From the Department of Biochemistry, Cornell University Medical College, New York, New York 10091

Transcription

1 THE Jomwn~ or BIO~GICAL CHEMIBTRY Vol. 248, No. 8, Issue of April 25, pp , 1973 Printed in U.S.A. y-glutamyl Cyclotransferase DISTRIBUTION, ISOZYMIC FORMS, AND SPECIFICITY * MARIAN ORLOWSKI AND ALTON MEISTER (Received for publication, December 11, 1972) From the Department of Biochemistry, Cornell University Medical College, New York, New York SUMMARY y-glutamyl cyclotransferase activity was found in homogenates of 11 rat tissues (kidney, liver, testes, spleen, brain, lung, heart, thymus, thyroid, skeletal muscle, and adrenal) ; two forms of the enzyme separable by starch gel electrophoresis were found in all tissues examined. L-y-Glutamyl- L-glutamine and L-y-glutamyl-L-methionine were the most active of nine substrates studied. The two enzyme forms present in rat liver were separated by chromatography on DEAE-cellulose and found to exhibit the same molecular weight (about 27,000) and similar substrate specificity. However, the substrate specificity of the two isolated y- glutamyl cyclotransferases was different from that of the liver homogenate and data were obtained indicating that the specificity of the enzyme changes during isolation and on storage at low temperature. Changes in the electrophoretic pattern are also observed after such storage. In general, the observed changes in specificity were decreases in activity toward y-glutamylglutamine and other y-glutamyl amino acids and either no change or a moderate increase in activity toward y-glutamyl-y-glutamyl-&nitroanilide. The findings suggest that the changes in specificity are associated with a process (possibly limited proteolysis) that modifies the enzyme in such a manner as to alter that portion of the active site that binds the amino acid moiety of the y-glutamyl amino acid substrate; the portion of the active site that binds the y-glutamyl moiety appears to be modiried relatively less. y-glutamyl cyclotransferase catalyzes the following reaction. L--r-Glutamyl-n-amino acid --) 5oxo-L-proline + L-amino acid It also catalyzes the conversion of y-glutamyl-y-glutamyl amino acids (and certain closely related compounds) to 5-oxoproline and y-glutamyl amino acids. Highly purified preparations of this enzyme have been obtained from human brain (1, 2), sheep brain (1, 2), and hog liver (3, 4). 5-Oxoproline was identified as a product of the enzymatic degradation of glutathione in kidney homogenates by Woodward and Reinhart in 1942 (5), and an enzyme activity (designated y-glutamyllactamase) that catalyzes the conversion of y-glutamylglycine to 5-oxoproline and glycine was reported in rat liver preparations by Connell and Hanes in 1956 (6). However, the significance of these observations remained uncertain largely because 5-oxoproline has generally been regarded as a nonenzymatic product derived from glutamate or glutamate derivatives. It is now evident that 5-oxoproline is an active metabolite and that it is an intermediate in the y-glutamyl cycle (7-9). The reactions of the y-glutamyl cycle, which account for the degradation and synthesis of glutathione, appear to be intimately associated with the active transport of amino acids. These findings and considerations, which emphasize the metabolic and physiological significance of y-glutamyl cyclotransferase, led us to the present study on this enzyme activity. The studies reported here have clarified several questions relating to the specificity and the distribution of this enzyme in mammalian tissues. The present work shows the existence of electrophoretically separable isozymic forms of the enzyme and also that the specificity of the enzyme can undergo substantial change during storage and isolation. EXPERIMENTAL Materials PROCEDURE n-y-glutamyl-l-y-glutamyl-p-nitroanilide, L-y-glutamyl-L-aaminobutyrate, L-y-glutamyl-n-glutamine, L-y-glutamyl-L-leutine, L-y-glutamylglycine were prepared as previously described (1, 10, 11). L-y-Glutamyl-L-methionine was prepared by the method described for the preparation of the L-y-glutamyl-a-aminobutyrate (10). The peptide crystallized directly on concentration of the 0.2 M acetic acid eluate from the Dowex 1 (acetate form) column; m.p The product gave a single spot on paper chromatography in Solvents A and B (see below) ; the corresponding RF values were 0.31 and On incubation with y-glutamyl cyclotransferase, the peptide was completely converted to 5-oxoproline and methionine. On acid hydrolysis (4 N HCl; 100 ; 4 hours) glutamate and methionine were found as the only amino acid products. * This research was supported in part by the National Science Foundation and the National Institutes of Health, United States Public Health Service. 1 The synonyms used are: L-pyroglutamic acid, Ld-oxopyrrolidine-2-carboxylic acid, L-2-pyrrolidone-5-carboxylic acid. Calculated: C 43.2, H 6.52, N 10.1 Found : C 43.3, H 6.53, N 9.96 Na-y-L-Glutamyl-N6-benzyloxycarbonyl-L-lysine was prepared 2836

2 by a modification ef the general procedure of King and Kidd (12). Phthaloyl-L-glutamic anhydride (13 g; 0.05 mole) (Sheehan and Bolhofer (13); King et al. (14)) was suspended in 50 ml of glacial acetic acid and 14 g (0.05 mole) of N6-benzyloxycarbonyl-L-lysine was added. The suspension was heated rapidly to boiling and then allowed to cool at 26. The solvent was removed by flash evaporation and the residual white solid was suspended in 100 ml of water. This suspension was placed in a flask equipped with a magnetic stirrer and adjusted to ph 7.0 by adding saturated sodium carbonate. Hydrazine hydrate (0.1 mole) was added and the suspension was stirred for 48 hours at 26. The ph was then adjusted to 3.0 by adding hydrochloric acid and the mixture was then filtered. The filtrate was concentrated in volume to about 100 ml and then applied to the top of a column (5 x 45 cm) of Dowex 1 (acetate form; AG l-x4; 200 to 400 mesh). The column was washed at 4 with 6 liters of water and elution was then carried out in sequence with 5 liters each of acetic acid solutions of the following concentrations: 0.1, 0.3, and 0.5 M. Fractions of 500 ml were collected and tested for ninhydrin-positive material. Na-L-y-Glutamyl- NG-benzyloxycarbonyl-L-lysine was eluted with 0.5 M acetic acid. The fractions containing the products were combined and concentrated by flash evaporation. The product crystallized on addition of methanol; the yield was 8.2 g (40%; m.p ). The product was homogeneous on paper chromatography; the RF values in Solvents A and B, respectively, were 0.57 and Calculated: C 55.8, H 6.64, N 10.3 Found : C 55.9, H 6.35, N 10.4 NQ-n-y-Glutamyl-L-lysine was prepared from the corresponding N6-bcnzyloxycarbonyl derivative by catalytic hydrogenation in 90y0 acetic acid over palladium on charcoal. The product, which was crystallized from aqueous methanol, was homogeneous on paper chromatography; the R,v values in Solvents A and B were, respectively, 0.11 and 0.14; m.p (decomposition). CIIE-IUN~O~ Calculated: C 48.0, H 7.68, N 15.3 Found : C 47.2, H 7.46, N 15.2 The following materials were obtained from Sigma: L-y-glutamyl-L-glutamate; hydrolyzed starch for electrophoresis; o- dianisidine (3,3 -dimethoxybcnzidinc), L-amino acid oxidase from Crotalus adamanteus venom (crude, type I), horseradish peroxidase (type II), bovine serum albumin, ovalbumin, chymotrypsinogcn, soybean trypsin inhibitor, ribonuclease A. Methods L)etermination of Enzyme Activity-The two methods described previously (1) for the determination of enzymatic activity were employed here. One of these involves the determination of L-Yglutamyl-p-nitroanilide released from L-y-glutamyl-r-y-glutamylp-nitroanilide. This procedure is convenient for studies involving assays on a large number of samples. Enzymatic activity toward the other substrates was followed by determining the formation of 5-oxoproline by measurement of its absorbance at 205 nm after passing the sample through a column of Dowel; 50 (H+). The assay conditions employed here were similar to those previously described (1). The enzyme solution (0.05 ml) was added to a mixture containing 0.1 ml of substrate (final concentration, 0.02 M) and 0.1 ml of Tris-HCI buffer (final concentration 0.08 M; ph 8.0); incubation was at 37. A unit of enzyme activ- ity is defined as the amount of enzyme that catalyzes the formation of 1 pmole of product per min under the conditions of assay. Specific activity is expressed in terms of units per mg of protein. Protein was determined by the method of Lowry et al. (15) with bovine serum albumin as the standard. Paper Chromatography-Descending paper chromatograms were carried out on Whatman No. 1 paper using two solvent systems. Solvent A consisted of l-butanol-pyridine-water (1: 1: 1, v/v). Solvent B consisted of I-butanol-acetic acid-water (60: 15 :25 v/v). The dried papers were sprayed with a 0.3% solution of ninhydrin in acetone. Preparation of Tissue Extracts-The fresh tissues were homogenized at 0 with 3 volumes of Tris-HCI buffer (0.05 M; ph 8.0) in a Potter-Elvehjem glass homogenizer equipped with a motordriven Teflon pestle. After homogenization for 2 lain, the homogenates were centrifuged for 30 min at 34,000 x g. The supernatant solutions thus obtained were employed for the determination of y-glutamyl cyclotransferase activity. Estimation of Molecular Weights-The apparent molecular weights of the y-glutamyl cyclotransferases were estimated by gel filtration as described by Andrews (16). Columns (2.5 x 45 cm) equilibrated at 4 with Tris-HCl buffer (0.05 M; ph 8.2) containing 0.15 M sodium chloride were used. The enzyme and standard proteins (2 mg in a final volume of 1.O ml) were applied to the top of the column; elution was carried out with the same buffer at a constant flow rate of 25 ml per hour. Fractions of 2.5 ml were collected and analyzed for enzyme activity and protein (from the absorbance at 235 nm). Bovine serum albumin, ovalbumin, chymotrypsinogen, soybean trypsin inhibitor, ribonuclease A, and Blue Dextran 2000 (Pharmacia) were used as markers. For the determination of the molecular weight of the enzyme in a crude liver homogenate, 2 g of rat liver were homogenized in a Potter-Elvehjem homogenizer at 0 with 6 ml of Tris-HCI buffer (0.05 M; ph 8.2), and then centrifuged at 0 for 45 min at 34,000 x g. An aliquot (1.O ml) of the supernatant solution was passed through a Sephadex G-75 column as described above. A single peak of activity (as determined both with L-Yglutamyl-L-y-glutamyl-p-nitroanilide and L-y-glutamyl-n-aaminobutyrate) was eluted from the column. The apparent molecular weights of the purified y-glutamyl cyclotransferase A and B preparations (see below) were estimated in a similar manner. Electrophoresis of y-glutamyl Cyclotransjerase on Starch Gel- Vertical starch gel electrophoresis was carried out according to the procedure of Smithies (17) using a commercially available apparatus (Buchler). The gels were buffered at ph 8.5 with a solution consisting of M Tris, M boric acid, and M EDTA (18). The anodic and cathodic compartments contained the same buffer system, except that the buffers were 4 and 2.85 times more concentrated, respectively. Samples of 30 to 40 ~1 of homogenates or purified enzyme preparations were applied to the gels and electrophoresis was carried out at 4 for 18 hours at 7.5 volts per cm. The gels were then sliced and each slice was covered with a reaction mixture suitable for localization of the enzyme. The procedure employed for localization of the enzyme in gel slices is based on the fact that y-glutamyl amino acids are relatively poor substrates of L-amino acid oxidase as compared to the free amino acids released by the action of the cyclotransferase. Thus, the enzyme can bc localized by a reaction sequence similar to that used by Lewis and Harris (19) for the detection of peptidases in human erythrocytes. The sequence of reactions used is as follows.

3 2838 cylclotransferase L-y-Glutamyl-L-amino acid f L-amino acid + 5-oxoproline n-amino acid oxidase L-Amino acid (2) HtOa + o-dianisidine peroxidase A oc-keto acid + NH3 + H202 oxidized dianisidine + H20 A brown deposit consisting of oxidized dianisidine is formed at the site of the enzyme in the gel. Only compounds that are substrates of y-glutamyl cyclotransferase and which yield an amino acid which is a substrate of n-amino acid oxidase can be used in this system. Both L-y-glutamyl-n-methionine and L-y-glutamyln-cY-aminobutyrate fulfill these requirements, the former being more suitable. The reaction mixture used for localization of the enzyme was prepared just before use by mixing 30 ml of a solution of 2% aqueous agar at 55 with 30 ml of a solution (at 26 ) containing 17 mg of L-y-glutamyl-n-methionine, 6 mg of n-amino acid oxidase, 6 mg of o-dianisidine, and 12 mg of horseradish peroxidase in 0.05 M Tris-HCl buffer (ph 8.0). The mixture was poured on the surface of the sliced starch gels; the gels were then covered with a plastic film (Saran Wrap), and incubated at 26 until distinct brown bands became visible. Since the reaction leading to the formation of oxidized dianisidine proceeds continuously, very long incubation periods lead to diffusion of the reaction products and thus blurring of the enzyme bands. For this reason, the gels were photographed at various times (0.5 to 2 hours) during incubation. The progress of the enzymatic reaction in the gel can be stopped by overlaying the gel with 2 M acetic acid. Gels treated in this manner can be stored for several months with good preservation of the enzyme bands provided that excess acet.ic acid is removed and that the gels are sealed in plastic film. RESULTS Studies on Distribution and Specijkity of y-glutamyl Cyclotransjerase in Rat Tissues Table I summarizes the findings on seven rat tissues in assays carried out with nine different substrates. In these studies freshly prepared tissue preparations were used. Under these conditions employed, enzymatic activity was proportional to the amount of tissue preparation present and also to the length of incubation. As indicated in Table I, kidney exhibited the highest specific activity, but considerable activity was found also in liver and testes. The values for the relative specific activities with various substrates are expressed in terms of the value obtained for the model substrate, L-y-glutamyl-L-y-glutamyl-p-nitroanilide. The rate of reaction varied greatly depending on the substrate. Thus, the highest activities were observed with the y-glutamyl derivatives of L-glutamine, L-methionine, and ~-aaminobutyrate; somewhat lower activities were observed with the other substrates, L-y-glutamyl-n-leucine being the least active. Glutathione is not a substrate. Comparison of the activities obtained with the various tissue preparations reveals that the pattern of specificity is remarkably similar for each of the tissues examined. On the other hand, this pattern of specificity differs very substantially from that observed with the highly purified preparation of y-glutamyl cyclotransferase obtained previously from human brain (I) (last column, 0) (3) Table I). Thus, the purified human brain enzyme preparation exhibited maximal activity toward the model substrate L-Yglutamyl-n-y-glutamyl-p-nitronanilide; the activity with n-y-glutamyl-n-glutamine was less than half of that observed with the model substrate and the other substrates examined were even less active. This very large difference in specificity induced us to carry out studies designed to explore the possibility that tissues might contain several y-glutamyl cyclotransferases which exhibit different substrate specificites. Electrophoretic Demonstration of y-glutamyl Cyclotransjerase Isozymes Centrifuged homogenates of various rat tissues were subjected to starch gel electrophoresis carried out as described under Methods. The enzyme was localized by the coupled system described above. A representative electrophoresis pattern is given in Fig. 1. In this study, two enzyme bands were found in studies on lung, thymus, thyroid, spleen, kidney, brain, testis, and liver. In similar studies on adrenal, skeletal muscle, and heart muscle, the two bands of activity were also found. The two forms of the enzyme will bc referred to here as y-glutamyl cyclotransferases A and B; Form A exhibits greater mobility toward the anode under the conditions employed. It is notable that these two enzyme forms were found in all of the tissue extracts that were examined. In the studies on kidney an additional slower moving band of activity was also seen (4, Fig. 1). The same electrophoretic patterns were obtained when either y-glutamylmethionine or y-glutamyl-cr-aminobutyrate was used for the localization of the enzyme. Other y-glutamyl substrates could not be used with this localization method because L-amino acid oxidase was not, under these conditions, sufficiently active toward the other amino acids (e.g. glutamate, glycine, lysine, glutamine). Isolation of y-glutamyl Cyclotransjerases A and B The following studies were undertaken to purify and separate the two electrophoretically separable forms of y-glutamyl cyclotransferase. Unless otherwise stated, all procedures were carried out at 4 ; ph determinations were done at 26. Step I-Fresh frozen rat livers obtained from Sprague-Dawley rats (300 to 350 g) were thawed and homogenized with 3 volumes of Tris-HCI buffer (0.05 M; ph 8.0) in a Waring Blendor for 3 min. The homogenate was centrifuged at 13,000 x g for 45 min and the supernatant solution was filtered through a layer of cotton and processed immediately as described below. Step %--The filtered solution was cooled to 0 and then adjusted to ph 4.5 by adding 1 M acetic acid (with mechanical stirring). The precipitate which formed was removed by centrifugation for 30 min at 13,000 x g. The supernatant solution obtained, which contained all of the y-glutamyl cyclotransferase activity, was adjusted to ph 7.8 by addition of 3 M ammonium hydroxide. Step S-Solid ammonium sulfate was added to the solution ob tained in the preceding step to achieve 507$ of ammonium sulfate saturation. The precipitate which formed was removed by centrifugation for 15 min at 13,000 x g, Additional solid ammonium sulfate was added to the supernatant solution to achieve 90% of ammonium sulfate saturation; the precipitate which formed was collected by centrifugation and dissolved in the minimal amount of Tris-HCI buffer (0.05 IN; ph 8.0). Step &-The solution obtained in Step 3 was applied to the top of a Sephadex G-75 column (5 x 100 cm) equilibrated with potassium phosphate buffer (0.005 M; ph 6.1). The column was

4 2839 Substrate -- I,-r-Glutamyl-n-r-glutamyl-p-nitroanilide.. r,-7..glutamyl-n-a-aminobutyrate. 1,.r-Glutamyl-n-methionine 1,.r-Glutamyl-n-glutamine I I,-r-Glutamyl-a-N-benzyloxycarbonyl-nlysine.... w-n-(n-r-glutamyl)-l-lysine r,-y-glutamyl glycine.. n-y-glutamyl-n-glutamate..... n-y-glutamyl-l-leucine.. - a Tissue extract, - I - T.USL~ I r-glutamyl cyclotransferase activity of several rat tissues Kidney Liver (1400)d (440) to 20 ~1, was used in the assays and the formation of 5-oxoproline was determined (see Methods ) after incubation for 5 to 300 min. * The values are expressed as relative specific activity (relative to that found with n-r-glutamyl-n-r-glutamy-p-nitroanilide, arbitrarily set at 100). Relative specific activity Testis Spleen Brain Lung Heart (175) (30) (50) (33) (30) Purified human from brainc c From Reference 1. d The values given in parentheses are the specific activities (units X 103 per min per mg) determined with n-r-glutamyl-nglutamine. ORIGIN FIG. 1. r-glutamyl cyclotransferase patterns in homogenates of rat tissues after separation by starch gel electrophoresis; localization of activity was carried out as described in the text. 1, liver; 2, testis; 3, brain; 4, kidney; 6, spleen; 8, thyroid; 7, thymus; 8. lung. cluted with the same buffer. The enzyme activity emerged iu two peaks (Fig. 2). The larger of these (Pecrk L) was processed further as described in Step 5; the smaller of these (Peak S) will be discussed further below. Step J-The pooled fractions from Peak L obtained in Step 4 were passed through a column of carboxymethylcellulose (2.5 x 15 cm; Whatman CM-52) previously equilibrated with potassium phosphate buffer (0.005 M; ph 6.1). The column was eluted with the same buffer. The enzyme activity did not bind to the column and the effluent fractions containing protein were pooled and further purified as described below. Step 6-The solution obtained in Step 5 was added to the top of a DEAE-cellulose column (Whatman DE-52 microgranular, preswollen, 17.5 x 1.2 cm) which had been equilibrated with potassium phosphate buffer (0.005 M; ph 6.1). The column was washed with 50 ml of the same buffer and elution was started with a linear gradient established between 250 ml of M potas- sium phosphate buffer (ph 6.1) and 250 ml of 0.2 M potassium phosphate buffer (ph 6.1). The flow rate was maintained at 30 ml per hour and fractions of 5 ml were collected and assayed for enzyme activity. As indicated in Fig. 3, chromatography on DEAE-cellulose led to the separation of two peaks of y-glutamyl cyclotransferase activity. The first of these to emerge from the column was found to exhibit electrophoretic mobility identical with cyclotransferase B and the second t,o y-glutamyl cyclotransferase A (Fig. 4). A summary of the purification procedure is given in Table II. The activity was followed during purification by assays with L-Yglutamyl-n-y-glutamyl-p-nitroanilide and L-y-glutamyl-L-c+ aminobutyrate. The ratio of the specific activities toward these substrates was 2.5 for the first two steps in the procedure and close to 1.0 for the remaining steps and for the isolated Components A and B. The purification procedure resulted in an increase in specific activity toward n-y-glutamyl-l-y-glutamyl-p-

5 12345 A280- FICJ. 2. Gel filtration on Sephadex G-75 (Step 4 of the purification procedure; see the text). I 'ACTIVIN CYCLOTRANSFERASE _I umts/ml A280nm _ - E d -- FRACTION FIN. 3. Chromatography of rat liver r-glutamyl cyclotransferase on DEAE-cellulose (Step 6 of the purification procedure; see the text). nitroanilide of about 900- and 200-fold, respectively, for Forms A and B. Dialysis of Forms A and B against buffer containing 5 mm EDTA did not affect the enzyme activities. Estimates of the molecular weight of the cyclotransferase activity present in rat liver homogenate and of the activities of cyclotransferases A and B were carried out as described under Methods using the gel filtration procedure. As indicated in Fig. 5, the elution volume obtained for rat liver homogenate y- glutamyl cyclotransferase corresponded to a molecular weight of 25,500 & 3,000. Studies of the A and B forms of cyclotransferase (obtained as described in Table II) gave values of 27,500 f 3,000. Thus, the apparent molecular weights of the separated forms of the enzyme and of the enzyme in liver homogenate are about the same. When Peak S (Fig. 2; Step 4 of the purification procedure) was subjected to chromatography on carboxymethylcellulose and on DE-52 (as described in Steps 5 and 6 of the purification procedure) two peaks were obtained that exhibited electrophoretic mobility identical with that observed with Components A and B obtained from Peak L (Fig. 4). Furthermore, the specificity of Peak S was identical with that of Peak L. These findings indicate that Peak S is probably not a separate form of y-glutamyl cyclotransferase; it may represent an artifact of chromatography possibly associated with overloading of the Sephadex G-75 column, or it may be the result of enzyme aggregation. The purification of Components A and B (Table II) was associated with less increase of the specific activity toward y-glutamyl-cy-aminobutyrate than toward y-glutamyl-y-glutamyl-p- ORIGIN FIN. 4. Starch gel electrophoresis of rat liver -r-glutamyl cyclotransferase. 1, Peak S from Sephadex G-75 column (details are given in the text) ; B, Peak L from Sephadex G-75 column; ponent A; 4, Component B; 5, rat liver homogenate. 8, Com- nitroanilide; this is reflected in the decrease of the ratio of the specific activities toward these substrates from 2.5 for the initial homogenate to about 1.0 for the isolated enzyme forms. It should be noted that while ratios of about 2.5 for these enzyme activities are found in homogenates prepared from fresh frozen rat liver, the ratio of these activities is about 4.0 for homogenates prepared from fresh rat liver (see Table I). Although the ratio of the specific activities toward these two substrates varies for homogenates of fresh liver, homogenates of fresh frozen liver, and for the isolated cyclotransferase Forms A and B, there is no significant difference between Components A and B in terms of their relative activities toward these substrates. We considered the possibility that the observed change in this ratio might be associated with removal during purification of a component with high activity toward y-glutamyl-oc-aminobutyrate. However, no evidence could be obtained for the existence of such an enzyme form. It appears more likely at this time that the enzyme is modified during purification (and freezing or thawing). Thus, although the isolated Components A and B do not differ in electrophoretic mobility from the forms present in fresh liver homogenates, they seem to have been changed in some manner so as to substantially affect their specificity. The marked difference between the specific activity ratios found for fresh liver homogenates and homogenates prepared from frozen liver led us to carry out a study on the specificity of the enzyme during storage at 0. It was found that when a solution of the enzyme (obtained after Step 3 of the purification procedure) was stored at 0, there was a significant increase of activity toward y-glutamyl-y-glutamyl-p-l?itroanilide and an appreciable decrease of activity toward y-glutamyl-a-aminobutyrate (Table III). Thus, the ratio of the two activities changed over a period of 25 days from an initial value of 1.08 to a value of It seemed of interest to determine whether this profound change in specificity of the enzyme was associated with any detectable change in its molecular properties. Therefore, an

6 2841 TABLE Summary of purijication of r-glutamyl cyclotransferase from rat livera II step 1. llat liver homogenate Precipitation at ph (NH&S04 fractionation Gel filtration Carboxymethylcellulose DEAE-cellulose Component A.... Component B.... Volume ml hcentration Protein Total w/ml ntg 32 15, , , pgluz- PNAb Activity Specific +X-abC v-gluz-pnab 7.Glu-abC units/mg Ratid r-gluab:r-glut-pna 696 1, , a From 150 g of liver. b Activity determined with L-r-glutamyl-n--y-glutamyl-p-nitroanilide (r-gh+pna). c Activity determined with n-r-glutamyl-l-or-aminobutyrate (7.Glu-ab). d Ratio of specific activities. vf VC 2.C I- 1.t I- l.f ;t I I I I I I I I I 4 5x104 I05 Molecular Weight FIG. 5. Estimation of the molecular weight of r-glutamyl cyclotransferase by gel filtration on Sephadex G-75 (see the text). TABLE III Change of specijicity of y-glutamyl cyclotransferase toward r-glutamyl-r-glutam$-p-nitroanilide (y-g&pna) and y-glutamyl-cu-aminobutyrate (r-glu-ab) during storage at 0 The enzyme (after Step 3 of the purification procedure) was stored at O, and the activity was determined as described under Methods. Days of storage r-glur-pna Activity units/ml -,-Glu-ab Ratio r-glut-ab:.y-glu-pna enzyme preparation that had been stored for 25 days at 0 was carried through Steps 4 and 5 of the purification procedure and then subjected to DEAE-cellulose chromatography (Step 6). As indicated in Fig. 6, three peaks (Peaks 1, 2, and 3) of activity /-IT- ( ii ACTMTY unifslml A28Onm I 0.6 I L FRACTION FIG. 6. Chromatography (on DEAE-cellulose) of rat liver y- glutamyl cyclotransferase after storage at 0 for 25 days. The experimental conditions were identical with those given in Step 6 of the purification procedure except that the elution gradient was established between buffers containing M and 0.15 M phosphate. emerged from the column. While all of these were active toward y-glutamyl-y-glutamyl-p-nitroanilide, Peaks 1 and 3 exhibited considerable activity toward y-glutamyl-cr-aminobutyrate while Peak 2 showed little activity toward this substrate. Comparisons between the data given in Fig. 6 with those given in Fig. 3 suggest that cyclotransferase B was converted in part to enzyme which elutes from the column (Fig. 6) with the Peak 2 fraction. When the three peaks of activity isolated from the stored preparation were subjected to electrophoresis on starch gel (Fig. 7), it was found that Peak 1 exhibited mobility identical with that of Component B of a fresh rat liver homogenate. Peak 2 separated into three components, two of which exhibited mobility intermediate between Components A and B, and a third, which migrated in the region of Component 13. Peak 3 separated on electrophoresis into a main band moving in the position of Component A and a minor band exhibiting migration intermediate between Components A and B. When cyclotransferases A and B (prepared as described in Table II) were stored at 0 for 2 weeks, there was no change in the ratio of activities toward y-glutamyl-a-aminobutyrate and y- glutamyl-y-glutamyl-p-nitroanilide; this contrasts with a change

7 2842 Specijicitfj of several forms of rat liver y-gluiamyl cyclotransferase Substrate - F resh live homogenate - r Relative FO;i b activitya Fb Peak Zc L-r-Glutamyl-L-y-glutamyl-pnitroanilide L-y-Glutamyl-L-a-aminobutyrate. L-~-Glutamyl-L-methionme.I.. n-r-glutamyl-l-glutsmine. L--y-Glutamyl-e-N-benzyloxycarbonyl-L-lysine a-n(l-r-glutamyl)-l-lysine L-r-Glutamyl-L-glutamate.. L-r-Glutamylglycine. L-r-Glutamyl-L-leucine. (W (100) (W (100) ORIGIN FIG. 7. Starch gel electrophoresis of rat liver -y-glutamyl cyclotransferase after storage at 0 and chromatography on DEAEcellulose (see Fig. 6). 1, Peak 1; 2, Peak 2; S, Peak 3; 4, Components A and B of rat liver homogenate. in ratio from 1.08 to about 0.2 observed on similar storage of the enzyme after Step 3 of the purification procedure (Table III). On further storage of the purified A and B fractions, there was a slow decrease in this activity ratio, reaching values of 0.3 to 0.4 after 48 days at 0. It thus appears that change in specificity occurs much more slowly with more purified preparations than with the less purified one. The enzymatic activities exhibited by cyclotransferases A and B (Fig. 3) and Peak 2 (Fig. 6) toward several y-glutamyl amino acids were compared with those obtained with freshly prepared rat liver homogenate (Table IV). The data indicate that Peak 2 showed only very little activity toward y-glutamyllysine and y- glutamylglycine and also that its activity t.oward the other y-glutamyl amino acids was quite low. In contrast to the cyclotransferase A and B preparations and to the fresh liver homogenate, the Peak 2 fraction was much more active toward y-glutamyl-y-glutamyl-p-nitroanilide than toward the other y-glutamyl substrates. The findings therefore indicate that storage of y-glutamyl cyclotransferase at 0 leads to marked changes in substrate specificity and also to substantial changes in the molecular properties of the enzyme as indicated by altered migration on electrophoresis and on ion exchange chromatography. It is also of interest that the apparent Km values for the various substrates tend to increase during purification. Thus, as indicated in Table V, the apparent Km values obtained for five substrates with the purified cyclotransferase A and B preparations were 2 to 4 times greater than those found with freshly prepared rat liver homogenate. DISCUSSION These studies provide additional evidence that y-glutamyl cyclotransferase activity is widely distributed in mammalian tissues. This work also shows that the two electrophoretically separable a Activity was determined as described under Methods. b Prepared as described in Table II (Fig. 3). c Prepared as described in Fig. 6. TABLE Apparent K, values for y-glutam$ cyclotransferases Substrate L-r-Glutamyl-L-~-glutamyl-p-nitroanilide...._..._...._. 0.2 n-r-glutamyl-l-a-aminobutyrate n-r-glutamyl-l-methionine. 1.4 n--r-glutamyl-l-glutamine... 6 or-n-(l-r-glutamyl)-l-lysine. 26 V %M mdd Form forms of the enzyme occur widely; these were found in all 11 of the rat tissues examined. The substrate specificity of the two forms of y-glutamyl cyclotransferase is virtually the samr. Studies on the y-glutamyl cyclotransferase activity of homogenates of seven rat tissues indicate that y-glutamylglutamine and y-glutamylmethionine are the most active bubstrates. This observation is of particular interest since glutamine and methionine are among the best acceptors in the reaction catalyzed by y- glutamyl transpeptidase. These observations are consistent with the view (7) that y-glutamyl transpeptidase and y-glutamyl cyclotransferase function together as catalytic components of the y-glutamyl cycle. Glutamine accounts for 20 to 25% of the total blood plasma amino acids and thus also of the amino acids transported across the kidney tubule. The findings that glutamine is one of the best substrates for y-glutamyl transpeptidase and that y-glutamylglutamine is the best substrate for y-glutamyl cyclotransferase are therefore in accord with the proposed function of the y-glutamyl cycle in amino acid transport. The present work seems to clarify several earlier observations on y-glutamyl cyclotransferase. For example, Kakimoto et al. (20) purified an enzyme (designated y-glutamylglutamine lactamase) about 14-fold from an extract of rat liver acetone powder and examined its activity toward eight y-glutamyl amino acids; these workers found that only y-glutamylglutamine, y-glutamylserine, and y-glutamylalanine were substrates. Since they found that their purified enzyme preparation was not active toward B

8 y-glutamylglycine, while a less purified preparation prepared by the procedure of Connell and Hanes (6) was active toward y- glutamylglycine, they concluded that the enzyme they had purified was different from that originally described by Connell and Hanes. The present studies, which indicate that the substrate specificity of y-glutamyl cyclotransferase may undergo marked change on purification and even on storage at 0, suggest that the preparation described by Kakimoto el al. (20) is probably not a separate y-glutamyl cyclotransferase. Thus, we obtained a modified form of the enzyme (Peak 2, Table IV) which exhibited very little activity toward y-glutamylglycine. Indeed, the specificity exhibited by this fraction (obtained from rat liver y-glutamyl cyclotransferase stored for 25 days at 0 ) is somewhat similar to that observed with the highly purified y-glutamyl cyclotransferases previously isolated in this laboratory from human and sheep brain acetone powders (1, 2). These considerations suggest that the brain enzymes previously studied in our laboratory (1, 2) may have undergone modification during isolation similar to that found in the present work on rat liver y-glutamyl cyclotransferase. However, we cannot exclude the possibility that species differences exist. In general, it appears that the specificity exhibited by freshly prepared homogenates is broader than that exhibited by various partially purified preparations of the enzyme. The possibility that there are different y-glutamyl cyclotransferases exhibiting different substrate specificities must be considered, but the present findings indicate that theoccurrenceof enzymemodification during purification presents a serious complication that would hinder studies on this point. The findings indicate that the observed changes in specificity are probably not due to removal during purification of catalytically active components, but rather to some type of enzyme modification. Thus, enzyme preparations obtained from frozen liver exhibited a substantially different specificity than did those obtained from fresh liver; furthermore, storage at 0 also was accompanied by dramatic changes in substrate specificity. While the nature of the process responsible for enzyme modification requires additional study, certain possibilities appear relatively unlikely. The isolated y-glutamyl cyclotransferases A and B exhibited virtually the same molecular weight as the enzyme present in the liver homogenate. Thus, it seems unlikely that interaction of the enzyme with other proteins or subunit interactions are involved in the observed alteration of specificity. While there is clear evidence for the existence of separate A and B isozymes, the data do not provide evidence of a relationship between the occurrence of isozymic forms of the enzyme and the observed specificity. Indeed, the substrate specificities of the A and B y-glutamyl cyclotransferases are very similar. The findings indicate that the changes in the specificity of the enzyme actually precede the changes that are observed by application of the electrophoretic method. Thus, the specificities of the isolated y- glutamyl cyclotransferases A and B are markedly different from the specificity of the y-glutamyl cyclotransferase activity of rat liver homogenates, while the electrophoretic behavior of the separated y-glutamyl cyclotransferases is about the same as that of the two forms of the enzyme in the homogenate. The differences in electrophoretic mobility of y-glutamyl cyclotransferases A and B may possibly be explained in terms of two enzymes containing similar but nonidentical subunits. However, the relatively low molecular weight of the enzyme would seem to argue against this possibility; furthermore, we have found only two forms, and one would expect to find three forms if the enzymes were composed of two subunits of different types. A more attractive possibility is that the A and B forms differ in amino acid 2543 composition or in the attachment of a carbohydrate moiety as has been found for pancreatic ribonuclease (21). The available data are consistent with the possibility that the observed modification of enzyme specificity is promoted enzymatically, perhaps by a mechanism involving limited proteolysis. In the course of this work we attempted to isolate y-glutamyl cyclotransferases A and B from kidney homogenates by the procedure described here for the liver activities. Although these studies failed to yield the separated A and B forms of the enzyme, several fractions were obtained after DEAE-cellulose chromatography which exhibited specificity similar to that shown by Peak 2 (Table IV). In some studies, fractions were obtained which exhibited specificity intermediate between that of Peak 2 and Forms A and B. These findings suggest that enzyme modification proceeds much more rapidly in kidney preparations than in preparations of liver, and would seem to be consistent with the possibility that enzyme modification is due to an enzyme-catalyzed process, perhaps involving cleavage of peptide bonds. Whatever the nature of such modification may be, its effect would seem to be to alter the conformation of that portion of the active site that binds the amino acid moiety of the y-glutamyl amino acid substrate. That the portion of the active site which binds the y-glutamyl moiety may be modified relatively less is suggested by the finding that the observed changes in substrate specificity are not accompanied by marked decreases in the activity toward the model substrate (y-glutamyl-y-glutamyl-pnitroanilide) ; indeed, the activity toward the model substrate increased during storage at 0 (Table III). It seems notable, however, that the apparent K, values for the model substrate as well as those for several other y-glutamyl substrates increased substantially during purification of y-glutamyl cyclotransferases A and B (Table V). The present communication thus describes a situation which is somewhat unusual (and fortunately atypical) in enzymology. Thus, it is usually possible to isolate enzymes from mammalian tissues in essentially unaltered form with respect to substrate affinity and specificity as compared to the activity present in homogenates. It would appear that purification and study of the native y-glutamyl cyclotransferases must await the development of procedures that will minimize or prevent altogether enzyme alteration of the type observed here. In this connection, one may speculate that such enzyme alteration could conceivably occur in viva and perhaps be controlled by genetic factors; it might thus be responsible for cert ain types of amino acid transport defects. Acknowledgment-We wish to thank Nss Charlene Michaud for her untiring and skillful assistance in many of these experiments. REFERENCES 1. ORLOWSKI, M., RICHMAN, P. G., AND MEISTER, A. (1969) Biochemistry 8, ORLOWSKI, M., AND MEISTER, A. (1970) Methods Enzymol. 17A, ADAMSON, E. D., SZEWCZUK, A., AND CONNELL, G. E. (1971) Can. J. Biochem. 49, ADAMSON, E. D., CONNELL, G. E., AND SZEWCZUK, A. (1970) Methods Enzymol. 19, WOODWARD,G. E., AND REINHART, F.E. (1942) J.BioZ. Chem. 146, CONNELL, G. E., AND HANES, C. S. (1956) Nature 177, ORLOWSKI,M., AND MEISTER, A. (1970) Proc. Nat. Acad. Sci. U. 5. A. 67,

9 ORLOWSKI, M., AND MEISTER, A. (1971) Enzymes 4, VAN DER WERF, P., ORLOWSKI, M., AND MEISTER, A. (1971) Proc. Nat. Acad. Sci. U. S. A. 68, ORLOWSKI, M., AND MEISTER, A. (1971) J. Biol. Chem. 246, ORLOWSICI, M., AND MEISTER, A. (1971) Biochemistry 10, KING, F. E., AND KIDD, D. A. (1949) J. Chem. Sot SHEEHAN, J. C., AND BOLHOFER, W. A. (1952) J. Amer. Chem. Sot. 72, KING, F. E., CLARK-LEWIS, J. W., AND WADE, R. (1957) J. Chem. Sot LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, ANDREWS, P., (1964) Biochem. J. 91, ; (1965) 96, SMITHIES, 0. (1959) Biochem. J. 71, BOYER, S. H., FAINER, D. C., AND NAUGHTON, M. A. (1963) Science 140, LEWIS, W. H. P., AND HARRIS, H. (1967) Nature 216, KAKIMOTO, Y., KANAZAWA, A., AND SANO, I. (1967) Biochim. Biophys. Acta 132, PLUMMER, T. H., JR., AND HIRS, C. H. W. (1964) J. Biol. Chem. 239,

10 γ-glutamyl Cyclotransferase: DISTRIBUTION, ISOZYMIC FORMS, AND SPECIFICITY Marian Orlowski and Alton Meister J. Biol. Chem. 1973, 248: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at