractions.

TABLE I Summary of subcellular distribution of glutamine synthetase, urea

cycle enzymes, and marker enzymes in spiny dogfish liver Subcellular fractionation and enzyme assays were carried out as

described in the text.

protein

Glutamate dehydrogenase

7.8 71.4 20.7 0.3 Carbamoyl-phosphate syn- 7.0 66.7 26.2 10.8 Arginase 7.5 65.9 26.6 20.2 Glutamine synthetase

97.3 2.0 17.3 j 0.7 Lactate dehydrogenase 2.0 75.5 2.1 22.5 Cytochrome oxidase 3.3 ' 70.3 17.1 ' 26.3

Ornithine carbamoyltrans- 3.3 21.5 73.2 5.3

Argininosuccinate synthe- 0.6 0.6

98.0 1.0 0.9 0.7 Argininosuccinate lyase

97.2 2.2

Protein 100 11.8 22.7 65.5 " Units are expressed as micromoles/min for enzyme ac

I' Glutamine- and acetylglutamate-dependent.

thetase"

ferase

tase

as milligrams for protein. t

106 62 97 87 69 82

90

84

93 88

ivity and

ratios &e similar (32). In mammalian ureotelic species, ammonia generated inside

the mitochondria is detoxified by conversion to citrulline, which then exits the mitochondria and ultimately gives rise to urea, which is excreted. This intramitochondrial detoxication is accomplished as a result of the specific location of ammonia- and acetylglutamate-dependent carbamoyl-phosphate synthetase and ornithine carbamoyltransferase in the mitochondrial matrix; the other three enzymes of the urea cycle, argininosuccinate synthetase, argininosuccinate lyase, and arginase, are located in the cytosol. Vorhaben and Campbell (25) have shown that glutamine synthetase performs a parallel function in uricotelic species. In avian species, ammonia formed inside the mitochondria is incorporated into glutamine, which then leaves the mitochondria and serves as a precursor in the cytosol for biosynthesis of uric acid, the major nitrogen excretory product of uricotelic species. This is accomplished as a result of the fact that the relatively high levels of glutamine synthetase in uricotelic species are located exclusively in the mitochondrial matrix (25, 33, 34), just as ammo-

I I I 1

t 2

a t 0

W E N z w

FRACTION

FIG. 2. Equilibrium density gradient centrifugation of intact mitochondria on a 30-60% sorbitol gradient. The arrow indicates the top of the gradient. Glutamate dehydrogenase (O), cytochrome oxidase (O), glutamine- and acetylglutamate-dependent carbamoyl- phosphate synthetase (.), arginase (A), and glutamine synthetase (B). Enzyme activity values have been normalized for comparison on a common relative scale.

8452 Glutamine Synthetase and Urea

nia- and acetylglutamate-dependent carbamoyl-phosphate synthetase and ornithine carbamoyltransferase are located in the mitochondrial matrix of ureotelic species. Glutamine synthetase in mammalian, ureotelic species is a cytosolic enzyme, and the activity in liver is relatively low (35, 36). In the case of teleost fishes, which are ammonotelic, there is little evidence for significant conversion of mitochondrial ammonia to either glutamine or citrulline, and present evidence indicates that most of the ammonia excreted at the gills originates directly from ammonia generated in liver, presumably by direct efflux from liver mitochondria (3, 37, 38). The levels of glutamine synthetase activity in liver of teleost fishes, like that of carbamoyl-phosphate synthetase activity, have been reported to be either very low or undetectable (8, 9, 36, 38, 39); in one teleost species (Ictalaruspunctutus), the low level of glutamine synthetase which is present in liver has been shown to be located in the c ~ ~ o s o ~ . ~

The presence of high concentrations of both glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase in mitochondria of spiny dogfish, therefore, is clearly a unique finding, but one which is consistent with the view that ammonia assimilation for urea synthesis in elasmobranchs proceeds through the formation of glutamine, which then serves as the nitrogen-donating substrate for carbamyl phosphate and, ultimately, urea synthesis. Very little is known about the process of ammonia formation in marine elasmobranchs. Blood glutamine levels in elasmobranchs are very low, while blood ammonia levels are relatively high (40, 41). Leech et al. (41) have shown that ammonia is released continuously from skeletal muscle of spiny dogfish, and that during starvation, both ammonia and alanine (but not glutamine) are released into the blood; these authors have suggested that ammonia released by this tissue may be a quantitatively important source of nitrogen available for urea synthesis by the liver.

The location of arginase in the mitochondrial fraction is also an unusual finding; this enzyme is located primarily in the cytosol in ureotelic species, and in the mitochondria of uricotelic species (28, 31,42). Aside from subcellular location, arginases in ureotelic species also appear to differ from arginases from uricotelic species on the basis of both K, and molecular size (31,43). Arginases from uricotelic species have molecular weights in the range of 220,000-280,000 and have high K, values for arginine (30-300 mM). In contrast, arginases from ureotelic species have lower molecular weights (100,- 000-120,000) and lower values of K,,, for arginine (1-10 mM). Arginases from both groups are activated by Mn2' and exhibit maximum activity at pH 9.5-10.0 (31). Estimation of the molecular weight of the arginase from spiny dogfish by gel fitration chromatography on Sephadex G-200 gave a value of 105,000. The sedimentation coefficient estimated by sucrose density gradient centrifugation was found to be 5.5, which corresponds to a molecular weight of about 100,000 for a spherical molecule (22). Although these are not precise values, it is clear that the molecular size is similar to those reported for ureotelic species. The K , for arginine was found to be 1.2 mM, which is much lower than that observed for uricotelic species and is similar to that reported for ureotelic species. The enzyme has a fairly broad pH optimum, with maximum activity obtained at pH 9.5-10.0, and the activity is activated by Mn2+. The properties of the arginase from spiny dogfish, therefore, appear to be similar to arginases from ureotelic species.

Thus, spiny dogfish appear to incorporate aspects of ammonia metabolism associated with both mammalian ureotelic

' C. Casey and J. W. Campbell, unpublished experiments.

Cycle Enzymes in Spiny Dogfish

species (mitochondrial carbamoyl-phosphate synthetase) and uricotelic species (mitochondrial glutamine synthetase and arginase). In the case of this elasmobranch species, glutamine synthesized in the mitochondria is probably not excreted, but would presumably be utilized for carbamyl phosphate synthesis catalyzed by the glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase. The significance of these unique properties is not yet apparent. Elasmobranchs are ureoosmotic and are also considered to be ureotelic (3,37, 44, 45). The unusual properties of the carbamoyl-phosphate synthetase (requirement for acetylglutamate as a positive allosteric effector and the utilization of glutamine instead of ammonia as the nitrogen-donating substrate) and the location of this enzyme along with glutamine synthetase and arginase in the mitochondrial fraction may be related to this dual role for urea synthesis, or to unique regulatory requirements associated with the relationship between urea synthesis and osmoregulation.

Acknowledgment-We would like to thank the University of Wash- ington, Friday Harbor Laboratories, for use of their facilities.

1. 2.

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4.

5.

6. 7. 8.

9.

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12. 13. 14.

15. 16.

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24. 25.

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29.

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subcellular location of glutamine synthetase and urea cycle

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