Acidic glutathione transferase from human heart

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

40,

123-132

(1988)

Acidic Glutathione Transferase from Human Heart Characterization

A. M. CACCURI,”

and N-terminal

Sequence

C. DI ILIO,* D. COMPAGNONE,

Department of Biology, lJniversit.v of Rome, Tor Vergata, University of Chieti, and fDepartmerlt of Biochemical La Sapienza, 1trcl.v

Received

January

12.

1988.

and

in revised

Determination

D. BARRA,? AND G. FEDERICI” *In.stitlrte Sciences.

form

March

of Biochemiccrl Sciences. University of Rome.

21.

1988

The glutathione transferases (GST; EC2.5.1.18) are a family of enzymes that promote the conjugation of GSH with a large array of electrophilic substances enabling the body to excrete the conjugated products or their derivatives through the bile or urine. Glutathione transferases also bind covalently/noncovalently a multitude of lipophilic compounds of either exogenous or endogenous origin (l3). In addition, certain isoenzymes of glutathione transferase possess seleniumindependent glutathione peroxidase activity toward organic hydroperoxides (3). Multiple forms of glutathione transferase have been demonstrated in most human tissues so far analyzed and in each case at least one anionic form (pl 4.4-4.9) is always present (3,4-9). However, the interrelationships among the anionic glutathione transferases of different human tissues are not as yet completely established. Structural studies indicate that the anionic glutathione transferases of placenta (5,10), lung (7), erythrocytes (5,10,1 l), breast (5), platelet (12), thyroid (13), skin (6), and uterus (14) are composed of two subunits of identical molecular mass (23,000 M,). In addition all the anionic glutathione transferases purified from the above-mentioned tissues cross-react with antibodies raised against the glutathione transferase of human placenta but not with antibodies raised against the “basic” (GST-a-e) or “near-neutral” (GST-p) form of human glutathione transferase. In contrast the anionic glutathione transferases of brain (S), liver (15), and cornea (16) are heterodimers composed of two subunits of 22,500 and 24,000 M,. Furthermore, these enzymes all cross-react with antibodies raised against both the anionic and the cationic human glutathione transferases. We have previously demonstrated by isoelectric focusing the presence of an anionic form of glutathione transferase in human heart extracts (17) whose structural nature was not well investigated. In the present paper, the anionic form of human heart glutathione transferase has been purified to homogeneity and its subunit composition as well as its catalytic and immunological properties is compared with those of other human anionic glutathione transferases. In addition, the amino 123 0885-4505/88

$3.00

Copyright 0 1988 by Academic Press. Inc. All right\ of reproduction m any form raewed.

124

CACCURI

ET AL.

acid sequence of the N-terminal region of heart glutathione transferase determined and compared with those of other available transferases.

was

MATERIALS AND METHODS Chemicals. DEAE-cellulose was obtained from Whatman Biochemicals, Maidstone, Kent, U.K. The Mono-P HR 5/20 chromatofocusing column and the electrophoresis calibration kit for protein molecular weight determination were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Ampholines were obtained from LKB Produkter, Bromma, Sweden. Hematin was obtained from Aldrich-Chemie, Steinheim, Germany. Cibacron blue and triphenyltin chloride were obtained from Fluka AG, Buchs, Switzerland. Epoxy-activated Sepharose 6B, NADH, GSH, glutathione reductase (Bakers yeast, type III), cumene hydroperoxide, I-chloro-2,Cdinitrobenzene, 1,2epoxy-3+nitrophenoxy)-propane, ethacrynic acid, p-nitrophenylacetate, bromosulfophthalein, and gossypol-acetic acid were purchased from Sigma Chemical Co., St. Louis, Missouri, All other chemicals were of the best grade available. Pun$ication of glutathione transferase from human heart. Human hearts obtained at autopsy were extensively washed with cold physiological saline solution to eliminate residual blood cells and stored at - 20°C until used. The heart samples were thawed, minced, and homogenized in 3 vol of 10 mM Tris/HCl buffer, pH 8.0, in a Waring blender for 2 min. The resulting homogenate was centrifuged (Beckman 52-21) at 12,000g for 1 hr. The supernatant was applied to a column (5 x 23 cm) of DEAE-cellulose equilibrated with 10 mM Tris/HCl buffer, pH 8.0. The column was rinsed with the equilibrating buffer until no absorption at 280 nm was observed. The enzyme activity retained on the column (acidic fraction) was eluted with 10 mM Tris/HCl buffer, pH 8.0, containing 0.5 M NaCl. The active fractions were pooled, dialyzed against 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM 2-mercaptoethanol, and applied to a GSH-affinity column (2.5 x 25 cm) (18) equilibrated with 10 mM potassium phosphate buffer, pH 7.0. After washing with 0.1 M potassium phosphate buffer, pH 7.0, until no absorption at 280 nm was recorded, the affinity column was eluted with 50 mM Tris/HCl buffer, pH 9.6, containing 5 mM GSH. The eluted fractions containing glutathione transferase activity were pooled, concentrated by ultrafiltration, dialyzed against 10 mM potassium phosphate buffer, pH 7.0, containing 3 mM dithiothreitol, and subjected to chromatofocusing by high-performance liquid chromatography on the LKB FPLC System. The Mono-P HR 5/20 prepacked column equilibrated with 0.025 M methylpiperazine/HCl buffer (pH 5.7) was used. The sample was eluted with 1: 10 diluted polybuffer 74 adjusted to pH 4 with HCl. Fractions of 0.5 ml were collected. The active fractions were pooled, dialyzed against 10 mM potassium phosphate buffer, pH 7.0, and used for further characterization. Efectrophoretic methods. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed according to the method of Weber and Osborn (19). The gel and the SDS concentrations were respectively 10% and 0.1% (w/v). Electrophoresis was carried out on a LKB Multiphor II horizontal unit, at 190 mA constant current for 5 hr at 10°C. Protein standards were phos-

HUMAN

HEART

ACIDIC

GLUTATHIONE

TRANSFERASE

125

phorylase b (M, 94,000), bovine serum albumin (M, 67,000), ovalbumin (M, 43,000), carbonic anhydrase (M, 30,000), soybean trypsin inhibitor (M, 20,100), and (Ylactalbumin (M, 14,400). After the run the slab gel was fixed and stained following the method reported in the LKB laboratory manual. The isoelectric focusing analysis was performed on ampholine PAG plate (pH range, 3.5-5.2) at 10°C using an LKB multiphor II apparatus. The run was carried out at 25 W constant power for 3 hr. Glutathione transferase activity on the gel was detected by the method of Ricci et al. (20). Amino acid analysis. Amino acid analysis was performed on an LKB 4151 Alha Plus amino acid analyzer. Hydrolysis was carried out in 6 N HCI in vacuumsealed tubes at 110°C for 24, 48, and 72 hr. Three independent samples were analyzed. Automated amino acid sequence analysis. N-terminal sequence analysis was performed on an Applied Biosystems Model 470A gas phase protein sequencer equipped with an Applied Biosystems Model 120A PTH analyzer for on-line detection of phenylthiohydantoin (PTH) amino acids. The native protein (50 pg) was loaded onto a TFA-treated glass-fiber filter coated with polybrene. Prior to sample application, the filter was subjected to three cycles of Edman degradation, using the Applied Biosystems 03R PRE program. ATZ amino acid derivatives were automatically converted to PTH amino acids and injected onto the on-line analyzer for the identification. Secondary-structure analysis. The secondary-structure analysis was predicted by the method of Chou and Fasman (21). Enzyme assays. Glutathione transferase activity with different substrates was determined essentially as reported by Habig et al. (22). The Se-independent glutathione peroxidase activity of glutathione transferase was determined as previously described (17), using 1.5 mM cumene hydroperoxide or 0.25 mM Hz02. Inhibition studies were performed according to Tahir et al. (23). Immunological studies. Antibodies raised in rabbits against GST V and GST III of human uterus (14), GST-pZ8.5 of human skin (6), as well as against placental transferase (GST-m) (5), were available in the laboratory. It was previously established that GST V and GST III of human uterus are similar to GST-7~ and GST-p (14), whereas GST-pZ 8.5 of human skin is similar to GST-a-& (6). RESULTS

By sequentially using DEAE-cellulose, affinity chromatography, and FPLC an anionic glutathione transerase was purified from human heart. Analytical isoelectric focusing of the pure enzyme on ampholine PAG plate in the 3.5-5.2 pH range revealed the presence of two bands of activity at pH 4.75 and pH 4.9 which were reduced essentially to a single activity band at pH 4.75 when the enzyme was preincubated for 30 min at 37°C with 3 mu dithiothreitol (Fig. 1). For comparison the isoelectric focusing pattern of the transferase of human placenta (GST-m) is also included. A parallel run on SDS-PAGE of the purified glutathione transferase of human heart with the glutathione transferase of human placenta, in the presence of 2-mercaptoethanol, revealed that they

126

CACCURI

ET AL. PH

-6.5

-5.5

-4.5

-4.0

FIG. I. Comparison of isoelectric focusing pattern on ampholine PAG plate (pH range 3.5-5.2) of human placenta and heart glutathione transferases. Placenta GST after (I) and before (2) treatment with 3 mM dithiothreitol; acidic heart GST after (3) and before (4) treatment with 3 mM dithiothreitol. The gel was stained for GST activity according to Ricci er al. (20). To determine the pH gradient the gel was cut into I-cm sections and placed in individual tubes containing 1 ml of distillated water.

migrated as a single band in the same position corresponding to M, of 23,000 (Fig. 2). The amino acid composition of human heart glutathione transferase is shown in Table 1. A good degree of homology was observed among the heart enzyme and the acidic enzymes purified from other human tissues. When, as a quantitative measure of similarities among the amino acid compositions, the difference index of Metzger et al. (24) was calculated, values of 5.9, 6.1, 6.0, and 6.5 were obtained comparing the human heart glutathione transferase with the acidic glutathione transferases of human placenta (5), erythrocytes (5), breast (5), and skin (6). In order to clarify better the relationship between the heart

HUMAN Mrx 10 94 -

HEART

ACIDIC GLUTATHIONE

TRANSFERASE

127

-3

67 -

20.1 -

14.4 -

FIG. 2. SDS/polyacrylamide gel electrophoresis of acidic glutathione transferase of human heart. (1) Standards; (2) heart GST: (3) placenta GST.

glutathione transferase and the other human glutathione transferases, the enzyme was subjected to sequence analysis by automated Edman degradation. The Nterminal amino acid sequence of the first 48 residues is shown in Table 2. This result was obtained by first running a preliminary analysis which gave a certain amount of sequence information. Then, a second more extended analysis was run after loading a larger amount of protein (2 nmole) onto the filter and treating it with o-phthalaldehyde after the 8th cycle, in order to reduce the background (25). The enzyme has proline as N-terminal amino acid. Although the acidic glutathione transferase of heart is composed of two subunits only a single polypeptide sequence was obtained. Comparison of the N-terminal sequence of the acidic glutathione transferase of human heart with those of placenta (26) and skin (6), for which 23 residues and 16 residues are respectively known, revealed complete identity. On the other hand, comparison of the heart enzyme partial

128

CACCURI

ET AL.

TABLE I Amino Acid Composition of the Acidic Glutathione Transferase of Human Heart”

Amino Acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine

Residues of amino acid/mole of enzyme 39.4 16.1 30.6 47.0 20.6 39.6 27.0 24.0 N.D. 2.0 11.6 54.7 23.3 13.1 N.D. 21.4 9.0 15.3

” The calculation was based on a molecular weight of 46,000. N.D., not determined.

sequence with the sequence of placental enzyme reported by Dao et al. (7), who determined 30 residues, revealed the presence of Trp (heart) instead of Arg (placenta) in the 28th position. Furthermore, the N-terminal sequence of the acidic enzyme of heart proved to be distinctly different from the N-terminal sequences of GST-p and GST-9 of liver (26,27) and of the most basic transferase (GST-pZ 9.9) of human skin (6) (Table 3). I-Chloro-2,Cdinirobenzene was found to be the best substrate (101 U/mg) of anionic heart enzyme. No activity was noted with cumene hydroperoxide whereas ethacrynic acid resulted to be the second best substrate (1.97 U/mg). Moderate yet significant activity was recorded for 1,2-epoxy-3-(p-nitrophenoxy)-propane (0.88 U/mg) and for p-nitrophenylacetate (0.26 U/mg). The ICsO values for characteristic inhibitors was also determined. The enzyme had a low I&, value for cibacron blue (0.55 FM) and high values for gossypol (>I50 PM) and bromosulfophthalein (185 PM). Intermediate values were found for hematin (33 PM) and for triphenyltin chloride (18 (uM). In the immunodiffusion experiment antibodies raised against the acidic transferase of human placenta (GST-m) and human uterus (GST V) precipitated (complete identity) the glutathione transferase purifed from human heart, whereas no crossreactivity was seen with the antibodies prepared against both the “basic” transferase (GST-pZ 8.5) of human skin (6) and the “near-neutral” transferase (GST III) of human uterus (14).

HUMAN

HEART

ACIDIC GLUTATHIONE

129

TRANSFERASE

TABLE 2 Automated Edman Degradation and Secondary Structure Prediction of Acidic Glutathione Transferase of Human Heart (2 nmole)” Cycle 1 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23 24

PTH-aa Pro Pro TY~ Thr Val Val ‘Or Phe Pro Val A% GUY Arg CYS Ala Ala Leu Arg Met Leu Leu Ala Asp Gln

Yield (pmole)

Cycle

1123 957 749 718 925 977 658 831 403 466 188 234 192 N.I. 262 285 208 109 227 215 247 251 107 153

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 49 41 42 43 44 45 46 47 48

Yield (pmole)

PTH-aa

157 162 91 22 82 72 76 112 121 54 87 55 61 13 75 58 86 40 73 49 47 53 22 15

GUY Gin Ser Trp LYS GIU GIU

Val Val Thr Val Glu Thr Trp Gin Glu GUY Ser Leu LYS Ala Ala Ser Met

a Repetitive yield, 94%; N.I., not identified.

--A

JJA&vWdJ)J&-.LLL! 40

20 structure

percentage

.fi : (Y -HELIX

43.7 96 22.9 %

n

: B-SHEET

18.7 %

J-l : fl -TURN

14.6 %

-

: RANDOM COIL

DISCUSSION

A previous report demonstrated the existence of an acidic isoenzyme of glutathione transferase in human heart (17). The present paper deals with the purification and characterization of this form of the enzyme. The enzyme is composed of two similar subunits of 23,000 M, and has an isoelectric point at pH 4,75. Thus the subunit composition of the acidic form of human heart appears to be similar to that of the anionic isoenzymes of placenta (5,10), lung (7), breast (5), erytrocytes (5,l l), thyroid (13), and skin (6) but different to the subunit composition of the acidic forms of liver (15), cornea (16), and brain (8). These latter proteins

130

CACCURI

Comparison of the N-terminal Anionic” heart GST

GST-T?

PI.0

pro

FYO

pro

Tyr

Tyr

Thr Val Val Tyr Phe

Thr Val Val Tv Phe Pro Val Arg GUY A% CYS Ala Ala Leu Arg Met Leu Leu Ala Asp GIR GUY GlR N.D. Arts LYS Glu

pro

Val Arg GlY Arg N.D.’ Ala Ala Leu Arg Met Leu Leu Ala Asp Gln GUY Gin Ser Trp LYS Glu Glu Val Vat Thr Val Ghl Thr Trp GlR Glu GUY Ser Leu LYS Ala Ala Ser Met ’ Present work. b Dao er al. (7).

ET AL.

TABLE 3 Amino Acid Sequence of Human Glutathione Transferases GST-/L’

GST+’

Most basic skin’ GST (~1 9.9)

Pro Met Be Leu GUY Tyr Trp Asp Be ‘4% GUY Leu Ala His Ala Ile Arg Leu Leu Leu Glu Vr Thr

Pro Met Be

Pro GUY LYS

L&U

pro

GUY

Val Leu His Tyr Phe Asp GUY Arg ‘JY Am Met Giu

Tv Trp

Asp Be Arg GUY Leu Ala His Ala Be Arg Leu Leu Leu Glu Vr Thr ASP

His Asp Tv Leu GUY LYS

” Alin et al., (26). ’ Singh et al. (27).

’ Del Boccio et al. (6). / N.D., not determined.

HUMAN

HEART

ACIDIC GLUTATHIONE

TRANSFERASE

131

are in fact dimers of 22,500 and 24,500 M,. The results of immunochemical studies clearly indicate that the anionic glutathione transferase of human heart is immunologically similar to the anionic glutathione transferase of placenta and uterus and immunologically distinct from the “cationic” form of the enzyme of human skin and the “near-neutral” enzyme of uterus. The evidence that the enzyme from human heart is very similar to the glutathione transferase of human placenta (GST-T) is clearly supported by the results obtained. These include amino acid composition, substrate specificities, and sensitivities to characteristic inhibitors. Definitive evidence of their close similarity is provided, however, by the results of N-terminal amino acid sequence analysis. With the exception of the 28th position, a complete identity of up to 30 residues between placenta (7) and heart enzymes resulted. In addition, position 27, which was not determined for the placental enzyme (7), was assigned to a serine residue in the heart enzyme. The cDNA-deduced amino acid sequence of a rat enzyme referred to as GST-P has previously been reported (28). It is worth of mentioning that the human acidic heart transferase shows more extensive similarity with rat GST-P (75% identity) than to any other rat transferases when the first 48 N-terminal amino acid residues are compared. SUMMARY

The anionic glutathione transferase of human heart has been purified to homogeneity by using DEAE-cellulose, affinity chromatography, and FPLC. The enzyme has an isoelectric point at pH 4.75 and has an electrophoretic mobility on SDS-PAGE identical to placental transferase V, indicating that the heart enzyme is formed by two similar subunits of 23,000 M,. Upon isoelectric focusing on ampholine PAG plates the enzyme recovered from FPLC gave two bands of activity at pH 4.75 and 4.9 which were reduced to essentially a single band at pH 4.75 after incubation with dithiothreitol. In the immunodiffusion experiment, the heart enzyme gave a positive precipitin line with the antibodies against transferase rr but not with antibodies prepared against the “basic” transferase of human skin or against the “near-neutral” transferase of human uterus. The substrate specificities, the sensitivities to characteristic inhibitors. the amino acid composition, together with the immunological studies, strongly indicate that the anionic enzyme of human heart is closely related to the transferase rr of human placenta. The N-terminal amino acid sequence of the first 48 residues was determined and compared with the N-terminal region of other reported human glutathione transferase sequences. The heart enzyme differs from the placental enzyme in a single residue (Trp instead of Arg in the 28th position) further supporting their similarity. ACKNOWLEDGMENT This work was supported by a grant from Consiglio Nazionale delle Ricerche. Progetto finalizzato di Medicina Preventiva e Riabilitativa. SP4.

REFERENCES 1. Jakoby, W. B., and Habig. W. H. “Enzymatic Academic Press, New York, 1980. 2. Chasseaud, L. F.. Adv. Cancer Res. 29, 175-293

Basis of Detoxification.” (1979).

Vol. 2. pp. 63-94.

132 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

CACCURI

ET AL.

Mannervik, B., Adv. Enzpnol. 57. 357-417. Guthenberg, C., Warholm, M., Rane, A., and Mannervik, B., Biochem. J. 235, 741-745 (1986). Di rho, C., Del Boccio, G., Massoud. R., and Federici, G.. Biochem. Int. 13, 263-269 (1986). Del Boccio, G., Di Ilio, C. Alin, P.. Jornvall, H., and Mannervik, B., fJio&em. J. 244, 21-25 (1987). Dao, D. D., Partridge, C. A., Kurosky, A., and Awasthi, Y. C., Biochem. J. 221, 33-41 (1984). Theodore, C., Singh, S. V., Hong, T. D., and Awasthi, Y. C., Biochem. J. 225, 375-382 (1985). Di Ilio, C., Del Boccio, G., Aceto, A., and Federici, G., Curcinogenesis 8, 861-864 (1987). Guthenberg, C., and Mannervik, B.. Biochim. Biophys. Acta 661, 255-260 (1981). Marcus, C. J., Habig, W. H., and Jakoby, W. B., Arch. Biochem. Biophys. 188, 287-293 (1978). Federici, G., Di Ilio, C., Sacchetta, P., Polidoro, G., and Bannister J. V., lnt. J. Biochem. 17, 653-656 (1985). Del Boccio, G., Di Ilio, C., Casalone, E., Pennelli, A. Aceto, A., Sacchetta, P., and Federici, G., Ital. J. Biochem. 36, 8-17 (1987). Di Ilio, C., Aceto A., Del Boccio, G., Casalone, E., Pennelli A., and Federici, G., Eur. J. Biochem. 171, 491-496 (1988). Singh, S. V., Dao, D. D., Partridge, C. A.. Theodore, C., Srivastava, S. K., and Awasthi, Y. C., Biochem. J. 232, 781-790 (1985). Singh. S. V., Hong, T. D., Srivastava, S. K.. and Awasthi, Y. C., Exp. Eye Res. 40, 431-437 (1985). Di Ilio, C., Sacchetta, P., Lo Belle, M.. Caccuri. A. M., and Federici, G., J. MO!. CeN. Cardiol. 18, 983-991 (1984). Simons, P. C., and Vander Jagt, D. L., Anal. Biochem. 82, 334-341 (1977). Weber, K., and Osborn, M., J. Biol. Chem. 244, 4406-4412 (1969). Ricci, G., Lo Bello, M., Caccuri, A. M., Galiazzo, F., and Federici, G., Anal. Biochem. 143, 226-230 ( 1984). Chou, P. Y., and Fasman, G. D., Annu. Rev. Biochem. 47, 251-276 (1978). Habig, W. H., Pabst, M. J., and Jakoby, W. B., .f. Biot. Chem. 249, 7130-7139 (1974). Than, M. K., Guthenberg, C.. and Mannervik, B. FEBS Left. 181, 249-252 (1985). Metzer, H., Shapiro, M. B., Mosiman, J. E., and Vinton, J. E., Nature (London) 219, 11661168 (1968). Brauer, A. W., Oman, C. L.. and Margolies, M. N.. Anal. Biochem. 137, 134-142 (1984). Alin, P.. Mannervik, B., and Jornvall, H., FEBS Left. 182, 319-322 (1985). Singh, S. V.. Kurosky, A.. and Awasthi, Y. C.. Biochem. J. 243, 61-67 (1987). Suguoka, Y., Kano, T., Okuda, A., Sakai. M., Kitagawa, T., and Muramatsu, M., Nucleic Acids Res. 13, 6049-6057 (1985).