Carcinogenesis vol.11 no. 12 pp.2267-2269, 1990

SHORT COMMUNICATION

Differential activity of tamae, rat, mouse and bacteria glutatMome transferase isoeezymes towards 4-nitroqeiMoMiie 1-oxide

Antonio Aceto, Carmine Di Dio, Mario Lo Bello1, Tonino Bucciarelli, Stefania Angelucci and Giorgio Federici1 Istituto di Scienze Biochimiche, Facolta' di Medicina, Universita' G D'Annunzio', 66100 Chieti and 'Dipartimento di Biologia, Universila' di Roma 'Tor Vergata', 00190 Roma, Italy

4-Nitroquinoline 1-oxide (4-NQO*) is known to produce cancer in a number of organs and tissues (1). The carcinogenic action of 4-NQO is probably due to its DNA-damaging properties (2). Glutathione transferases (GSTs) are dimeric proteins which catalyse the nucleophilic attack of GSH on the electrophilic center of a wide variety of organic molecules (3—6). Given that several substrates of the GSTs are known to be carcinogens (3), it seems likely that these enzymes play an important role in the protection of cells from the toxicity of these compounds. At least three classes of cytosolic GSTs, i.e. alpha, mu and pi, common to several mammalian species, have been identified (7). Recently GSTs have also been purified and characterized from bacteria (8). It has been demonstrated that the cytosolic fractions prepared from rat liver as well as from mouse, human and monkey livers catalyse the GSH-dependent liberation of NO2 from 4-NQO (4,9,10), and the conjugate of 4-NQO formed in this reaction has also been identified (11). A spectrophotometric assay for the study of the reaction between GSH and 4-NQO has been developed (9). In the cytosolic fraction, however, a mixture of GSTs is present and differences in the specificity between the isoenzymes in the GSH conjugation of 4-NQO might occur. In order to investigate this, the GSH conjugation of 4-NQO was studied with the GSTs purified from a number of mammalian and microbial sources. We provide evidence that both pi and mu classes GSTs have maximum activity toward 4-NQO. The catalytic efficiency of these latter enzymes has also been measured.

Table I. Glutathione conjugation activity of purified human, rat, mouse and bacterial glutathione transferases toward 4-NQO Enzyme

Activity°(/imol/min/mg) CDNB

Rat GST GST GST GST GST GST GST GST

1-1 1-2 2-2 3-3 3-4 +4 6-6 8-8

Mouse MI Mil Mill Human GST^t 3 (liver) GST^t 6 (skin) GST4.5 (prostate) G S T - T (placenta)

GST- e (RBC) GST4.5 (uterus) GST-4.6 (nasal mucosa) GST-4.8 (heart) GST-n (liver) GST-^ (liver) GST-5.9 (liver) GST-6 6 (liver) GST-9.9 (testis) GST B|B, (liver) GST B,B2 (liver) GST B ^ (liver) GST-8.6 (heart) GST-8.5 (kidney)

11 10 5 5.0 4.7 51 46 9.2 7.4

4-NQO 0 0 0 4.8 4.8 5.1 9.9 2.6

alpha alpha alpha mu mu mu mu alpha

22 75 22

1 33 20

alpha Pi mu

4.6 13 39 52 54 90 44 30 91 20 6.8 18 13 50 55 60 4.5 25

2.7 5.9 15 25 47 31 12 20 0.9 0.3 0.09 0.14 0 0 0 0 0.09 0

Pi Pi P> P> P> P' Pi P' mu mu alpha alpha alpha alpha alpha alpha alpha alpha

4-NQO was purchased from Fluka Chemie AG, Buchs, Switzerland. Human, rat, mouse and bacteria GSTs were purified according to previous published procedures (8,12-22). The

Bacteria Pm-GST-6.0 (P.mirabilis) Sm-GST-7.3 (5.marcescens)

•Abbreviations: 4-NQO, 4-nrtroquinoline 1-oxide; GST, glutathione transferase; CDNB, l-chloro-2,4-dinitrobenzene.

"The concentration of substrates in the assay mixture was 1 mM GSH, 1 mM CDNB, 0.1 mM 4-NQO.

© Oxford University Press

Class

3.1 0.4

0 0

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The conjugation capacity of 4-iutroquinoline 1-oxide (4-NQO) with GSH by a number of human, rat, mouse and bacteria glutathione transferases (GSTs) was investigated. Pi and mu classes GSTs exhibited maximum conjugation capacity. Alpha class glutathione transferases as well as bacteria glutathione transferases were found to be unable to conjugate GSH to 4-NQO. The l^ values as well as the catalytic efficiency (Kj^/K^ for most of the GSTs investigated were also determined. Mouse liver GST MTU (class mu) was the most efficient of the various isoenzymes tested. Its K^/Km value was 162 times higher than that of mouse liver GST MI (class alpha). The relatively high catalytic efficiency exhibited by GST-g (class pi) is prevalently due to its low affinity for 4-NQO.

enzymes were homogeneous as judged by SDS-PAGE. GST activity toward 4-NQO was measured spectrophotometrically at 25 °C as reported by Stanley and Benson (9). The assay system (2 ml; pH 6.5) contained 0.1 M potassium phosphate buffer, 1 mM GSH, 0.1 mM of 4-NQO dissolved in ethanol and an appropriate amount of enzyme. GST activity with 1-chloro2,4-dinitrobenzene (CDNB) was measured as described by Habig (23). Specific activities were based on protein concentration measured with the method of Bradford (24). Catalytic efficiency (KJKm) was derived from and Km values were estimated by non-linear regression analysis of data in

A.Aceto el al.

experiments where the concentration of 4-NQO was varied between 0.01 and 0.1 mM. The values listed in Table II are the means of five separate experiments; corresponding standard deviations are in general < 10% of the means. Each measurement was made in triplicate. The sp. act. towards 4-NQO of a number of rat, human, mouse and microbial GSTs are reported in Table I. For comparison the activity values towards the standard and highly reactive substrate CDNB are also given. Data in Table I indicate that the human acidic GSTs as well as mouse liver GST Mil have a relatively high capacity to conjugate GSH to 4-NQO- The activity with 4-NQO, as a percentage of the CDNB activity, ranged from - 2 5 % of nasal mucosa GST-4.6 to 87% of GST-e. It has to be noted that the relative activity for ethacrinic acid, which is considered to be the second best substrate of these GSTs, is < 5 % when the activity toward CDNB was taken as 100% (5). As

20 40 60 80 COMB-6ST octivity (U/ntg)

100

30

20

B

10

r=1

10 20 CDHB-6ST octlvity (U/rog)

30

Fig. 1. The relationship between GST activity measured with CDNB and with 4-NQO for human pi class (A, Y = 3.69 + 0.39-V) and for rat mu class (B, Y = 0.92 + O.88X).

Table II. Kinetic parameters of various glutathione transferases towards 4-NQO Enzyme and source

K^, (min" 1 )

Km (mM)

(mM" 1 min" 1 )

GST-4.5 (human prostate) GST-4.7 (human uterus) G S T - T (human placenta) GST-p (human RBC) MI (mouse liver) MU (mouse liver) Mil (mouse liver) GST 3-3 (rat liver) GST 3-4 (rat liver) GST 4-4 (rat liver)

1144 326 4892 242 168 2333 1632 235 170 28

0.47 0.19 0.89 0.015 0.59 0.32 0.036 0.056 0.05 0.019

2 434 1 715 5 497 16 165 285 7 290 45 332 4 201 3 407 1 489

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0

previously reported, the human acidic GSTs and mouse liver GST Mil have similar chemico-physical, structural and immunological properties and were included in pi family in the classification scheme proposed by Mannervik et al. (7). The bacterial enzymes as well as the human alpha class GSTs were not able to conjugate efficiently GSH to 4-NQO in our experimental conditions. It has to be noted that bacterial enzymes do not fall (8,25) into the classification scheme introduced by Mannervik et al. (7). The inability of Pm-GST-6.0 and Sm-GST-7.3 to conjugate GSH to 4-NQO confirms that bacterial GSTs do not have as broad a substrate specificity pattern as most mammalian GSTs (8). Additional alpha class GSTs from rat and mouse liver, i.e. GST 1-1, GST 1-2, GST 2-2 and MI, did not show any significant activity toward 4-NQO. High sp. act. with organic hydroperoxides is a characteristic feature of these isoenzymes (5). Relatively high catalytic activity towards 4-NQO is also associated with GST 3-3, GST 3 ^ , GST AA and GST 6-6 of rat liver and with GST Mill of mouse liver. All these enzymes are mu class GSTs (7). Within the mu family, the human enzymes GST \p and GST /x appear to be less active in catalysing the conjugation of GSH to 4-NQO than mouse and rat enzymes. A strong positive correlation was found between CDNB activity versus 4-NQO activity for rat mu class GST (r = 1.0) and for human pi class GST (r = 0.73) (Figure 1). When the kinetics of the formation of GS-4-NQO adduct were studied for the most active GSTs, the results reported in Table II were obtained. Owing to the low activities of some of the isoenzymes listed in Table I their Km and K^ could not be determined. The highest affinity was found for GST-e (Km = 0.015 mM) and rat liver GST AA (Km = 0.019 mM) followed by mouse liver GST Mm (Km = 0.036 mM), rat liver GST 3 ^ (Km = 0.05 mM) and rat liver GST 3-3 (Km = 0.056 mM). The Km values for the other GSTs were found to be ~ 10-60 times higher than GST-e. This latter result indicates that most of the sp. acts reported in Table I have been determined at a substrate concentration below Km. Therefore these sp. acts are not a reflection of a maximal enzymatic activity but merely a relative value related to substrate concentration. The ratio of Kat to Km describes the specificity of an enzyme for its substrate. The catalytic specificities for several GSTs toward 4-NQO have been determined and the results reported in Table II. It can be seen that the most efficient enzyme in catalysing the conjugation of GSH to 4-NQO is the mouse liver GST Mm (K^l^ = 45 332 mM"' min"1) followed by GST-e (^cat^m = 16 165 mM"' min"'). Mouse liver GST MI has a poor catalytic capacity to conjugate GSH to 4-NQO as indicated by its low KalIKm value. From the results presented in Table II it can be also noted that there is a significant different catalytic efficiency between the human acidic GSTs, even though they all belong to the same class (class pi). However, the higher catalytic efficiency of GST-e is mainly due to its high affinity for 4-NQO rather than to a high V^^ value. On the other hand, the lower rate of metabolism of GSTT for 4-NQO was a result of a low affinity, with the V^ being relatively high. The differences in the kinetic characteristics of these isoenzymes towards 4-NQO are particularly noteworthy and suggest that they are distinct, although closely related enzymes. In agreement with our results, studies from other laboratories also indicate significant differences in the inhibition of GST-JT and GST-e by plant phenols (26). In addition, fine structural differences between G S T - T and GST-e have been also reported (27,28). In summary, we have shown that 4-NQO is rapidly conjugated with GSH in the presence of purified GSTs of pi and mu classes, whereas alpha class GSTs as well as microbial GSTs have a very low activity with this substrate.

Activity of GSTs towards 4-NQO

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Differential activity of human, rat, mouse and bacteria glutathione transferase isoenzymes towards 4-nitroquinoline 1-oxide.

The conjugation capacity of 4-nitroquinoline 1-oxide (4-NQO) with GSH by a number of human, rat, mouse and bacteria glutathione transferases (GSTs) wa...
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