- Email: [email protected]
γ-Glutamyl transferase of swine kidney
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
FGlutamyl
127,292-301
(1968)
Transferase
F. H. LEIBACH”
AND
of Swine FRANCIS
Kidney’
BINKLEY
Department of Biochemistry, Division of Basic Health Sciences, Emory University, Atlanta, Georgia 30322 Received
December
26, 1967; accepted
March
22, 1968
The enzyme of renal tissue responsible for the transfer of the r-glutamyl grouping of glutathione to various acceptors was solubilized by a brief digestion with ficin and purified to apparent homogeneity by fractionation with ethanol and ammonium sulfate, chromatography on DEAE-cellulose, gel filtration with Sephadex, and gradient centrifugation. The enzyme was found to establish an equilibrium between products and reactants. Glutamic acid was not a product of the reaction and the enzyme was entirely without activity in the absence of an acceptor. The purified enzyme did not hydrolyze -r-glutamylnapthylamide and did not transfer the y-glutamyl grouping of this compound to potential acceptors. An enzyme responsible for the hydrolysis of y-glutamylnaphthylamide was separated from renal tissue and was found to be not influenced in its activity by the addition of acceptors. This enzyme was without activity in the hydrolysis of glutathione or of arylamide derivatives of other amino acids.
These studies were designed to investigate the mode of action of the enzyme of renal tissue responsible for the transfer of the r-glutamyl grouping of glutathione to appropriate acceptors. We had previously reported that the requirement for an acceptor was absolute; there was no hydrolysis of glutathione in the absence of an appropriate amino acid or peptide (2). The absolute requirement for an acceptor presented the possibility that the transfer was an equilibrium reaction and various methods were devised to test this possibility. Reduced glutathione presented many problems in chromatography and the product of the transfer, cysteinylglycine, was difficult to obtain in adequate purity and, in solution, was ’ These studies were supported by grant AM06089 of the United States Public Health Service. This is publication No. 886 of the Division of Basic Health Sciences. A preliminary report has appeared (1). ‘This work was taken from a thesis submitted by F. H. Leibach to the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Spring, 1964. Present address, Department of Biochemistry, Medical College of Georgia, Augusta.
converted to the diketopiperazine (3). Thus, it was not possible to study the reaction mixture over extended periods of time with glutathione as the substrate. Oxidized glutathione was found to be an equivalent substrate for the transfer enzyme and the product of the transfer from oxidized glutathione, cystinyldiglycine, was readily prepared and was stable in solution under the conditions employed. At the time these studies were in progress, the y-glutamylarylamides were introduced as substrates for the detection of enzymes active in the hydrolysis of -,-glutamyl compounds and it was inferred by several authors that the enzymes responsible for the release of arylamines from such compounds were identical with the transfer enzyme (4, 5, 6). Thus, it is of interest to report our results with an enzyme active in the hydrolysis of y-glutamylnaphthylamide that was clearly separated from the transferase. METHODS
AND
MATERIALS
Reduced glutathione was used in the routine assay of transferase activity as in the previous publication (2). The assay was with 0.012 M glycylglytine as the acceptor with 0.003 M glutathione. 0.001 292
r-GLUTAMYL magnesium acetate at pH 8.0 in 0.01 M Tris buffer; the time of incubation was 30 minutes and the reaction was stopped by the addition of trichloroacetic acid to a final concentration of 5rr. Cysteinylglycine was determined by the method of Sullivan and Hess (7). Units were expressed as pmol per min and specific activity as units per mg total nitrogen of the preparation (micmKjeldah1). For chromatographic studies, oxidized glutathione was substituted at the same concentration of y-glutamyl groupings (0.0015 M oxidized glutathione). In the studies of the reverse reaction cystinyldiglycine (cystinyl-bis-glycine) was used at a molarity of 0.0033 M. The appearance or disappearance of cystinyldiglycine (appearance in the forward reaction and disappearance in the reverse reaction with yglutamylglycylglycine as the donor of the y-glutamyl grouping) was followed by the method of Sullivan and Hess (7); it should be noted that cystinyldiglycine yields half as much color in this reaction as does cysteinylglycine (2). Reduced and oxidized glutathione were obtained from Schwarz BioResearch and r-glutamylglycylglycine, glycylglycine, and cystinyldiglycine were obtained from Cycle Chemical Corporation. Chromatography of the reaction mixtures with the solvent system butanol : acetic acid : water (60 : 15 : 25) with Whatman No. 3 filter paper in a descending system was found to separate all the components of the test system (oxidized glutathione, glycylglycine, cystinyldiglytine, and r-glutamylglycylglycine) as well as glycine and glutamic acid. In order to provide additional information as to the mechanism of action of the en“C-glycylglycine (“C-1-4) (California Corpo zyme, ration for Biochemical Research) was employed in studies with oxidized glutathione and also with yglutamylglycylglycine as the donor of -/-glutamy groupings. The reaction mixture for the routine assay of the enzyme active in the hydrolysis of yglutamylnaphthylamide (hydmlase activity) conmined r-glutamylnaphthylamide, 0.00125 M, 0.1 ml of an appropriately diluted enzyme solution, and Tris buffer, final concentration 0.1 M and pH 8.0, in a total volume of 2 ml. The solution was incubated at 37’ for lo-30 minutes and the liberated naphthylamine was determined as described by Goldberg and Rutenberg (8). Analyses for carbohydrate were made at various stages of purification by the methods of Winzler (9) for protein-bound hexose and by the anthrone method of Fales (10) for total carbohydrate. Polyacrylamide disc electrophoresis, by adaptations of the procedures of Omstein (11) and Davis (12), was used to check the products at each stage of the purification. Sucrose density gmdients. Gradients were prepared according to the method of Bolton et al. (13) and fractions were collected with drop counting after centrifugation by puncturing the base of the M
TRANSFERASE
293
centrifuge tube with a hypodermic needle in an apparatus similar to that of Martin and Ames (14). The gradients were prepared with 14.7 ml of a 30’;. and 15.3 ml of a 7.5?( solution of sucrose. One ml of sample containing 10 mg or less of the preparations was layered onto the gradient and centrifugation was in the SW-25 swinging bucket rotor of the Spinco Model L centrifuge. Purification of the tmmfeme. Solubilization of the transferase may be achieved by treatment of renal microsomes with deoxycholate or detergents (15), but these materials complicate the purification and the products are often highly contaminated with extraneous glycoproteins. Brief digestion with ficin was found to release most of the activity into solution and the activity thus released was easily purified by conventional methods. Ficin was found to have no activity in the release of cysteinylglycine from glutathione or cystinyldiglycine from oxidized glutathione in the absence or in the presence of acceptors. The purification is outlined in Fig. 1 and summarized in Table I. Frozen swine kidneys were allowed to thaw partially and the cortical tissue was removed and dissected free of connective tissue and fat. Ten-pound batches of the cortical tissue were ground in a meat grinder and homogenized in a Waring Blendor with 25 liters of 0.1 M MgCb at room temperature. Crude &in (Nutritional Biochemical Corp.) was added to the homogenate to yield a hnal concentration of 3 mg per ml. Toluene was added to prevent bacterial growth and the homogenate was incubated at 37” for 4 hours. Suspended material was removed by centrifugation with the Intemational PR-2 centrifuge for 30 min at O” and at 2300 rpm. Cold ethanol (4/5 volume) was added to the SUMMARY
OF PURIFICATION
KIDNEY
CORTEX HOMOGENIZE DIGEST wth
PREClblTATE
FICIN
SUPERAATANT
PRECIPITATE (DISCARD)
415 VeLUME ETHANOL
I
I
SUPERNATANT
PREClPlTAiE
'"'s;D'
IIN
BUFFER1
;~;$C$N,"M
SUPERNATANT
PRECIPITATE IDISCARD)
80% AMMONIUM SULFATE
1 SUPERNATANT IDISCARD)
FIG. 1. Scheme transterase.
1 PRECIPITATE
of preliminary
iIN
purification
BUFFERI
of the
294
LEIBACH
AND TABLE
PURIFICATION
Homogenate” Supernatant Ethanol precipitate Ammonium sulfate 80% DEAE step-wiseb Sephadex G-100’ Sephadex G-100” second Sucrosec gradient
I
OF THIS: TRANSFERASIC Volume (ml)
step
BINKLEY
9,500 8,200
i,200 1,200 1,100
Nitrogen (W)
52,250 32,800 7,560 1,080 48
Total activity (pmole/min)
Specific activity C,mmle/mg NJ
139,000 134,009 79 ) 000 22,800 15,580
2.66 4.10 10.4 21.1 333 700 5,ooo 5,000
time
a Four pounds of renal cortex in 10 1. b Five columns as described in Fig. 2. c Yields were essentially quantitative for amounts supematant solution and after 8 hours at 4” the precipitate was removed by centrifugation and the supematant solution was discarded. The precipitate was dissolved in a minimal amount of 0.01 M TrisHCl buffer, pH 8.0, and was dialyzed against the same buffer until free of ethanol. The solution was clarified by centrifugation and ammonium sulfate was added to the supematant solution to produce 60:; saturation. The solution was permitted to stand at 4’ for 6 hours and was centrifuged at 2300 rpm for 1 hour. The precipitate was discarded and solid ammonium sulfate was added to the supematant solution to produce 80% saturation. The solution was permitted to stand at 4’ for 6 hours and was centrifuged at 2300 rpm for 1 hour. The precipitate was discarded and solid ammonium sulfate was added to the supematant solution to produce 80:; saturation. The solution was permitted to stand at 4’ for 8 hours and the precipitate was collected by centrifugation and dissolved in a minimal amount of the 0.01 M Tris buffer. The solution was dialyzed against the same buffer until free of ammonium sulfate, lyophilized, and stored in the dry state in the cold. This material is essentially free of carbohydrate and is an excellent starting material for the isolation of the transferase, the r-glutamylamidase, leucylarylamidase and the solubilized aminopeptidase of Robinson et 01. (16). Chromatogmphic purification. All chromatography was carried out at room temperature. 1.0 gm of the lyophilized material was dissolved in 100 ml of the 0.01 M Tris buffer and placed on a column of DEAE-cellulose (S & S type 40, 2.5 X 50 cm) equilibrated with the same buffer. The column was washed successively with 0.01 M, 0.08 M, and 0.08 M Tris-HCl plus 0.15 M NaCl all at pH 8.0. The starting buffer eluted three inactive fractions and the transferase was eluted with 0.08 M Tris buffer; the r-glutamylnaphthylamidase (hydrolase) was eluted with the salt solution (Fig. 2). The discontinuous
of material
up to 10 mg protein
nitrogen
elution was very effective in the resolution of the two activities whereas continuous gradients were relatively ineffective either in separation of the two activities or in further purification of the transferase. Thirty ml of a solution of the transferase (up to 10 mg) was re-equilibrated with 0.01 M Tris-HCl buffer and placed on a column of Sephadex G-100, 2.5 X 150 cm, equilibrated with the same buffer; elution was with the same buffer. The transferase was retarded (Fig. 3) and, after a second passage through the column of Sephadex, was homogeneous as judged by gradient centrifugation (Fig. 4) and by disc electrophoresis (Fig. 5). The -y-glutamylnaphthylamidase was not retarded by Sephadex G-100 but was further purified by gradient centrifugation; curve II (Fig. 4) is for material recovered from preliminary gradient centrifugation. Curve I is for a leucyl-8-naphthylamidase prepared from endoplasmic membranes by similar procedures. In Table II results with several renal enzymes are summarized; all materials were run and the gradients were calibrated with proteins of known molecular weights under identical conditions. The overall purification of the napthylamide hydrolase is not described because we have not prepared the enzyme except from the eluate from DEAE-cellulose. The material at that stage had a specific activity of about 10 pmole naphthylamide per minute per mg protein (N X 6.25). Subsequently, the yield was essentially quantitative in the gel filtration with Sephadex and with gradient centrifugation. No claim is made for the homogeneity of the preparation; specific activity of about 100 pmole per minute per mg protein (N X 6.25) was obtained for the material from gradient centrifugation in the assay with 1.25 rmole naphthyiamide per ml at pH 8.0 and 37’. Other workers have reported a specific activity near 109 with a higher level of substrate and at a higher pH (4) for what may be a preparation of the same enzyme.
y-GLUTAMYL 0.01 M
TRANSFERASE
TRIS
0.08
M
295
TRIS
0.15 0.08 1
i
M M
NaCl TRIS
TRANSFERASE
ML
ELUATE
FIG. 2. Elution of the activities from DEAE-cellulose with a discontinuous gradient. Activity was assayed as described in the text with reduced glutathione plus glycylglycine and with r-glutamylnaphthylamide. There was no cross activity in the two peaks and there was no activity in the hydrolysis of reduced or oxidized glutathione in the absence of glycylglycine in any of the samples (8 ml per tube).
TRANSFERASE SEPHADEX
VOIDVOLUME /
I 20
with dex.
1 60
40
TUBE FIG.
G/O0 2x
NUMBER
3. Further purification Sephadex G-100; second
of time
the transferase through Sepha-
Preparations of the y-glutamylarylamidases by others (4) have been reported to contain a high level of carbohydrate; carbohydrate was not detectable (less than lS,,) in the final preparations of either activity. Absorbancy in the ultraviolet and changes in absorbancy upon the addition of alkali or of acid were those commonly observed with simple proteins and there was no evidence for the presence of materials other than protein in either preparation. Mechanism of action of the trunsfemse. In the studies of activity described here only the transferase from gradient centrifugation with a specific ac-
4
8
12
16
20
24
28
32
ML
FIG. 4. Gradient centrifugation of three renal enzymes. Curve 1, leucyl-p-naphthylamidase isolated from endoplasmic membranes, Curve II, r-glutamylnaphthylamidase recentrifuged after a preliminary gradient centrifugation, and Curve III, the transferase. In all cases activity tracked completely with the absorbancy.
tivity of 5,000 @mole per minute per mg N was used. Amounts of enzyme up to 10 rg per ml were used in certain experiments, but the usual concentration of enzyme was 0.15 rg per ml. In the absence of the acceptor, there was no reaction with the transferase with oxidized glutathione or reduced glutathione as judged by the formation of material reacting in the method of Sullivan and Hess (7). With oxidized glutathione as the substrate but in the absence of
296
LEIBACH
AND
BINKLEY TABLE
II
SUMMARY OF SUCROSE GRADIENTS WITH SOME RENAL ENZYMES Thirty ml sucrose, 30-7~7~ (w/v), with 1 ml sample was centrifuged for 19 hours in the SW-25 rotor of the Spinco Model L centrifuge at 25,000 rpm with a thermostat setting of 21”. Thirty-two samples were collected by drop counting and the location of the enzyme was determined by assay for the activity and, when possible, by absorbancy at 280 w. The samples contained from 1 to 10 mg protein. Enzyme
Leucine aminopeptidase” Leucyl-fi-naphthylamidasec r-glutamylnaphthylamidase r-glutamyl transferase LeucylgIycinased
FIG. 5. Disc electrophoresis of, left to right, yglutamyl transferase from DEAE-cellulose, middle, the transferase after twice through Sephadex, and, right, the r-glutamyl naphthylamidase after gradient centrifugation. All were at pH 8.0 with 20 ~1 of a 1% solution. acceptor glycylglycine, there was no reaction with the transferase as judged by the appearance of cystinyldiglycine in amounts detectable by paper chromatography; glutamic acid could not be detected after long periods of incubation (3 hours) with amounts of enzyme one hundred fold greater than that necessary to give maximal transfer in 10 minutes in the presence of the acceptor. No naphthylamine was released and no materials reactive with ninhydrin, other than the substrate, could be detected in paper chromatography after incubation of a lc; solution of the transferase with the r-glutamylnaphthylamide (1.25 pmole per ml) for up to 3 hours. But, in the complete system, with oxidized glutathione and glycylglycine, the reaction proceeded with the formation of y-glutamylglycylglytine and cystinyldiglycine. As is illustrated in Fig. 6, as the intensity of the reaction of the oxidized glutathione and glycylglycine with ninhydrin de-
Peak tube
Molecularweight estimate
17 21 23
240,000* 170,000 110,000
26 26
80,000 80,000
a Soluble enzyme active in the hydrolysis of leucinamide and leucylglycine but not leucyl-flnaphthylamide; manganous ion required for activity. * This is for the main form of the enzyme; other fully active but minor components were found to have apparent molecular weights of 150,000 and 300,000. These minor components when recentrifuged in the gradient gave the same distribution as the original material. c Microsomal enzyme active in the hydrolysis of leucyl-p-naphthylamide and leucylglycine but not Ieucinamide; no metal ion requirement. d Microsomal enzyme active in the hydrolysis of leucylglyciue but not leucinamide or leucyl-pnaphnaphthylamide; no metal ion requirement,. This appears to be the solubilized aminopeptidase of Robinson et al. (16). creased, there was a corresponding increase in the reaction with glutamylglycylglycine and cystinyldiglycine. At the concentration of substrate used, there was no detectable change as judged by the reaction with ninhydrin on the paper chmmatograms in the relative concentration of the four components in samples taken after a few minutes of incubation. There was no indication of the presence of glutamic acid or of other materials reacting with ninhydrin in any of the chromatograms even with extended incubation. Chromatograms revealing only the same four components were obtained when cystinyldiglytine (0.0033 M) was incubated with y-glutamylglycylglycine (0.012 M) under the same conditions; the chromatogram, after 30 minutes, was indistinguishable from that given in the last column of Fig. 6.
-/-GLUTAMYL
product 0’
Tronspeptldose occumulatfon 5l
reaction with time 15’
30’
‘Jygly
0000 I 0
I
I
I
5
15
30
Time
TRANSFERASE
in minutes
FIG. 6. Accumulation of products at indicated times after addition of enzyme under standard conditions of assay with 0.15 pg protein N per ml, 0.012 M glycylglycine, 0.003 M oxidized glutathione, pH 8.0 in 0.01 M Tris buffer. 100 ~1 samples containing 5’( TCA were subjected to chromatography and were sprayed with ninhydrin. With a migration of the solvent front of 40 cm, the approximate RF values were oxidized glutathione 5, cystinyldiglycine 8, y-glutamylglycylglycine 16, glycylglycine 24, glytine 29, and glutamic acid 33. The latter two compounds were not detected but would be at the end of the section of the chromatogram reproduced here. The origin is indicated by the dark line. As is pointed out in the studies of the equilibrium detailed below, there is reason to believe that the product in the reverse direction is the mixed disulfide of glutathione and cysteinylglycine but there was no indication of the separation of this compound from the oxidized glutathione in the chromatographic system employed. Although there was no reason to suspect that glylcylglycine might be hydrolyzed in some reaction such that r-glutamylglycine and free glycine might be formed, the possibility was checked with “Cglycylglycine as the acceptor. The reaction mixture (after 30 minutes) was subjected to chromatography and the strip was scanned with a strip counter (Actigraph, Model II, Nuclear-Chicago) and, as is illustrated in Fig. 7, there was an accumulation of labeled r-glutamylglycylglycine without the appearance of glycine. Thus, ,the glycylglycine was not cleaved in the reaction. Then, since the same products are formed from oxidized glutathione plus glycylglycine as from y-glutamylglycylglycine and cys-
297
tinyldiglycine, it should be possible to obtain evidence that the y-glutamyl grouping is transferred back to the product remaining from the transfer of the glutamyl grouping to the enzyme. ‘“C-Glycylglycine was incubated with the transferase together with y-glutamylglycylglycine and, as was anticipated, there was an exchange of the labeled glycylglycine into the r-glutamylglycylglycine (Fig. 8). It is planned to test directly the possibility of the transfer of the glutamyl grouping to a stable position on the enzyme. Parallel studies were made with the naphthylamide hydrolase but at a concentration of enzyme of 10 pg per ml (or about 100-fold that of the transferase as detailed above); oxidized glutathione, glycylglycine, and the other materials were present at the levels described for the above studies. The experiments were ali negative in terms of evidence for a transfer reaction to an added acceptor. With oxidized glutathione or reduced glutathione as the substrate, there was no release of cystinyldiglycine or cysteinylglycine as measured by the method of Sullivan and Hess (7) either in the presence or in the absence of glycylglycine. When “C-glycylglytine was added together with oxidized glutathione with the naphthylamide hydrolase, there was no indication of the formation of any labeled product in the chromatograms prepared from the incubation mixture and there was no incorporation of the labeled glycylglycine into y-glutamylglycylglycine by the hydrolase. Thus, there was no evidence of any activity of the hydmlase with oxidized or reduced glutathione as the substrate. Then, similar studies were undertaken with the naphthylamide hydrolase with y-glutamylnaphthylamide as the substrate to determine if there might be some transfer reaction to an added acceptor along with the obvious hydrolysis. The amount of enzyme employed was about 10 rg per ml or an amount far in excess of that necessary to catalyze the release of 95-100’; of the available naphthylamine from 1.25 pmole of naphthylamide per ml at pH 8.0 in 10 minutes. The solubility of the naphthylamide precluded studies at higher levels of substrate. With the naphthylamide as the substrate, chromatograms were developed as above at various times; the only compounds detected that reacted with ninhydrin were the substrate and glutamic acid with no indication of other products. Then, glycylglycine was added together with the naphthylamide. With 1.25 pmole naphthylamide and 12.0 pmole glycylglycine per ml there was no effect on the rate of appearance of naphthylamine over the controls with the naphthylamide alone and the chromatograms revealed a small amount of substrate, glutamic acid, and the added glycylglycine as the materials reacting with ninhydrin. Then “C-glycylglycine was substituted for the unlabeled glycylglycine, chromatograms were
298
LEIBACH Transpeptidase
AND -
BINKLEY Actigraph
y-Glu Glygly
CllYg’Y
-
Scanning
Cys digly
GSSG
“C-glycylglycine. Conditions were as in FIG. 7. Labeling of y-glutamylglycylglycine with Fig. 6 with incubation for 30 minutes at 37” and solvent migration in chromatography (to the left) was approximately 30 cm. The origin is indicated by the dark line. prepared and the strips were scanned with the strip counter as above. The only radioactivity was found at the position of glycylglycine as in the zero time samples with the transferase with oxidized glumthione and labeled glycylglycine as in Fig. 7. Thus, we were unable to demonstrate any reaction with the naphthylamide hydrolase other than the hydrolysis of the naphthylamide with an essentially theoretical release of naphthylamine. These studies may be criticized because of the relative low level of substrate as compared with the work of others (4) and the much shorter period of incubation. The major point to be made is that we have been unable to demonstrate the release of less than very nearly the theoretical amount of naphthylamine with or with-
out an acceptor present and no evidence was obtained for any transfer of a glutamyl grouping to glycylglycine by this enzyme either from glutathione or from the naphthylamide; this behavior is in complete contrast to the behavior of the transferase. Otherwise, we have little knowledge of the behavior of the naphthylamide hydrolase; it is inactive in the hydrolysis of alanyl-, leucyl-, and prolylnaphthylamides and simple dipeptides of leucine and alanine. Equilibrium studies. From the studies of the products of the reaction and from the experiments with ‘“C-glycylglycine it appeared evident that the transferase established an equilibrium between a pair of donors and a pair of acceptors. Thus, the re-
y-GLUTAMYL Transpeptidose
299
TRANSFERASE -
Actigraph
Scanning
y-
Glygb
Glu - glyglycine
y - w3lYillY
FIG. 8. Labeling of r-glutamylglycylglycine with ‘“C-glycylglycine with r-glutamylglycylglycine, 5 pmole per ml, substituted for oxidized glutathione under standard conditions with 0.15 pg protein N per ml. Incubation was for 30 minutes at 37’ at pH 8.0. action with oxidized might be written as: GSSA
+ 2 glygly
glutathione
and
glycylglycine
z cystinyldiglycine + 2 -y-glutamylglycylglycine
However, since oxidized glutathione might be considered to have two equal sites of donor groups and cystinyldiglycine to have two acceptor sites (two cystinyl amino groupings) the reaction was studied and calculations were made as: half oxidized glutathione half cystinyldiglycine
+ glycylglycine z + -r-glutamylglycylglycine.
The equilibrium was studied from the right with a fixed level of oxidized glutathione and varied levels of glycylglycine and in the reverse direction with a
and
fixed level of cystinyldiglycine and varied levels of y-glutamylglycylglycine. In the forward reaction the appearance of cystinyldiglycine was measured and in the reverse reaction the disappearance of cystinyldiglycine was measured by the method of Sullivan and Hess (7). Equilibrium was found to be established under the conditions employed (1.5 fig protein N per ml) between 30 minutes for the lower levels and 2 hours for the higher levels of substrate, but all reactions were followed for at least 24 hours. With the higher levels of substrate, it was established that the addition of large amounts of extra enzyme, after equilibrium was established, was without effect on the equilibrium. Representative results are given in Table III. The forward reaction, half oxidized glutathione plus glycylglycine, was found to have an equilibrium constant of slightly
300
LEIBACH TABLE STUDIES
AND
III
OF THE EQUILIBRIUM TRaNSFERaSE RE.~CTION
OF THE
Three and three-tenths pmole half-oxidized glutathione or half cystinyldiglycine and the indicated number of rmole of glycylglycine or yglutamylglycylglycine were present in each milliliter of incubation mixture together with 1.5 pg of protein N at pH 8.0 in 0.1 M Tris buffer and incubation was at 30” for up to 24 hours; equilibrium was found to be established in from 30 minutes to 2 hours. Balf cystinyldiglycine formed pm&/ml
Glycylglycine added qnole/ml Forward
reaction,
oxidized glycylglycine
2.5 5.0 8.0 12.0 20.0
glutathione
1.9 2.3 2.7 2.9 3.1
2.3 2.0 2.2 2.2 2.4
Avg. y-glutamylglycylglycine added, pm&/ml Reverse
1.0 5.0 10.0 Avg.
plus
2.2 Half cystinyldiglycine used, pm&/ml
K-2 3.3 pm& basis
reaction, cystinyldiglycine y-glutamylglycylglycine 0.50 1.05 1.20
5.0 8.3 12.5
KW 1.65 #mole basis plus
1.9 2.1 2.3 2.1
more than two indicating that the reaction favored the transfer from glutathione. Studies of the reverse reaction gave peculiar results in that the equilibrium constant increased as the concentration of yglutamylglycylglycine (up to 50 @mole per ml); the disappearance of cystinyldiglycine approached very closely to but never exceeded 1.65 pmole or half of the total possible in terms of the reaction as formulated above. When the constants for the reverse reaction were recalculated on the basis that cystinyldiglycine accepted only one r-glutamyl grouping, the same constants were obtained at all the tested levels of r-glutamylglycylglycine and these constants were very nearly the same as found for the forward reaction. While there may be explanations other than the preferential transfer of only one giutamyl grouping to cystinyldiglycine, they are not
BINKLEY readily apparent. It would be desirable to prepare the mixed disulfide of glutathione and cysteinylglytine for further studies but direct attempts at such preparations have failed to produce the desired product in an unequivocal manner.
DISCUSSION Aside from the earlier report of the absolute requirement for an acceptor for the activity of the transferase, there are a number of points of evidence that the transferase is distinct from the material described as an arylamide “transpeptidase” (4). The transferase is totally inactive with r-glutamylnaphthylamide as a substrate under a spectrum of conditions of time, pH, and concentration of enzyme or substrate and with or without potential acceptors. The incubation of a 1:; solution of the transferase with 1.25 pmole per ml of naphthylamide for 3 hours at 37” did not result in the release of detectable amounts of naphthylamine. Glutamic acid could not be detected as a product of the action of the transferase on reduced or oxidized glutathione with or without acceptors present. Also, the transferase does not hydrolyze glutamine and does not form hydroxamates with reduced or oxidized glutathione as substrates. The transferase has a sharp optimal pH near 8.0, whereas the reported optimum for the arylamide “transpeptidase” was 8.8 (4). The transferase is devoid of carbohydrate (less than 1 s‘(‘), whereas the arylamide “transpeptidase” was reported to contain about 20Yr carbohydrate. Also, the transferase was eluted from DEAE cellulose (pH 8.0-9.0) with a low concentration of salt (0.08 M Tris-HCl) whereas it was reported that 0.05 M Tris-citrate buffer plus 0.15 M NaCl was required to elute the arylamide In addition treatment “transpeptidase.” with heat can not be used in the purification of the transferase for almost all activity is destroyed by very short treatment at 55O and treatment with organic solvents (such as butanol or chloroform and octanol) was found to destroy most of the activity. Thus, there seems to be no relationship between the transferase and those enzymes active in the hydrolysis of the arylamides.
y-GLUTAMYL
On the other hand, we cannot be certain that the arylamide hydrolase isolated by us is not at least a component of the system described as “transpeptidase” (4). A major discrepancy is that we find the hydrolase to be totally inactive with reduced or oxidized glutathione as substrates either in the presence or absence of potential acceptors. No evidence could be obtained for the formation of any products other than glutamic acid and naphthylamine in the hydrolysis of the naphthylamide by the hydrolase; however, we did not employ the levels of substrate employed by others (4) and long periods of incubation. Our substrate levels for the study of products was 1.25 Fmole per ml and the time of incubation was up to 30 minutes (as contrasted with 5 hours). The naphthylamide is sparingly soluble and we could see little or no purpose in working with a two-phase system. We have not investigated the formation of hydroxamates by our preparation of the hydrolase and can state only that it is totally inactive in the hydrolysis of leucyl- , alanyl-, and prolyl naphthylamides and simple dipeptides. The complications encountered in the determination of the equilibrium constants in the system utilizing oxidized glutathione are such that it would be premature to place any emphasis on a possible change in free energy in the transfer of the glutamyl grouping. Later studies made with the transferase with r-glutamylglycylglycine and y-glutamylmethionine as the donors and with glycylglycine and methionine as the acceptors gave equilibrium constants near unity. Sometime after the conclusion of these studies, the inhibitory effects of bromcresol green upon the arylamide hydrolase were studied; bromcresol green is markedly inhibitory of the transferase in con-
301
TRANSFERASE
centrations of 2 x 10e5 M and the inhibition is removed competitively by glycylgiycine (2). Bromcresol green at a concentration of 10 -’ M was only slightly inhibitory of the hydrolase and glycylglytine was ineffective in the removal of the inhibition. Thus, the additional studies confirm the conclusion that the transferase is distinct from the activities responsible for the hydrolysis of the yglutamylarylamides. REFERENCES 1. LEIBACH, F. H., AND BINKLEY, F., Fed. Proc. 23 (2), 524 (1964). 2. BINKLEY, F., J. Biol. Chem. 236, 1075 (1961). 3. OLSON, C. K., AND BINKLEY, F., J. Biol. Chem. 186,731(1950). 4. ORLOWSKI, M., AND MEISTER, A., J. Biol. Chem. 240, 338 (1965). 5. SZEWCZUK, A., AND BARANOWSKI, T., Biochem. Z. 338, 317 (1963). P. J. 6. GLENNER, G. G., FOLK, J. E., AND MCMILLAN, J. Histochem. Cytochem. 10, 481 (1962). SULLIVAN, M. X., AND HESS, W. C., J. Biol. Chem. 116, 221 (1936). 8. GOLDBARG, d. A., AND RUTENBURG, A. M., Cancer 11, 283 (1958). 9. WINZLER, R. J., in “Methods of Biochemical Analysis” (David Glick, ed.), Vol. II., Interscience Publishers, New York (1955). 10. FALES, F., J. Biol. Chem. 193, 113 (1951). 11. ORNSTEIN, L., Ann. N.Y. Acad. Sci. 121, 321 (1964). 12. DAVIS, B. .J., Ann. N.Y. Acad. Sci. 121, 404 (1964). 13. BOLTON, E. T., BRITTEN, R. J., COWIE, D. B., MCCARTHY, B. J., MCQUILLEN, K., AND ROBERTS, R. B., “Carnegie Institution of’ Washington Yearbook,” No. 58, p. 259. The Carnegie Institution, Washington, D.C. (1959). 14. MARTIS, R. G., AND AMES, B. N., J. Biol. Chem. 236, 1372 (1964). 15. BINKLEY, F., DAVENPORT, J., AND EASTALL, F., Biothem. Biophys. Commun. 1, 206 (1959). 16. ROBINSON, D. S., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 202, 1 (1953).
OF BIOCHEMISTRY
AND BIOPHYSICS
FGlutamyl
127,292-301
(1968)
Transferase
F. H. LEIBACH”
AND
of Swine FRANCIS
Kidney’
BINKLEY
Department of Biochemistry, Division of Basic Health Sciences, Emory University, Atlanta, Georgia 30322 Received
December
26, 1967; accepted
March
22, 1968
The enzyme of renal tissue responsible for the transfer of the r-glutamyl grouping of glutathione to various acceptors was solubilized by a brief digestion with ficin and purified to apparent homogeneity by fractionation with ethanol and ammonium sulfate, chromatography on DEAE-cellulose, gel filtration with Sephadex, and gradient centrifugation. The enzyme was found to establish an equilibrium between products and reactants. Glutamic acid was not a product of the reaction and the enzyme was entirely without activity in the absence of an acceptor. The purified enzyme did not hydrolyze -r-glutamylnapthylamide and did not transfer the y-glutamyl grouping of this compound to potential acceptors. An enzyme responsible for the hydrolysis of y-glutamylnaphthylamide was separated from renal tissue and was found to be not influenced in its activity by the addition of acceptors. This enzyme was without activity in the hydrolysis of glutathione or of arylamide derivatives of other amino acids.
These studies were designed to investigate the mode of action of the enzyme of renal tissue responsible for the transfer of the r-glutamyl grouping of glutathione to appropriate acceptors. We had previously reported that the requirement for an acceptor was absolute; there was no hydrolysis of glutathione in the absence of an appropriate amino acid or peptide (2). The absolute requirement for an acceptor presented the possibility that the transfer was an equilibrium reaction and various methods were devised to test this possibility. Reduced glutathione presented many problems in chromatography and the product of the transfer, cysteinylglycine, was difficult to obtain in adequate purity and, in solution, was ’ These studies were supported by grant AM06089 of the United States Public Health Service. This is publication No. 886 of the Division of Basic Health Sciences. A preliminary report has appeared (1). ‘This work was taken from a thesis submitted by F. H. Leibach to the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Spring, 1964. Present address, Department of Biochemistry, Medical College of Georgia, Augusta.
converted to the diketopiperazine (3). Thus, it was not possible to study the reaction mixture over extended periods of time with glutathione as the substrate. Oxidized glutathione was found to be an equivalent substrate for the transfer enzyme and the product of the transfer from oxidized glutathione, cystinyldiglycine, was readily prepared and was stable in solution under the conditions employed. At the time these studies were in progress, the y-glutamylarylamides were introduced as substrates for the detection of enzymes active in the hydrolysis of -,-glutamyl compounds and it was inferred by several authors that the enzymes responsible for the release of arylamines from such compounds were identical with the transfer enzyme (4, 5, 6). Thus, it is of interest to report our results with an enzyme active in the hydrolysis of y-glutamylnaphthylamide that was clearly separated from the transferase. METHODS
AND
MATERIALS
Reduced glutathione was used in the routine assay of transferase activity as in the previous publication (2). The assay was with 0.012 M glycylglytine as the acceptor with 0.003 M glutathione. 0.001 292
r-GLUTAMYL magnesium acetate at pH 8.0 in 0.01 M Tris buffer; the time of incubation was 30 minutes and the reaction was stopped by the addition of trichloroacetic acid to a final concentration of 5rr. Cysteinylglycine was determined by the method of Sullivan and Hess (7). Units were expressed as pmol per min and specific activity as units per mg total nitrogen of the preparation (micmKjeldah1). For chromatographic studies, oxidized glutathione was substituted at the same concentration of y-glutamyl groupings (0.0015 M oxidized glutathione). In the studies of the reverse reaction cystinyldiglycine (cystinyl-bis-glycine) was used at a molarity of 0.0033 M. The appearance or disappearance of cystinyldiglycine (appearance in the forward reaction and disappearance in the reverse reaction with yglutamylglycylglycine as the donor of the y-glutamyl grouping) was followed by the method of Sullivan and Hess (7); it should be noted that cystinyldiglycine yields half as much color in this reaction as does cysteinylglycine (2). Reduced and oxidized glutathione were obtained from Schwarz BioResearch and r-glutamylglycylglycine, glycylglycine, and cystinyldiglycine were obtained from Cycle Chemical Corporation. Chromatography of the reaction mixtures with the solvent system butanol : acetic acid : water (60 : 15 : 25) with Whatman No. 3 filter paper in a descending system was found to separate all the components of the test system (oxidized glutathione, glycylglycine, cystinyldiglytine, and r-glutamylglycylglycine) as well as glycine and glutamic acid. In order to provide additional information as to the mechanism of action of the en“C-glycylglycine (“C-1-4) (California Corpo zyme, ration for Biochemical Research) was employed in studies with oxidized glutathione and also with yglutamylglycylglycine as the donor of -/-glutamy groupings. The reaction mixture for the routine assay of the enzyme active in the hydrolysis of yglutamylnaphthylamide (hydmlase activity) conmined r-glutamylnaphthylamide, 0.00125 M, 0.1 ml of an appropriately diluted enzyme solution, and Tris buffer, final concentration 0.1 M and pH 8.0, in a total volume of 2 ml. The solution was incubated at 37’ for lo-30 minutes and the liberated naphthylamine was determined as described by Goldberg and Rutenberg (8). Analyses for carbohydrate were made at various stages of purification by the methods of Winzler (9) for protein-bound hexose and by the anthrone method of Fales (10) for total carbohydrate. Polyacrylamide disc electrophoresis, by adaptations of the procedures of Omstein (11) and Davis (12), was used to check the products at each stage of the purification. Sucrose density gmdients. Gradients were prepared according to the method of Bolton et al. (13) and fractions were collected with drop counting after centrifugation by puncturing the base of the M
TRANSFERASE
293
centrifuge tube with a hypodermic needle in an apparatus similar to that of Martin and Ames (14). The gradients were prepared with 14.7 ml of a 30’;. and 15.3 ml of a 7.5?( solution of sucrose. One ml of sample containing 10 mg or less of the preparations was layered onto the gradient and centrifugation was in the SW-25 swinging bucket rotor of the Spinco Model L centrifuge. Purification of the tmmfeme. Solubilization of the transferase may be achieved by treatment of renal microsomes with deoxycholate or detergents (15), but these materials complicate the purification and the products are often highly contaminated with extraneous glycoproteins. Brief digestion with ficin was found to release most of the activity into solution and the activity thus released was easily purified by conventional methods. Ficin was found to have no activity in the release of cysteinylglycine from glutathione or cystinyldiglycine from oxidized glutathione in the absence or in the presence of acceptors. The purification is outlined in Fig. 1 and summarized in Table I. Frozen swine kidneys were allowed to thaw partially and the cortical tissue was removed and dissected free of connective tissue and fat. Ten-pound batches of the cortical tissue were ground in a meat grinder and homogenized in a Waring Blendor with 25 liters of 0.1 M MgCb at room temperature. Crude &in (Nutritional Biochemical Corp.) was added to the homogenate to yield a hnal concentration of 3 mg per ml. Toluene was added to prevent bacterial growth and the homogenate was incubated at 37” for 4 hours. Suspended material was removed by centrifugation with the Intemational PR-2 centrifuge for 30 min at O” and at 2300 rpm. Cold ethanol (4/5 volume) was added to the SUMMARY
OF PURIFICATION
KIDNEY
CORTEX HOMOGENIZE DIGEST wth
PREClblTATE
FICIN
SUPERAATANT
PRECIPITATE (DISCARD)
415 VeLUME ETHANOL
I
I
SUPERNATANT
PREClPlTAiE
'"'s;D'
IIN
BUFFER1
;~;$C$N,"M
SUPERNATANT
PRECIPITATE IDISCARD)
80% AMMONIUM SULFATE
1 SUPERNATANT IDISCARD)
FIG. 1. Scheme transterase.
1 PRECIPITATE
of preliminary
iIN
purification
BUFFERI
of the
294
LEIBACH
AND TABLE
PURIFICATION
Homogenate” Supernatant Ethanol precipitate Ammonium sulfate 80% DEAE step-wiseb Sephadex G-100’ Sephadex G-100” second Sucrosec gradient
I
OF THIS: TRANSFERASIC Volume (ml)
step
BINKLEY
9,500 8,200
i,200 1,200 1,100
Nitrogen (W)
52,250 32,800 7,560 1,080 48
Total activity (pmole/min)
Specific activity C,mmle/mg NJ
139,000 134,009 79 ) 000 22,800 15,580
2.66 4.10 10.4 21.1 333 700 5,ooo 5,000
time
a Four pounds of renal cortex in 10 1. b Five columns as described in Fig. 2. c Yields were essentially quantitative for amounts supematant solution and after 8 hours at 4” the precipitate was removed by centrifugation and the supematant solution was discarded. The precipitate was dissolved in a minimal amount of 0.01 M TrisHCl buffer, pH 8.0, and was dialyzed against the same buffer until free of ethanol. The solution was clarified by centrifugation and ammonium sulfate was added to the supematant solution to produce 60:; saturation. The solution was permitted to stand at 4’ for 6 hours and was centrifuged at 2300 rpm for 1 hour. The precipitate was discarded and solid ammonium sulfate was added to the supematant solution to produce 80% saturation. The solution was permitted to stand at 4’ for 6 hours and was centrifuged at 2300 rpm for 1 hour. The precipitate was discarded and solid ammonium sulfate was added to the supematant solution to produce 80:; saturation. The solution was permitted to stand at 4’ for 8 hours and the precipitate was collected by centrifugation and dissolved in a minimal amount of the 0.01 M Tris buffer. The solution was dialyzed against the same buffer until free of ammonium sulfate, lyophilized, and stored in the dry state in the cold. This material is essentially free of carbohydrate and is an excellent starting material for the isolation of the transferase, the r-glutamylamidase, leucylarylamidase and the solubilized aminopeptidase of Robinson et 01. (16). Chromatogmphic purification. All chromatography was carried out at room temperature. 1.0 gm of the lyophilized material was dissolved in 100 ml of the 0.01 M Tris buffer and placed on a column of DEAE-cellulose (S & S type 40, 2.5 X 50 cm) equilibrated with the same buffer. The column was washed successively with 0.01 M, 0.08 M, and 0.08 M Tris-HCl plus 0.15 M NaCl all at pH 8.0. The starting buffer eluted three inactive fractions and the transferase was eluted with 0.08 M Tris buffer; the r-glutamylnaphthylamidase (hydrolase) was eluted with the salt solution (Fig. 2). The discontinuous
of material
up to 10 mg protein
nitrogen
elution was very effective in the resolution of the two activities whereas continuous gradients were relatively ineffective either in separation of the two activities or in further purification of the transferase. Thirty ml of a solution of the transferase (up to 10 mg) was re-equilibrated with 0.01 M Tris-HCl buffer and placed on a column of Sephadex G-100, 2.5 X 150 cm, equilibrated with the same buffer; elution was with the same buffer. The transferase was retarded (Fig. 3) and, after a second passage through the column of Sephadex, was homogeneous as judged by gradient centrifugation (Fig. 4) and by disc electrophoresis (Fig. 5). The -y-glutamylnaphthylamidase was not retarded by Sephadex G-100 but was further purified by gradient centrifugation; curve II (Fig. 4) is for material recovered from preliminary gradient centrifugation. Curve I is for a leucyl-8-naphthylamidase prepared from endoplasmic membranes by similar procedures. In Table II results with several renal enzymes are summarized; all materials were run and the gradients were calibrated with proteins of known molecular weights under identical conditions. The overall purification of the napthylamide hydrolase is not described because we have not prepared the enzyme except from the eluate from DEAE-cellulose. The material at that stage had a specific activity of about 10 pmole naphthylamide per minute per mg protein (N X 6.25). Subsequently, the yield was essentially quantitative in the gel filtration with Sephadex and with gradient centrifugation. No claim is made for the homogeneity of the preparation; specific activity of about 100 pmole per minute per mg protein (N X 6.25) was obtained for the material from gradient centrifugation in the assay with 1.25 rmole naphthyiamide per ml at pH 8.0 and 37’. Other workers have reported a specific activity near 109 with a higher level of substrate and at a higher pH (4) for what may be a preparation of the same enzyme.
y-GLUTAMYL 0.01 M
TRANSFERASE
TRIS
0.08
M
295
TRIS
0.15 0.08 1
i
M M
NaCl TRIS
TRANSFERASE
ML
ELUATE
FIG. 2. Elution of the activities from DEAE-cellulose with a discontinuous gradient. Activity was assayed as described in the text with reduced glutathione plus glycylglycine and with r-glutamylnaphthylamide. There was no cross activity in the two peaks and there was no activity in the hydrolysis of reduced or oxidized glutathione in the absence of glycylglycine in any of the samples (8 ml per tube).
TRANSFERASE SEPHADEX
VOIDVOLUME /
I 20
with dex.
1 60
40
TUBE FIG.
G/O0 2x
NUMBER
3. Further purification Sephadex G-100; second
of time
the transferase through Sepha-
Preparations of the y-glutamylarylamidases by others (4) have been reported to contain a high level of carbohydrate; carbohydrate was not detectable (less than lS,,) in the final preparations of either activity. Absorbancy in the ultraviolet and changes in absorbancy upon the addition of alkali or of acid were those commonly observed with simple proteins and there was no evidence for the presence of materials other than protein in either preparation. Mechanism of action of the trunsfemse. In the studies of activity described here only the transferase from gradient centrifugation with a specific ac-
4
8
12
16
20
24
28
32
ML
FIG. 4. Gradient centrifugation of three renal enzymes. Curve 1, leucyl-p-naphthylamidase isolated from endoplasmic membranes, Curve II, r-glutamylnaphthylamidase recentrifuged after a preliminary gradient centrifugation, and Curve III, the transferase. In all cases activity tracked completely with the absorbancy.
tivity of 5,000 @mole per minute per mg N was used. Amounts of enzyme up to 10 rg per ml were used in certain experiments, but the usual concentration of enzyme was 0.15 rg per ml. In the absence of the acceptor, there was no reaction with the transferase with oxidized glutathione or reduced glutathione as judged by the formation of material reacting in the method of Sullivan and Hess (7). With oxidized glutathione as the substrate but in the absence of
296
LEIBACH
AND
BINKLEY TABLE
II
SUMMARY OF SUCROSE GRADIENTS WITH SOME RENAL ENZYMES Thirty ml sucrose, 30-7~7~ (w/v), with 1 ml sample was centrifuged for 19 hours in the SW-25 rotor of the Spinco Model L centrifuge at 25,000 rpm with a thermostat setting of 21”. Thirty-two samples were collected by drop counting and the location of the enzyme was determined by assay for the activity and, when possible, by absorbancy at 280 w. The samples contained from 1 to 10 mg protein. Enzyme
Leucine aminopeptidase” Leucyl-fi-naphthylamidasec r-glutamylnaphthylamidase r-glutamyl transferase LeucylgIycinased
FIG. 5. Disc electrophoresis of, left to right, yglutamyl transferase from DEAE-cellulose, middle, the transferase after twice through Sephadex, and, right, the r-glutamyl naphthylamidase after gradient centrifugation. All were at pH 8.0 with 20 ~1 of a 1% solution. acceptor glycylglycine, there was no reaction with the transferase as judged by the appearance of cystinyldiglycine in amounts detectable by paper chromatography; glutamic acid could not be detected after long periods of incubation (3 hours) with amounts of enzyme one hundred fold greater than that necessary to give maximal transfer in 10 minutes in the presence of the acceptor. No naphthylamine was released and no materials reactive with ninhydrin, other than the substrate, could be detected in paper chromatography after incubation of a lc; solution of the transferase with the r-glutamylnaphthylamide (1.25 pmole per ml) for up to 3 hours. But, in the complete system, with oxidized glutathione and glycylglycine, the reaction proceeded with the formation of y-glutamylglycylglytine and cystinyldiglycine. As is illustrated in Fig. 6, as the intensity of the reaction of the oxidized glutathione and glycylglycine with ninhydrin de-
Peak tube
Molecularweight estimate
17 21 23
240,000* 170,000 110,000
26 26
80,000 80,000
a Soluble enzyme active in the hydrolysis of leucinamide and leucylglycine but not leucyl-flnaphthylamide; manganous ion required for activity. * This is for the main form of the enzyme; other fully active but minor components were found to have apparent molecular weights of 150,000 and 300,000. These minor components when recentrifuged in the gradient gave the same distribution as the original material. c Microsomal enzyme active in the hydrolysis of leucyl-p-naphthylamide and leucylglycine but not Ieucinamide; no metal ion requirement. d Microsomal enzyme active in the hydrolysis of leucylglyciue but not leucinamide or leucyl-pnaphnaphthylamide; no metal ion requirement,. This appears to be the solubilized aminopeptidase of Robinson et al. (16). creased, there was a corresponding increase in the reaction with glutamylglycylglycine and cystinyldiglycine. At the concentration of substrate used, there was no detectable change as judged by the reaction with ninhydrin on the paper chmmatograms in the relative concentration of the four components in samples taken after a few minutes of incubation. There was no indication of the presence of glutamic acid or of other materials reacting with ninhydrin in any of the chromatograms even with extended incubation. Chromatograms revealing only the same four components were obtained when cystinyldiglytine (0.0033 M) was incubated with y-glutamylglycylglycine (0.012 M) under the same conditions; the chromatogram, after 30 minutes, was indistinguishable from that given in the last column of Fig. 6.
-/-GLUTAMYL
product 0’
Tronspeptldose occumulatfon 5l
reaction with time 15’
30’
‘Jygly
0000 I 0
I
I
I
5
15
30
Time
TRANSFERASE
in minutes
FIG. 6. Accumulation of products at indicated times after addition of enzyme under standard conditions of assay with 0.15 pg protein N per ml, 0.012 M glycylglycine, 0.003 M oxidized glutathione, pH 8.0 in 0.01 M Tris buffer. 100 ~1 samples containing 5’( TCA were subjected to chromatography and were sprayed with ninhydrin. With a migration of the solvent front of 40 cm, the approximate RF values were oxidized glutathione 5, cystinyldiglycine 8, y-glutamylglycylglycine 16, glycylglycine 24, glytine 29, and glutamic acid 33. The latter two compounds were not detected but would be at the end of the section of the chromatogram reproduced here. The origin is indicated by the dark line. As is pointed out in the studies of the equilibrium detailed below, there is reason to believe that the product in the reverse direction is the mixed disulfide of glutathione and cysteinylglycine but there was no indication of the separation of this compound from the oxidized glutathione in the chromatographic system employed. Although there was no reason to suspect that glylcylglycine might be hydrolyzed in some reaction such that r-glutamylglycine and free glycine might be formed, the possibility was checked with “Cglycylglycine as the acceptor. The reaction mixture (after 30 minutes) was subjected to chromatography and the strip was scanned with a strip counter (Actigraph, Model II, Nuclear-Chicago) and, as is illustrated in Fig. 7, there was an accumulation of labeled r-glutamylglycylglycine without the appearance of glycine. Thus, ,the glycylglycine was not cleaved in the reaction. Then, since the same products are formed from oxidized glutathione plus glycylglycine as from y-glutamylglycylglycine and cys-
297
tinyldiglycine, it should be possible to obtain evidence that the y-glutamyl grouping is transferred back to the product remaining from the transfer of the glutamyl grouping to the enzyme. ‘“C-Glycylglycine was incubated with the transferase together with y-glutamylglycylglycine and, as was anticipated, there was an exchange of the labeled glycylglycine into the r-glutamylglycylglycine (Fig. 8). It is planned to test directly the possibility of the transfer of the glutamyl grouping to a stable position on the enzyme. Parallel studies were made with the naphthylamide hydrolase but at a concentration of enzyme of 10 pg per ml (or about 100-fold that of the transferase as detailed above); oxidized glutathione, glycylglycine, and the other materials were present at the levels described for the above studies. The experiments were ali negative in terms of evidence for a transfer reaction to an added acceptor. With oxidized glutathione or reduced glutathione as the substrate, there was no release of cystinyldiglycine or cysteinylglycine as measured by the method of Sullivan and Hess (7) either in the presence or in the absence of glycylglycine. When “C-glycylglytine was added together with oxidized glutathione with the naphthylamide hydrolase, there was no indication of the formation of any labeled product in the chromatograms prepared from the incubation mixture and there was no incorporation of the labeled glycylglycine into y-glutamylglycylglycine by the hydrolase. Thus, there was no evidence of any activity of the hydmlase with oxidized or reduced glutathione as the substrate. Then, similar studies were undertaken with the naphthylamide hydrolase with y-glutamylnaphthylamide as the substrate to determine if there might be some transfer reaction to an added acceptor along with the obvious hydrolysis. The amount of enzyme employed was about 10 rg per ml or an amount far in excess of that necessary to catalyze the release of 95-100’; of the available naphthylamine from 1.25 pmole of naphthylamide per ml at pH 8.0 in 10 minutes. The solubility of the naphthylamide precluded studies at higher levels of substrate. With the naphthylamide as the substrate, chromatograms were developed as above at various times; the only compounds detected that reacted with ninhydrin were the substrate and glutamic acid with no indication of other products. Then, glycylglycine was added together with the naphthylamide. With 1.25 pmole naphthylamide and 12.0 pmole glycylglycine per ml there was no effect on the rate of appearance of naphthylamine over the controls with the naphthylamide alone and the chromatograms revealed a small amount of substrate, glutamic acid, and the added glycylglycine as the materials reacting with ninhydrin. Then “C-glycylglycine was substituted for the unlabeled glycylglycine, chromatograms were
298
LEIBACH Transpeptidase
AND -
BINKLEY Actigraph
y-Glu Glygly
CllYg’Y
-
Scanning
Cys digly
GSSG
“C-glycylglycine. Conditions were as in FIG. 7. Labeling of y-glutamylglycylglycine with Fig. 6 with incubation for 30 minutes at 37” and solvent migration in chromatography (to the left) was approximately 30 cm. The origin is indicated by the dark line. prepared and the strips were scanned with the strip counter as above. The only radioactivity was found at the position of glycylglycine as in the zero time samples with the transferase with oxidized glumthione and labeled glycylglycine as in Fig. 7. Thus, we were unable to demonstrate any reaction with the naphthylamide hydrolase other than the hydrolysis of the naphthylamide with an essentially theoretical release of naphthylamine. These studies may be criticized because of the relative low level of substrate as compared with the work of others (4) and the much shorter period of incubation. The major point to be made is that we have been unable to demonstrate the release of less than very nearly the theoretical amount of naphthylamine with or with-
out an acceptor present and no evidence was obtained for any transfer of a glutamyl grouping to glycylglycine by this enzyme either from glutathione or from the naphthylamide; this behavior is in complete contrast to the behavior of the transferase. Otherwise, we have little knowledge of the behavior of the naphthylamide hydrolase; it is inactive in the hydrolysis of alanyl-, leucyl-, and prolylnaphthylamides and simple dipeptides of leucine and alanine. Equilibrium studies. From the studies of the products of the reaction and from the experiments with ‘“C-glycylglycine it appeared evident that the transferase established an equilibrium between a pair of donors and a pair of acceptors. Thus, the re-
y-GLUTAMYL Transpeptidose
299
TRANSFERASE -
Actigraph
Scanning
y-
Glygb
Glu - glyglycine
y - w3lYillY
FIG. 8. Labeling of r-glutamylglycylglycine with ‘“C-glycylglycine with r-glutamylglycylglycine, 5 pmole per ml, substituted for oxidized glutathione under standard conditions with 0.15 pg protein N per ml. Incubation was for 30 minutes at 37’ at pH 8.0. action with oxidized might be written as: GSSA
+ 2 glygly
glutathione
and
glycylglycine
z cystinyldiglycine + 2 -y-glutamylglycylglycine
However, since oxidized glutathione might be considered to have two equal sites of donor groups and cystinyldiglycine to have two acceptor sites (two cystinyl amino groupings) the reaction was studied and calculations were made as: half oxidized glutathione half cystinyldiglycine
+ glycylglycine z + -r-glutamylglycylglycine.
The equilibrium was studied from the right with a fixed level of oxidized glutathione and varied levels of glycylglycine and in the reverse direction with a
and
fixed level of cystinyldiglycine and varied levels of y-glutamylglycylglycine. In the forward reaction the appearance of cystinyldiglycine was measured and in the reverse reaction the disappearance of cystinyldiglycine was measured by the method of Sullivan and Hess (7). Equilibrium was found to be established under the conditions employed (1.5 fig protein N per ml) between 30 minutes for the lower levels and 2 hours for the higher levels of substrate, but all reactions were followed for at least 24 hours. With the higher levels of substrate, it was established that the addition of large amounts of extra enzyme, after equilibrium was established, was without effect on the equilibrium. Representative results are given in Table III. The forward reaction, half oxidized glutathione plus glycylglycine, was found to have an equilibrium constant of slightly
300
LEIBACH TABLE STUDIES
AND
III
OF THE EQUILIBRIUM TRaNSFERaSE RE.~CTION
OF THE
Three and three-tenths pmole half-oxidized glutathione or half cystinyldiglycine and the indicated number of rmole of glycylglycine or yglutamylglycylglycine were present in each milliliter of incubation mixture together with 1.5 pg of protein N at pH 8.0 in 0.1 M Tris buffer and incubation was at 30” for up to 24 hours; equilibrium was found to be established in from 30 minutes to 2 hours. Balf cystinyldiglycine formed pm&/ml
Glycylglycine added qnole/ml Forward
reaction,
oxidized glycylglycine
2.5 5.0 8.0 12.0 20.0
glutathione
1.9 2.3 2.7 2.9 3.1
2.3 2.0 2.2 2.2 2.4
Avg. y-glutamylglycylglycine added, pm&/ml Reverse
1.0 5.0 10.0 Avg.
plus
2.2 Half cystinyldiglycine used, pm&/ml
K-2 3.3 pm& basis
reaction, cystinyldiglycine y-glutamylglycylglycine 0.50 1.05 1.20
5.0 8.3 12.5
KW 1.65 #mole basis plus
1.9 2.1 2.3 2.1
more than two indicating that the reaction favored the transfer from glutathione. Studies of the reverse reaction gave peculiar results in that the equilibrium constant increased as the concentration of yglutamylglycylglycine (up to 50 @mole per ml); the disappearance of cystinyldiglycine approached very closely to but never exceeded 1.65 pmole or half of the total possible in terms of the reaction as formulated above. When the constants for the reverse reaction were recalculated on the basis that cystinyldiglycine accepted only one r-glutamyl grouping, the same constants were obtained at all the tested levels of r-glutamylglycylglycine and these constants were very nearly the same as found for the forward reaction. While there may be explanations other than the preferential transfer of only one giutamyl grouping to cystinyldiglycine, they are not
BINKLEY readily apparent. It would be desirable to prepare the mixed disulfide of glutathione and cysteinylglytine for further studies but direct attempts at such preparations have failed to produce the desired product in an unequivocal manner.
DISCUSSION Aside from the earlier report of the absolute requirement for an acceptor for the activity of the transferase, there are a number of points of evidence that the transferase is distinct from the material described as an arylamide “transpeptidase” (4). The transferase is totally inactive with r-glutamylnaphthylamide as a substrate under a spectrum of conditions of time, pH, and concentration of enzyme or substrate and with or without potential acceptors. The incubation of a 1:; solution of the transferase with 1.25 pmole per ml of naphthylamide for 3 hours at 37” did not result in the release of detectable amounts of naphthylamine. Glutamic acid could not be detected as a product of the action of the transferase on reduced or oxidized glutathione with or without acceptors present. Also, the transferase does not hydrolyze glutamine and does not form hydroxamates with reduced or oxidized glutathione as substrates. The transferase has a sharp optimal pH near 8.0, whereas the reported optimum for the arylamide “transpeptidase” was 8.8 (4). The transferase is devoid of carbohydrate (less than 1 s‘(‘), whereas the arylamide “transpeptidase” was reported to contain about 20Yr carbohydrate. Also, the transferase was eluted from DEAE cellulose (pH 8.0-9.0) with a low concentration of salt (0.08 M Tris-HCl) whereas it was reported that 0.05 M Tris-citrate buffer plus 0.15 M NaCl was required to elute the arylamide In addition treatment “transpeptidase.” with heat can not be used in the purification of the transferase for almost all activity is destroyed by very short treatment at 55O and treatment with organic solvents (such as butanol or chloroform and octanol) was found to destroy most of the activity. Thus, there seems to be no relationship between the transferase and those enzymes active in the hydrolysis of the arylamides.
y-GLUTAMYL
On the other hand, we cannot be certain that the arylamide hydrolase isolated by us is not at least a component of the system described as “transpeptidase” (4). A major discrepancy is that we find the hydrolase to be totally inactive with reduced or oxidized glutathione as substrates either in the presence or absence of potential acceptors. No evidence could be obtained for the formation of any products other than glutamic acid and naphthylamine in the hydrolysis of the naphthylamide by the hydrolase; however, we did not employ the levels of substrate employed by others (4) and long periods of incubation. Our substrate levels for the study of products was 1.25 Fmole per ml and the time of incubation was up to 30 minutes (as contrasted with 5 hours). The naphthylamide is sparingly soluble and we could see little or no purpose in working with a two-phase system. We have not investigated the formation of hydroxamates by our preparation of the hydrolase and can state only that it is totally inactive in the hydrolysis of leucyl- , alanyl-, and prolyl naphthylamides and simple dipeptides. The complications encountered in the determination of the equilibrium constants in the system utilizing oxidized glutathione are such that it would be premature to place any emphasis on a possible change in free energy in the transfer of the glutamyl grouping. Later studies made with the transferase with r-glutamylglycylglycine and y-glutamylmethionine as the donors and with glycylglycine and methionine as the acceptors gave equilibrium constants near unity. Sometime after the conclusion of these studies, the inhibitory effects of bromcresol green upon the arylamide hydrolase were studied; bromcresol green is markedly inhibitory of the transferase in con-
301
TRANSFERASE
centrations of 2 x 10e5 M and the inhibition is removed competitively by glycylgiycine (2). Bromcresol green at a concentration of 10 -’ M was only slightly inhibitory of the hydrolase and glycylglytine was ineffective in the removal of the inhibition. Thus, the additional studies confirm the conclusion that the transferase is distinct from the activities responsible for the hydrolysis of the yglutamylarylamides. REFERENCES 1. LEIBACH, F. H., AND BINKLEY, F., Fed. Proc. 23 (2), 524 (1964). 2. BINKLEY, F., J. Biol. Chem. 236, 1075 (1961). 3. OLSON, C. K., AND BINKLEY, F., J. Biol. Chem. 186,731(1950). 4. ORLOWSKI, M., AND MEISTER, A., J. Biol. Chem. 240, 338 (1965). 5. SZEWCZUK, A., AND BARANOWSKI, T., Biochem. Z. 338, 317 (1963). P. J. 6. GLENNER, G. G., FOLK, J. E., AND MCMILLAN, J. Histochem. Cytochem. 10, 481 (1962). SULLIVAN, M. X., AND HESS, W. C., J. Biol. Chem. 116, 221 (1936). 8. GOLDBARG, d. A., AND RUTENBURG, A. M., Cancer 11, 283 (1958). 9. WINZLER, R. J., in “Methods of Biochemical Analysis” (David Glick, ed.), Vol. II., Interscience Publishers, New York (1955). 10. FALES, F., J. Biol. Chem. 193, 113 (1951). 11. ORNSTEIN, L., Ann. N.Y. Acad. Sci. 121, 321 (1964). 12. DAVIS, B. .J., Ann. N.Y. Acad. Sci. 121, 404 (1964). 13. BOLTON, E. T., BRITTEN, R. J., COWIE, D. B., MCCARTHY, B. J., MCQUILLEN, K., AND ROBERTS, R. B., “Carnegie Institution of’ Washington Yearbook,” No. 58, p. 259. The Carnegie Institution, Washington, D.C. (1959). 14. MARTIS, R. G., AND AMES, B. N., J. Biol. Chem. 236, 1372 (1964). 15. BINKLEY, F., DAVENPORT, J., AND EASTALL, F., Biothem. Biophys. Commun. 1, 206 (1959). 16. ROBINSON, D. S., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 202, 1 (1953).