DISTRIBUTION OF GAMMA-GLUTAMYL TRANSPEPTIDASE AND GLUTAMINASE ISOENZYMES IN THE RABBIT SINGLE NEPHRON Hajime SHIMADA*, Hitoshi ENDOU** and Fuminori SAKAI Department of Pharmacology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Accepted October 22, 1981 Abstract-"Phosphate-independent maleate-stimulated glutaminase'" was investigated as a function of gamma-glutamyl transpeptidase (gamma-GTP). The activity of gamma-GTP in brush border membranes was found to be four times higher than that in the microsomal fraction of the renal cortex. This gamma-GTP activity was exclusively located in the proximal tubule of isolated single nephrons. Specific activity of gamma-GTP was 105 U/g protein (19.8 ,tU/mm length) in the first 2 mm portion of the proximal tubule and 1352 U/g protein (209 /tU/mm) in the last 2 mm portion of the proximal straight tubule. Activity of phosphate independent glutaminase (PIG) was distributed in the same patterns as those of gamma-GTP, not only in the subcellular fractions, but also in the isolated nephron segments. On the other hand, phosphate dependent glutaminase (PDG) was dis tributed highly in the papillary mitochondrial fraction and in the distal tubule. Observations on the effect of pH on the enzyme activities of gamma GTP and PDG showed that these enzyme activities were decreased signifi cantly when the pH of the assay mixture was lowered. In the case of PIG, however, the effect of pH was just reversed. From these findings, it may he possible to interpret that gamma-GTP may play an important role in ammonia production in the brush border membrane of the proximal tubule as a function of glutaminase. It has been recognized that the activity of nephron was primarily found in the straight gamma-glutamyl transpeptidase (gamma portion of the proximal tubule (4). Gamma GTP), clinically evaluated in obstructive liver GTP catalyzes the transfer of the gamma disease, is much higher in the renal cortex glutamyl group of glutathione to free amino than in other organs, such as the liver, spleen acid to form the dipeptide, cysteinyl glycine and brain (1 ). Histochemically, gamma and gamma-glutamyl acceptor (5). GTP has been shown to be located in the Glutaminase I has at least two isoenzymes, brush border membrane of the proximal phosphate dependent glutaminase (PDG), tubule (2, 3). Heinle et al. reported that requiring inorganic phosphate as an activator; gamma-GTP activity in the lyophilized rat and phosphate independent glutaminase (PIG), activated by maleate (6). Tate and 'Present address: Department of Medicine , Kitasato Meister (7) have found that gamma-GTP University School of Medicine, Kitasato, Sagami hara, Kanagawa 228, Japan purified from rat kidney hydrolyzed glutamine '"To whom all correspondence should be addressed . to glutamate. Curthoys and Kuhlenschmidt (8) have chemicals used were of analytical grade. investigated both glutaminase and gamma Methods GTP reactions using PIG purified from rat (1) Preparation of subcellular fractions of kidney, reporting that the PIG reaction was kidney slices: Male New-Zealand white catalyzed by the same enzyme catalyzing the rabbits (2.5-3.0 kg, B.W.), maintained on gamma-GTP reaction. Therefore, these two normal food intake and water ad libitum, independent studies (7. 8) have indicated were used. The rabbit was anesthetized that "maleate-stimulated phosphate-inde intravenously with sodium pentobarbital 40 pendent glutaminase" is a catalytic function mg/kg. The left kidney was perfused with of gamma-GTP. physiological saline via the renal artery, then Glutaminase and glutamate dehydrogenase removed. The following steps were carried are considered to function in the main out on ice. The kidney was cut into three pathway of ammonia production in the kidney. parts (cortex, medulla, and papilla), and each Curthoys et al. (9) emphasized the importance section was homogenized in 9 volumes of of PDG in the proximal tubules of the chronic 0.25 M sucrose. The homogenates were acidotic rat. They showed a 20-fold increase centrifuged 900xg for 10 min and the pellets of PDG activity only in the early proximal were discarded. The supernatants were convoluted tubules of these rats. recentrifuged at 8,000xg for 10 min and the Additionally, in the acute acidotic state, resultant pellets were resuspended (mito ammonia excretion was reported to be chondrial fractions). Centrifugation of the increased prior to enzymatic induction, while remaining supernatant at 105,000xg for 60 the activity of PDG was somewhat decreased min y!elded microsomal fractions (pellets) (10). Several possible explanations have and soluble fractions. been proposed for these findings: increase of (2) Preparation of brush border mem an activator such as maleate (11 ), increase of branes: Brush border membranes were plasma glutamine (12), and change of the obtained from the renal cortex of New pathway producing ammonia (13, 14). Zealand white rabbits using the Liang and In order to clarify possible functional Sacktor method (15) with slight modifications relationships in renal ammonia production (3) Preparation of isolated nephron from glutamine, the present study was segments: Male New-Zealand white rabbits designed to investigate the distribution of (1 .5-2.0 kg B.W.) were used. Micro gamma-GTP and glutaminase isoenzymes in dissection of collagenase treated kidney isolated single nephrons of rabbit. slices was done by the procedure of Morel et al. (16). The isolated nephron segments MATERIALSAND METHODS were dissected in ice-cold Hanks' solution Materials under a stereoscopic microscope. The L-glutamine, L-glutamate, alpha-keto identification of each nephron segment was glutarate, L-gamma-glutamyl-p-nitroandilide, according to Maunsbach (17) and Morel glycylglycirie and 2-amino-2-methyl-propan et al. (16). The microdissected nephron 1,3-diol (Amediol) were obtained from Sigma. segments were collected into microcapillary Triethanolamine hydrochloride, nicotinamide pipettes under phase contrast microscopy adenine dinucleotide (NAD), iodonitrote (x100). trazolium chloride (INT), diaphorase, and (4) Enzyme assays: glutamate dehydrogenase (GLDH) were Gamma-glutamyl transpeptidase: Gamma obtained from Boehringer. All other GTP activity was measured spectrophoto metrically at 25'C using 4.0 mM L-gamma RESULTS glutamyl-p-nitroanilide as a substrate (15, 18) in 48 mM ammediol buffer, pH 8.2, Figure 1 shows the specific activity of containing 50 mM glycylglycine and 10 mM gamma-GTP in the subcellular fractions of MgCl2, except when the pH optimum was the rabbit kidney slices (cortex medulla and studied. Gamma-GTP activity was measured papilla). Gamma-GTP activity was highly on samples of kidney slices and brush border concentrated in the microsomal fraction of membranes in 2.1 ml final assay mixture. the cortex with a specific activity of 2375 U/g Each nephron segment, collected into a glass protein. This value was about 4 times as capillary pipette, was solubilized with 1 % high as the activity of the total homogenate deoxycholate. This sample solution was of the cortex. Soluble fractions (super divided into two parts: for enzyme assay and natants) revealed almost no activity. for protein determination. Usual protein Brush border membranes possessed high amounts used were 100 to 300 ng. The final gamma-GTP activity; 10 420 U/g protein assay volume was 105 /el. Enzyme activity (average of two independent experiments). showed linearity with respect to time, 0 to 10 min, and with protein content, 50 to 1 000 ng. There was no detectable loss of gamma-GTP activity during collagenase treatment at 37'C for 50 min and micro dissection at 0°C within at least 6 hours. Glutaminase isoenzymes: Glutaminase isoenzymes were determined by measuring glutamate formation as described by Curthoys and Lowry (9), with modifications in micro scale preparations. Glutaminase activity showed linearity with respect to time, 0 to 60 min incubation, and with protein content, 100 to 2,000 ng. Glutamate dehydrogenase: Glutamate dehydrogenase activity was photometrically Fig. 1. Localization of gamma-Glutamyl transpepti measured at 25°C according to the usual dase (gamma-GTP) activities in the subcellular fractionations of the kidney slices (cortex, method (19). The final assay volume was medulla, and papilla). The columns are the 205 /Ll. mean values and vertical lines indicate f1 S.D. (5) Protein determination: Protein content of 4 preparations. Numbers in the abscissa was determined by the Lowry procedure (20) represent the following centrifuged fractions: (1) 8,000xg for 10 min pellet (mitochondrial in a final assay volume of 22 or 65 ial using fraction), (2) 105,000xg for 60 min pellet an ultra-microcuvette having 0.5 mm (microsomal fraction), and (3) supernatant of diameter x 7 mm light path. Protein content the final centrifugation (soluble fraction). on the order of 10 ng was detectable. One Gamma-GTP activity was measured spectro photometrically at 25'C using L-gamma unit (U) was defined as the enzyme activity glutamyl-p-nitroanilide as a substrate. After that transforms 1 ;imole of substrate in adding the sample to the gamma-GTP assay 1 min at 25'C. mixture (final volume 2.1 ml), the O.D. change at 405 nm wavelength was measured. Enzyme activity is expressed as unit/gram protein at 25'C. In the isolated nephron segments, gamma 105 U/g protein in the first 2 mm portion of GTP activity was high in the proximal tubules the proximal convoluted tubule (PS,) and and very low in the other segments. Figure 2 1,352 U/g protein in the last part of the shows the distribution of gamma-GTP proximal straight tubule (PS3). activity in the 7 different nephron segments. The enzyme activities can be calculated as Specific activity of gamma-GTP was highly a function of tubule length (mm) or a concentrated in the proximal tubules, glomerulus. Table 1 shows protein contents especially in the last portion of the proximal of each nephron segment. Within the straight tubules.