ARCHIVES
OF BIOCHEMISTRY
Subcellular
AND
BIOPHYSICS
Localization
167, 51%%!‘i (1975)
of Rat Kidney
Phosphate
Independent
Glutaminase’ THERESA KUHLENSCHMIDT Department
of Biochemistry,
School
of Medicine,
AND University
Received September
NORMAN P. CURTHOYS of Pittsburgh,
Pittsburgh,
Pennsylvania
15261
4, 1974
The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and y-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16-1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.
Increased :renal synthesis and excretion of ammonia is a major element of the kidneys’ horneostatic response to metabolic acidosis (1, 2). Urinary ammonia is derived primarily from the amide and amine nitrogens of glutamine which the kidney extracts from the plasma (3). Previous experiments (4, 5) have suggested that renal ammonia synthesis from glutamine is primarily init.iated by a glutaminase acti+ity. But, rat kidney contains two distinct glutaminase activities: the phosphate dependent glutaminase, originally reported by Carter and Greenstein (6); and a phosphate independent glutaminase, described by Katunuma (7), which is not effected by phosphate, but is strongly activated by maleate. The two isoenzymes have different pH optima, K, for glutamine and heat stabilities. They also have a very complementary distribution in the various tubular structures of the kidney (8). Both gluta‘This investigation was supported in part by Research Grant AM 16651 from National Institute of Arthritis, Metabolism, and Digestive Diseases.
minase isoenzymes are particulate, and therefore knowledge of their subcellular localization is basic to understanding their role in renal ammoniagenesis. Errera and Greenstein (9) were the first to suggest that the phosphate dependent glutaminase is contained in mitochondria. More recently, Curthoys and Weiss (10) have shown, by means of digitonin-Lubrol and swell-shrink sonication submitochondrial fractionation procedures that this activity in rat kidney is a component of the inner mitochondrial membrane. Kalra and Brosnan (ll), using slightly more vigorous conditions, came to the conclusion that this glutaminase is a soluble component of the mitochondrial matrix. Neither of these reports provided definitive evidence as to the subcellular localization of the phosphate independent glutaminase. In this manuscript, we report that the phosphate independent glutaminase is a component of the kidney brush border membrane. A preliminary report of this work has been presented (12). 519
Copyright 0 All
rights
1975 by Academic Press, Inc. of reproduction in any form reserved.
520
KUHLENSCHMIDT MATERIALS
AND
METHODS
Materials White male Sprague-Dawley rats (ZOO-400 g) were obtained from Zivic Miller and were maintained on Purina rat chow. Glutamic dehydrogenase in 50% glycerol was obtained from Boehringer. All other biochemicals were obtained from Sigma Chemical Co.
Methods Differential fractionation. The various fractions obtained by differential centrifugation were resuspended in a standard buffer solution which contained 0.25 M sucrose, 10 rnM Tris, and 1 mM EDTA, pH 7.5. Rats were decapitated and the kidneys were excised immediately. The tissue was weighed, minced finely and then homogenized in a loose fitting Dounce homogenizer with 8 vol of hypotonic buffer prepared by diluting standard buffer to 30%. The crude homogenate was then centrifuged in a Sorvall RC-2B centrifuge until the SS-34 rotor reached a speed of 4500 rpm (20 s). The resulting pellet was resuspended with 2 vol of 30% buffer, rehomogenized and recentrifuged to 4500 rpm. The resuspended pellet from this centrifugation constituted the nuclear fraction. The combined supematants of the first two centrifugations were then centrifuged at 7006g for 10 min in order to pellet the mitochondria. The supernatant was then centrifuged at 40,OOOgfor 30 min. The resulting pellet contained the heavy microsomes and the supernatant was then centrifuged at 144,OOOg for 1 h to separate light microsomes (pellet) and cytosol (supernatant). Zsopycnic centrifugation. Sucrose solutions prepared in 10 mM Tris-1 mM EDTA buffer, pH 7.5, were used to prepare 28-ml linear gradients (25-45%). On top of a gradient was layered 0.5 ml of either the mitochondrial or heavy microsomal fraction obtained by differential centrifugation. The gradients were then centrifuged at 25,000 rpm for 5 h in an SW 25.1 rotor. Gradients were then pumped off from the bottom and 0.7-ml fractions were collected. Sucrose densities were determined with a Bausch and Lomb refractometer. Isolation of kidney cells. In order to obtain a preparation of intact, individual kidney cells, we modified the procedure of Nagata and Rasmussen (13) which yields a preparation of rat kidney cortical tubules. Their procedure involves infusion of a closed kidney system with a collagenase solution. Our major modification has been to completely flush the kidney free of plasma with a collagenase-papain perfusate, then close the system and allow the kidney to swell until rupture. To perform the perfusion, the rat is anesthetized with ether. The rat is then opened with a midline abdominal incision and the aorta and vena cava are cleared and separated. The descending aorta above the right renal artery, the anterior mesenteric artery and the vena cava below the left renal vein are
AND CURTHOYS ligated. Then a 20-ml syringe containing 0.04% collagenase, 0.1% hyaluronidase, 0.02% papain, and 0.25 mg/ml each of penicillin and streptomycin in a Ca*+ free Hanks’ solution (14) is inserted, using a 20 gauge needle, into the descending aorta below the left renal artery and tied in place. The two kidneys are then flushed with the enzyme solution until they become light tan in color (approximately 15 ml). The vena cava above the right renal vein is then tied off and the remainder of the enzyme solution is used to swell the kidneys. The kidneys are then excised, trimmed of fat and minced finely. The tissue is then incubated at 37°C with slight agitation in a metabolic shaker bath in 10 ml of the perfusion solution. After 15 min the cells are suspended further by drawing up into a large bore pipette. After a second 15-min incubation, the cell suspension is washed 3 times by repeated centrifugation at ZOOg for 1 min and resuspension in CaZ+ free Hanks’ solution. The final cell preparation was resuspended to 5 ml with Ca2+ free Hanks’ solution. The final cell suspension contains 150-200 mg protein. Examination under a light microscope indicates that the final suspension consists of more than 98% individual cells and of these more than 95% exclude trypan blue. Enzyme assays. The phosphate independent glutaminase was assayed as described previously (10). The standard mixture contained 20 mM glutamine, 60 mM maleate, 0.2 mM EDTA, pH 6.6. After stopping the reaction with acid, the amount of glutamate formed was measured enzymatically. These conditions were not optimal for the phosphate independent glutaminase, but were selected to minimize interference from the phosphate dependent glutaminase activity (less than 3% of the maximal phosphate dependent glutaminase activity is expressed under these conditions) (8). Cytochrome oxidase (15), TPNH cytochrome c reductase (16), and lactate dehydrogenase (17) were assayed as described previously. Acid phosphatase (18) and alkaline phosphatase (19) were assayed using p-nitrophenylphosphate as substrate. But, because of the large amounts of alkaline phosphatase in kidney tissue, the acid phosphatase reaction was stopped with 6 N KOH and the absorbance at 410 nm was determined immediately. 5’-Nucleotidase was assayed (20) by using the method of Lowry and Lopez (21) to determine phosphate released from AMP. y-Glutamyltranspeptidase (22) was assayed by following the appearance of p-nitroaniline from a solution containing 5 mM L-y-glutamyl-p-nitroanilide, 0.2 mM EDTA, and 50 mM imidazole, pH 7.2. All enzyme activities in this manuscript are reported in terms of pmoles OF product formed per min. Protein was determined by the method of Lowry (23). RESULTS
Differential centrifugation. The fractions obtained by differential centrifugation
RAT KIDNEY
GLUTAMINASE
SUBCELLULAR
were assayed for phosphate independent glutaminase activity and for various subcellular marker activities (Table I). About 30% of this glutaminase activity was recovered in tbe mitochondrial fraction and the bulk of the remaining activity was recovered in the heavy microsomes. The glutaminase activity did not correlate with marker activities for mitochondria, lysosomes or microsomes. Of the activities tested only the 5’-nucleotidase, alkaline phosphatase and y-glutamyltranspeptidase showed the largest percent recovery in the heavy microsomal fraction. The close correlation between the percent recoveries for the phosphate independent glutaminase and for alkaline phosphatase and yglutamyltranspeptidase activities in the various fractions suggests that this glutaminase is also a constituent of the brush border membrane of the proximal tubule cells. Sucrose gradients. The results of isopycnit centrifugation of the mitochondrial fraction obtained by differential centrifugation are shown in Fig. 1. The phosphate independent glutaminase activity banded as a single symmetrical peak which had a mean density of 1.165. The cytochrome oxidase activity banded in a region of greater density. The phosphate independent glutaminase activity showed a very close correlation with alkaline phosphatase
521
LOCALIZATION
activity. Whereas, the acid phosphatase activity spread over the entire gradient. The heavy microsomal fraction was also characterized by isopycnic centrifugation (Fig. 2). The TPNH cytochrome c reductase and acid phosphatase activities in this fraction behaved similarly. Neither produced a sharp peak, but instead both activities were spread over the entire gradient. In contrast, the glutaminase, alkaline phosphatase and y-glutamyltranspeptidase activities all banded as a single symmetrical peak of mean density of 1.163. The close correlation supports the conclusion that the phosphate independent glutaminase is contained in the brush border membrane. The fact that the mean densiI165 -40 AlPhOl ; ” ~0~~0 ;C
OF BIOCHEMISTRY
Subcellular
AND
BIOPHYSICS
Localization
167, 51%%!‘i (1975)
of Rat Kidney
Phosphate
Independent
Glutaminase’ THERESA KUHLENSCHMIDT Department
of Biochemistry,
School
of Medicine,
AND University
Received September
NORMAN P. CURTHOYS of Pittsburgh,
Pittsburgh,
Pennsylvania
15261
4, 1974
The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and y-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16-1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.
Increased :renal synthesis and excretion of ammonia is a major element of the kidneys’ horneostatic response to metabolic acidosis (1, 2). Urinary ammonia is derived primarily from the amide and amine nitrogens of glutamine which the kidney extracts from the plasma (3). Previous experiments (4, 5) have suggested that renal ammonia synthesis from glutamine is primarily init.iated by a glutaminase acti+ity. But, rat kidney contains two distinct glutaminase activities: the phosphate dependent glutaminase, originally reported by Carter and Greenstein (6); and a phosphate independent glutaminase, described by Katunuma (7), which is not effected by phosphate, but is strongly activated by maleate. The two isoenzymes have different pH optima, K, for glutamine and heat stabilities. They also have a very complementary distribution in the various tubular structures of the kidney (8). Both gluta‘This investigation was supported in part by Research Grant AM 16651 from National Institute of Arthritis, Metabolism, and Digestive Diseases.
minase isoenzymes are particulate, and therefore knowledge of their subcellular localization is basic to understanding their role in renal ammoniagenesis. Errera and Greenstein (9) were the first to suggest that the phosphate dependent glutaminase is contained in mitochondria. More recently, Curthoys and Weiss (10) have shown, by means of digitonin-Lubrol and swell-shrink sonication submitochondrial fractionation procedures that this activity in rat kidney is a component of the inner mitochondrial membrane. Kalra and Brosnan (ll), using slightly more vigorous conditions, came to the conclusion that this glutaminase is a soluble component of the mitochondrial matrix. Neither of these reports provided definitive evidence as to the subcellular localization of the phosphate independent glutaminase. In this manuscript, we report that the phosphate independent glutaminase is a component of the kidney brush border membrane. A preliminary report of this work has been presented (12). 519
Copyright 0 All
rights
1975 by Academic Press, Inc. of reproduction in any form reserved.
520
KUHLENSCHMIDT MATERIALS
AND
METHODS
Materials White male Sprague-Dawley rats (ZOO-400 g) were obtained from Zivic Miller and were maintained on Purina rat chow. Glutamic dehydrogenase in 50% glycerol was obtained from Boehringer. All other biochemicals were obtained from Sigma Chemical Co.
Methods Differential fractionation. The various fractions obtained by differential centrifugation were resuspended in a standard buffer solution which contained 0.25 M sucrose, 10 rnM Tris, and 1 mM EDTA, pH 7.5. Rats were decapitated and the kidneys were excised immediately. The tissue was weighed, minced finely and then homogenized in a loose fitting Dounce homogenizer with 8 vol of hypotonic buffer prepared by diluting standard buffer to 30%. The crude homogenate was then centrifuged in a Sorvall RC-2B centrifuge until the SS-34 rotor reached a speed of 4500 rpm (20 s). The resulting pellet was resuspended with 2 vol of 30% buffer, rehomogenized and recentrifuged to 4500 rpm. The resuspended pellet from this centrifugation constituted the nuclear fraction. The combined supematants of the first two centrifugations were then centrifuged at 7006g for 10 min in order to pellet the mitochondria. The supernatant was then centrifuged at 40,OOOgfor 30 min. The resulting pellet contained the heavy microsomes and the supernatant was then centrifuged at 144,OOOg for 1 h to separate light microsomes (pellet) and cytosol (supernatant). Zsopycnic centrifugation. Sucrose solutions prepared in 10 mM Tris-1 mM EDTA buffer, pH 7.5, were used to prepare 28-ml linear gradients (25-45%). On top of a gradient was layered 0.5 ml of either the mitochondrial or heavy microsomal fraction obtained by differential centrifugation. The gradients were then centrifuged at 25,000 rpm for 5 h in an SW 25.1 rotor. Gradients were then pumped off from the bottom and 0.7-ml fractions were collected. Sucrose densities were determined with a Bausch and Lomb refractometer. Isolation of kidney cells. In order to obtain a preparation of intact, individual kidney cells, we modified the procedure of Nagata and Rasmussen (13) which yields a preparation of rat kidney cortical tubules. Their procedure involves infusion of a closed kidney system with a collagenase solution. Our major modification has been to completely flush the kidney free of plasma with a collagenase-papain perfusate, then close the system and allow the kidney to swell until rupture. To perform the perfusion, the rat is anesthetized with ether. The rat is then opened with a midline abdominal incision and the aorta and vena cava are cleared and separated. The descending aorta above the right renal artery, the anterior mesenteric artery and the vena cava below the left renal vein are
AND CURTHOYS ligated. Then a 20-ml syringe containing 0.04% collagenase, 0.1% hyaluronidase, 0.02% papain, and 0.25 mg/ml each of penicillin and streptomycin in a Ca*+ free Hanks’ solution (14) is inserted, using a 20 gauge needle, into the descending aorta below the left renal artery and tied in place. The two kidneys are then flushed with the enzyme solution until they become light tan in color (approximately 15 ml). The vena cava above the right renal vein is then tied off and the remainder of the enzyme solution is used to swell the kidneys. The kidneys are then excised, trimmed of fat and minced finely. The tissue is then incubated at 37°C with slight agitation in a metabolic shaker bath in 10 ml of the perfusion solution. After 15 min the cells are suspended further by drawing up into a large bore pipette. After a second 15-min incubation, the cell suspension is washed 3 times by repeated centrifugation at ZOOg for 1 min and resuspension in CaZ+ free Hanks’ solution. The final cell preparation was resuspended to 5 ml with Ca2+ free Hanks’ solution. The final cell suspension contains 150-200 mg protein. Examination under a light microscope indicates that the final suspension consists of more than 98% individual cells and of these more than 95% exclude trypan blue. Enzyme assays. The phosphate independent glutaminase was assayed as described previously (10). The standard mixture contained 20 mM glutamine, 60 mM maleate, 0.2 mM EDTA, pH 6.6. After stopping the reaction with acid, the amount of glutamate formed was measured enzymatically. These conditions were not optimal for the phosphate independent glutaminase, but were selected to minimize interference from the phosphate dependent glutaminase activity (less than 3% of the maximal phosphate dependent glutaminase activity is expressed under these conditions) (8). Cytochrome oxidase (15), TPNH cytochrome c reductase (16), and lactate dehydrogenase (17) were assayed as described previously. Acid phosphatase (18) and alkaline phosphatase (19) were assayed using p-nitrophenylphosphate as substrate. But, because of the large amounts of alkaline phosphatase in kidney tissue, the acid phosphatase reaction was stopped with 6 N KOH and the absorbance at 410 nm was determined immediately. 5’-Nucleotidase was assayed (20) by using the method of Lowry and Lopez (21) to determine phosphate released from AMP. y-Glutamyltranspeptidase (22) was assayed by following the appearance of p-nitroaniline from a solution containing 5 mM L-y-glutamyl-p-nitroanilide, 0.2 mM EDTA, and 50 mM imidazole, pH 7.2. All enzyme activities in this manuscript are reported in terms of pmoles OF product formed per min. Protein was determined by the method of Lowry (23). RESULTS
Differential centrifugation. The fractions obtained by differential centrifugation
RAT KIDNEY
GLUTAMINASE
SUBCELLULAR
were assayed for phosphate independent glutaminase activity and for various subcellular marker activities (Table I). About 30% of this glutaminase activity was recovered in tbe mitochondrial fraction and the bulk of the remaining activity was recovered in the heavy microsomes. The glutaminase activity did not correlate with marker activities for mitochondria, lysosomes or microsomes. Of the activities tested only the 5’-nucleotidase, alkaline phosphatase and y-glutamyltranspeptidase showed the largest percent recovery in the heavy microsomal fraction. The close correlation between the percent recoveries for the phosphate independent glutaminase and for alkaline phosphatase and yglutamyltranspeptidase activities in the various fractions suggests that this glutaminase is also a constituent of the brush border membrane of the proximal tubule cells. Sucrose gradients. The results of isopycnit centrifugation of the mitochondrial fraction obtained by differential centrifugation are shown in Fig. 1. The phosphate independent glutaminase activity banded as a single symmetrical peak which had a mean density of 1.165. The cytochrome oxidase activity banded in a region of greater density. The phosphate independent glutaminase activity showed a very close correlation with alkaline phosphatase
521
LOCALIZATION
activity. Whereas, the acid phosphatase activity spread over the entire gradient. The heavy microsomal fraction was also characterized by isopycnic centrifugation (Fig. 2). The TPNH cytochrome c reductase and acid phosphatase activities in this fraction behaved similarly. Neither produced a sharp peak, but instead both activities were spread over the entire gradient. In contrast, the glutaminase, alkaline phosphatase and y-glutamyltranspeptidase activities all banded as a single symmetrical peak of mean density of 1.163. The close correlation supports the conclusion that the phosphate independent glutaminase is contained in the brush border membrane. The fact that the mean densiI165 -40 AlPhOl ; ” ~0~~0 ;C
