[9] INDIVIDUAL GPxs 101

[91 Estimation of Individual Types of Glutathione Peroxidases

B y REGINA BRIGELIUS-FLOHt~, KIRSTEN WINGLER, and CORDULA MULLER

I n t r o d u c t i o n

For more than a decade the classic glutathione peroxidase (cGPx), 1 the gene product of gpxl, remained not only the only type of glutathione peroxidase but also the only selenoprotein identified in mammals. At present 19 mammalian selenoproteins have been described, 2 among them 4 distinct glutathione per- oxidases: cGPx, the plasma GPx (pGPx), the phospholipid hydroperoxide GPx (PHGPx), and the gastrointestinal GPx (GI-GPx). This redundancy, at first glance, has led to the impression that nature considers removal of hydroperoxides so im- portant that it has created multiple lines of defense to make sure that every single hydroperoxide molecule is destroyed immediately. Several aspects, however, argue against this assumption.

1. The individual types of glutathione peroxidases are not evenly distributed in the organism, cGPx is the most ubiquitous GPx. pGPx is an extracellular enzyme found in blood plasma; it is released from the kidney into the plasma. Other sites of synthesis are placenta, large intestine, or lung (reviewed by Brigelius-Floh63). It thus might be responsible for extracellular hydroperoxide removal, especially at interfaces protecting tissues from environmental influences. This interpretation is, however, problematic because of the low extracellular concentration of glutathione, which cannot guarantee a sustained reduction of hydroperoxides. PHGPx is pref- erentially expressed in testis. It has, therefore, been postulated that this particular enzyme is responsible for the protection of sperm from oxidative damage. High PHGPx activity is, however, found only in immature spermatids, not in mature sperm cells. 4 Sperm cells contain a high concentration of PHGPx protein, which obviously is inactive. A putative role for GI-GPx was deduced from its prefer- ential expression in the gastrointestinal epithelium 5: prevention of hydroperoxide absorption. The identification of GI-GPx in tumor cell lines not derived from the intestine, 6 the increase in its mRNA 7 and protein s in colon cancer ceils, its

1 G. C. Mills, Z Biol. Chem. 229, 189 (1957). 2 L. Floh6, J. R. Andreesen, R. Brigelius-Floht, M. Maiorino, and E Ursini, 49, 411 (2000). 3 R. Brigelius-Floht, Free Radic. Biol. Med. 27, 951 (1999). 4 M. Maiorino, J. B. Wissing, R. Bfigelius-Floh~, K Calabrese, A. Roveri, R Steinert, E Ursini, and

L. Ftoh~, FASEB ./. 12, 1359 (1998). 5 E E Chu, J. H. Doroshow, and R. S. Esworthy, J. Biol. Chem. 268, 2571 (1993). 6 R. S. Esworthy, M. A. Baker, and E E Chu, CancerRes. 55, 957 (1995).

Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.

METHODS IN ENZYMOLOGY, VOL. 347 0076-6879/02 $35.00

102 SELENOPROTEINS [9]

localization in Paneth cells in human ileum, 8 and its specific intracellular local- ization in cells in the apical part of colon crypts, 8,9 however, hardly support the barrier hypothesis.

2. The individual glutathione peroxidases do not respond equally to selenium supply. This phenomenon has been addressed in the "hierarchy of selenoproteins." Selenoproteins ranking low in the hierarchy respond to selenium deprivation with a rapid loss of activity, protein, and even mRNA levels. Those ranking high in the hierarchy respond slowly in terms of activity and protein, and the mRNA levels remain stable. The hierarchy of glutathione peroxidases is as follows 3: GI-GPx > PHGPx > pGPx = cGPx.

3. Reverse genetics ruled out the notion that cGPx is a vital enzyme.l° cGPx-/- mice developed and grew normally. The hypothesis that cGPx represents a major antioxidant device, however, could be corroborated, cGPx -/- mice were more sen- sitive to poisoning with the redox cycling herbicides paraquat 11,12 and d iquat ) 3

cGPx -/- mice died within hours irrespective of selenium supplementation as did selenium-deficient wild-type mice, whereas selenium-adequate wild-type mice survived. This indicates that none of the other selenoproteins, notably none of the other glutathione peroxidases, can protect mice against oxidative stress as effi- ciently as cGPx. Thus, only cGPx functions as an antioxidative enzyme; the other glutathione peroxidases must have different roles.

This has been convincingly demonstrated for PHGPx. Apart from modulating intracellular signaling, reviewed by Brigelius-Floh6 e t al., 14 it has been discovered as an enzymatical ly inactive structural protein in spermatozoa.15 Whether the other

types of ghitathione peroxidases, pGPx and GI-GPx, similarly exert surprising

7 H. M6rk, O. H. al-Taie, K. Bghr, A. Zierer, C. Beck, M. Scheurlen, F. Jakob, and J. K6hrle, Nutr. Cancer 37, 108 (2000).

8 S. Florian, K. Wingler, K. Schmehl, G. Jacobasch, O. J. Kreuzer, W. Meyerhof, and R. Brigelius- Floh6, Free Rad., in press (2001).

9 R. Brigelius-Floh6, C. Miiller, J. Menard, S. Florian, K. Schmehl, and K. Wingler, Biofactors 14, 101 (2001).

10 y. S. Ho, J. L. Magnenat, R. T. Bronson, J. Cao, M. Gargano, M. Sugawara, and C. D. Funk, J. Biol. Chem. 272, 16644 (1997).

11 j. B. de Haan, C. Bladier, R Griffiths, M. Kelner, R. D. O'Shea, N. S. Cheung, R. T. Bronson, M. J. Silvestro, S. Wild, S. S. Zheng, E M. Beart, E J. Hertzog, and I. Kola, J. Biol. Chem. 273, 22528 (1998).

12 W. H. Cheng, Y. S. Ho, B. A. Valentine, D. A. Ross, G. E Combs, Jr., and X. G. Lei, J. Nutr. 128, 1070 (1998).

13 y. Fu, W. H. Cheng, J. M. Porres, D. A. Ross, and X. G. Lei, Free Radic. Biol. Med. 27, 605 (1999). 14 R. Brigelius-Floh6, F. Ursini, M. Maiorino, and L. Floh6, in "Handbook of Antioxidants: Biochem-

ical, Nutritional, and Clinical Aspects" (E. Cadenas and L. Packer, eds.). Marcel Dekker, New York, 2001.

15 E Ursini, S. Heim, M. Kiess, M. Malorino, A. Roveri, J. Wissing, and L. Floh6, Science 285~ 1393 (1999).

[9] INDIVIDUAL GPxs i03

functions remains to be elucidated. One of the prerequisites to identifying the roles of these enzymes is the availability of assays to differentiate the isotypes, by either activity, RNA, or protein level.

D e t e r m i n a t i o n b y Act iv i ty

Assay System

Estimation of glutathione peroxidase activity is not simple. Several test systems have been described and discussedl6-1Sand are not to be repeated here in detail. Instead, some important points are noted that must be considered in order to obtain a reliable determination. Activity is usually measured in a test coupled to the glutathione reductase-catalyzed reduction of oxidized glutathione (GSSG) at the expense of NADPH

ROOH + 2GSH ~ ROH + GSSG + H20 (1)

GSSG + NADPH + H + CR 2GSH + NADP + (2)

where GR is glutathione reductase, and GSH is reduced glutathione. Reaction (1) is a two-substrate reaction characterized as a ping-pong mechanism. 19 ROOH is reduced first, and then the resulting alcohol is released. The oxidized enzyme (containing a selenenic acid in the active center) is reduced back by a stepwise reaction with two molecules of GSH.19 For bovine cGPx infinite limiting maximum velocities and Michaelis constants for both substrates, hydroperoxide and GSH, have been determined. 16 In consequence, conditions for estimation of international units requiring "saturating" concentrations of all substrates are not applicable. This is a general phenomenon characteristic of all glutathione peroxidases tested in this respect so far. 2°'21 The implication of the peculiar kinetics of glutathione peroxidases must be considered in the choice of proper substrate concentrations for the assay and in the definition of the unit of activity.

Reaction (1) is dependent not only on the concentration of the hydroperoxide but also on the concentration of GSH. The apparent maximum velocities and Michaelis constants for one substrate are linear functions of the co- substrate concentration. Under such conditions, a reasonable compromise

~6 L. Floh6, G. Loschen, W. A. Gtinzler, and E. Eichele, Hoppe Seylers Z. PhysioL Chem. 353, 987 (1972).

17 L. FloM and W. A. Gtinzler, Methods. Enzymol. 105, 114 (1984). 18 R. Brigelius-Floh6, K. L6tzer, S. Maurer, M. Schultz, and M. Leist, Biofactors 5, 125 (t995). 19 L. Floh6, in "Glntathione: Chemical, Biochemical and Medical Aspects" (D. Dolphin, R. Poutson,

and O. Avramovic, eds.), Part A, p. 643. John Wiley & Sons, New York, 1989. 2o F. Ursini, M. Maiorino, and C. Gregolin, Biochim. Biophys. Acta 839, 62 (1985). 21 R. S. Esworthy, F.-E Chu, A. W. Girotti, and J. H. Doroshow, Arch. Biochem. Biophys. 3117, 29

(1993).

104 SELENOPROTEINS [9]

is to measure GPx activities at a fixed regenerated GSH concentration and at a hydroperoxide concentration that is high enough not to substantially influence the initial reaction rate.

Reaction (1) strongly depends on the pH. The pH optimum is pH 8.7, with a steep increase in the pH dependence of the reaction rate starting from pH 7.16 Slight variations in the pH of the assay system thus result in major variations in activities.

The temperature optimum is near the stability limit at 430, 22 with a steep increase between 30 and 43 °. This means that a minor deviation from a given temperature results in major differences in activities.

Hydroperoxides react with GSH spontaneously. The rate of the spontaneous reaction is different for different hydroperoxides and also increases with pH. It must be estimated separately and subtracted from the overall reaction rate obtained in the presence of enzyme.

Assay conditions (final concentrations) providing reproducible results both for tissue extracts and cultured cells, for example, are as follows: 100 mM Tris- HC1, pH 7.6; 5 mM EDTA; 1 mM sodium azide; 3 mM GSH; 0.1 mM NADPH; 0.1% (v/v) peroxide-free Triton X- 100; and 600 mU of glutathione reductase (GR), for example, from Sigma (St. Louis, MO) type III baker's yeast (117 U/ml). GSH, NADPH, and GR must be freshly prepared daily. The reaction is started by the addition of 10/zl of 5 mM hydroperoxide (final concentration, 50 #M). These assay conditions result in a pseudo zero-order rate because GSH remains constant due to regeneration and the hydroperoxide concentration is close to the apparent Vmax (see comments to Table I). The turnover of hydroperoxides is calculated from the decrease in NADPH measured at 340 nm, using Beer's law.

Comments. GPx activities are comparable only if measured under identical conditions. As the reaction rate depends on the steady state level of GSH, the as- say coupled with the regeneration of GSH by glutathione reductase, reaction (2), is most suitable for reproducible results. To avoid misinterpretations, enzymatic units expressed as ANADPH per minute must be given together with clearly de- fined conditions applied in the test system. As hydroperoxide is a substrate for all glutathione peroxidases, H202 or tert-butyl hydroperoxide (t-BOOH) can be used. t-BOOH has a lower spontaneous reaction rate with GSH than H202 and therefore is the preferred substrate to measure low GPx activities.

Rate constants for the reaction with both substrates differ between GPx isotypes (Table I). The implications of the kinetic constants compiled in Table I with respect to GPx determinations are as follows.

1. If equivalent concentrations of the oxidizing and the reducing substxate are chosen, the enzyme is present close to 100% as the oxidized form. In consequence,

22 ]7. Schneider and L. Floh6, Hoppe Seylers Z. Physiol. Chem. 348, 540 (1967).

[9] INDWIDUAL GPxs 105

TABLE I RATE CONSTANTS FOR REACTIONS OF GLUTATHIONE PEROXIDASES a

k+~ k+2

Substrate H 2 0 2 t -BOOH Cumene-OOH PCOOH b (GSH) Ref.

cGPx 59 7.5 12.8 - - 0.45 16 c 50 11.7 17 - - 0.80 27 '~ 86 - - 0.25 21 e

PHGPx 3.0 1.2 1.8 12.1 0.006-0.15 20 f pGPx 33 ND 0.075 21 e

a Apparent second-order rate constant k+l defined by v = k+l[ROOH][GPxred] for the ! reaction of glutathione peroxidases with different hydroperoxides, and k+z defined as

k+2 q- k+3 in the rate equation v = k+2 [GPxox][GSH] + k+3[GPxox - GSH][GSH], which describes the two successive reactions of glutathione peroxidases with GSH. The rate constants are given in/zM -1 sec -1.

b --, No reaction; ND, Not determined. c cGPx from bovine blood measured at pH 6.7. d cGPx from hamster liver measured at pH 7.6. e pGPx from human plasma and cGPx from human erythrocytes (Sigma) measured at pH

7.6. YPHGPx from pig heart measured at pH 7.6.

the activity measurement is determined by k~2; more precisely the activity is then

defined as v = k~_2[GSH][GPxox]. For practical purposes equimolar substrate concentrations are not feasible, for example, because of solubility limits of most

organic hydroperoxides. In most cases, constant substrate consumption is never-

theless obtained if the hydroperoxide concentration is not lower than 2% of the

GSH concentration and the reaction is made pseudo first order by keeping the GSH

concentration constant through regeneration. At lower relative concentrations of

ROOH, the activity becomes codetermined by k+b which means the slopes deviate from linearity.

2. Appropriate test conditions are difficult to recommend without reliable knowledge of the kinetic constants, as, for example, for GI-GPx. Differences of

rate constants between species also cannot be excluded. A suitable choice of sub-

strate concentrations according to the rules outlined above must be validated in each case.

3. The differences in the rate constants between the isotypes of GPx preclude

an estimation of enzyme molarities in mixtures of unknown relative compositions. 4. Because activity measurements ideally reflect the k+2 values, identical

"units" represent more moles of pGPx or PHGPx than cGPx.

Differentiation by Choice of Substrates

Individual glutathione peroxidases exert different levels of specificity for

hydroperoxide substrates. All glutathione peroxidases, however, use H202 and

106 SELENOPROTEINS [91

t-BOOH, which are the most commonly used substrates for the estimation of GPx activity. The respective kinetic constants (k+l and k~_2) for different types of glu- tathione peroxidases differ (see Table I), but are not pronounced enough to allow differential analysis. With some precautions, PHGPx can be measured individu- ally by using complex hydroperoxides such as phosphatidylcholine hydroperoxide (PCOOH). 1s,23'24 pGPx has also been shown to react with PCOOH. 25 As an extra- cellular enzyme it is present only when GPx activities are to be measured in crude tissue homogenates. An unequivocal differentiation can only be made in samples solely containing cGPx and pGPx. Other glutathione peroxidases cannot be dif- ferentiated by activity thus far. There are, however, hints of a certain specificity of GI-GPx for 13-hydroperoxyoctadecadienoic acid (13-HPODE). 26 In GI-GPx expressing cells (HepG2) a GPx activity with 13-HPODE could be determined in selenium deficiency. Selenium-deficient HepG2 cells express reasonable amounts of GI-GPx, whereas cGPx is completely lost. 26 In ECV cells, which do not ex- press GI-GPx, little activity with 13-HPODE was detected. PHGPx activity was comparable in both cell types. This might indicate that 13-HPODE is a substrate preferentially used by GI-GPx. As long as purified GI-GPx is not available this can, however, not be stated with certainty.

Comments . PCOOH is synthesized as described in Maiorino et al., 23 a pro- cedure that requires oxygenation of phosphatidylcholine by soybean lipoxyge- nase in the presence of bile salts, for example, 3 mM deoxycholate (DOC). At the end of the reaction DOC must be removed, because it is a strong inhibitor of PHGPx. Whereas cGPx is not inhibited by deoxycholate up to a concen- tration of 10 mM, PHGPx is inhibited by 50% at 1 mM DOC. 23 Thus, care must be taken that the deoxycholate concentration is kept below 0.1 mM in the test system. Every freshly prepared batch of PCOOH should be checked to determine that the DOC concentration is low enough. This can be done by comparing the initial velocities of the enzymatic reaction obtained with differ- ent dilutions of the respective PCOOH batch. The concentration of PCOOH is easily determined by letting the coupled test run to completion in the presence of purified PHGPx or, alternatively, a testis extract. DOC concentrations are low enough if the initial velocities of the reaction at different dilutions of PCOOH are the same or similar (-4-10% might be acceptable). Only if the initial slopes are comparable, is the DOC concentration low enough not to interfere with PHGPx activity.

23 M. Maiorino, C. Gregolin, and E Ursini, Methods EnzymoL 186, 448 (1990). 24 S. Maurer, C. Friedrich, M. Leist, M. Maiorino, and R. Brigelius-Floh6, Z. Erngihrungswiss. 37, 1 I0

(1998). 25 y. Yamamoto and K. Takahashi, Arch. Biochem. Biophys. 305, 541 (1993). 26 K. Wingler, C. MUller, K. Schmehl, S. Florian, and R. Brigelius-Floh6, Gastroenterology 119, 420

(2000). 27 j. Chaudi6re and A. L. Tappel, Arch. Biochem. Biophys. 226, 448 (1983).

[9] INDIVIDUAL GPxs !07

D e t e r m i n a t i o n b y R N A A n a l y s i s

Northern Blotting

Northern blots have been widely used for qualitative as well as quantitative de- termination of GPx expression. Northern blots are less sensitive than, for example, polymerase chain reaction (PCR)-based methods, and the specificity depends on the probe used for hybridization. In our hands, DNA probes spanning almost the complete coding sequence plus the 3'-untranslated region provided the best results 28:

cGPx (human): nt 76-629 (accession no. M21304), 554 bp PHGPx (pig): nt 42-773 (accession no. X76009), 732 bp GI-GPx (human): nt 29-925 (accession no. X68314), 897 bp

The probe for pig PHGPx also hybridizes with human and rat PHGPx and can be used to detect PHGPx expression in species different from pig. Probes can be obtained by reverse transcriptase (RT)-PCR and subsequent cloning of the PCR products. 28

Comments. The low sensitivity of Northern analysis implies that low-level transcription may be overlooked. For example, GI-GPx mRNA was not detected in rodent liver by Northern blots.5 RT-PCR, however, revealed that GI-GPx mRNA is indeed expressed in rat liver (Fig. 1), as was also confirmed by Western blotting (see below).

600 bp -~

300 bp -~

1 2 a 4 5 6 7

FIG. 1. GI-GPx expression in human cell lines and various rat tissues as detected by RT-PCR. Total RNA was isolated by using the SV total RNA isolation system (Promega, Madison, WI) according to the manufacturer instructions. PCR products from human cell types HepG2 (lane 2), CaCo-2 (lane 3), and ECV (lane 4) were obtained with the human GI-GPx primer pair, resulting in the 602-bp fragment. PCR products from rat tissues, colon (lane 5), spleen (lane 6), and liver (lane 7), were obtained by means of the mouse GI-GPx primer pair, resulting in the 570-bp fragment. Other conditions were as described in Procedures under Determination by RNA Analysis.

108 SELENOPROTEINS [9]

Reverse Transcriptase-Polymerase Chain Reaction

The structural relatedness between the various mammalian GPx isotypes is so low that they can easily be differentiated by PCR by choosing suitable primers. Appropriate PCR primers are as follows.

cGPx (human) (M21304) Forward primer: 51-AGTCGGTGTATGCCTTCTCG-3 ', nt 76-95 Reverse primer: Y-TTGAGACAGCAGGGCTTCGAT-3 I, nt 629-609,

554 bp GI-GPx (human) (X68314)

Forward primer: Y-TCACTCTGCGCTTCACCATG-T, nt 18-37 Reverse primer: Y-AGCAGTTCACATCTATATGGC-3', nt 619-599,

602 bp GI-GPx (mouse) (X91864)

Forward primer: Y-GGCTTACATTGCCAAGTCGTTC-3', nt 42-63 Reverse primer: 51-CTAGATGGCAACTTTGAGGAGCCGT-3 ', nt 611-

587, 570 bp PHGPx (human) (X71973)

Forward primer: 5~-ATGAGCCTCGGCCGCCTTTG-3 ', nt 81-100 Reverse primer: 5'-AGCTAGAAATAGTGGGGCAGG-3 ~, nt 676-656,

596 bp

Procedures Any method for the isolation of total RNA can be used. Care should be taken

that the RNA does not contain DNA, that is, use methods that contain a DNA digestion step.

Appropriate conditions for RT-PCR (e.g., Access RT-PCR system from Promega, Madison, WI) are as follows: 10/zl of 5 x reaction buffer, dNTPs (0.2 mM each), 3 mM MgSO4, 2% (v/v) dimethyl sulfoxide (DMSO), 50 pmol of each primer, 5 U of avian mycloblastosis virus (AMV) reverse transcriptase, and 5 U of Thermusflavus DNA polymerase, in a final volume of 50/zl. Overlay the samples with mineral oil (PerkinElmer, Weiterstadt, Germany). Program a thermal cycler [e.g., a Biometra (GOttingen, Germany) thermal cycler T3] to 45 min at 48 °, 2 min at 95 °, 45 sec at 94 °, 1 min at 63 °, and 1 rain at 68 °, (40 cycles) and a final elon- gation step of 7 rain at 68 °. An aliquot of the RT-PCR sample can then be run on a 1% (w/v) agarose gel and the fragments stained with ethidium bromide.

The human and mouse primers (which work also for rat GI-GPx) listed above were used to analyze GI-GPx mRNA in human cell lines and various rat tissues according to the described procedure (Fig. 1). GI-GPx mRNA was clearly de- tectable in HepG2 (Fig. 1, lane 2) and CaCo-2 (Fig. 1, lane 3) cells but not in ECV

28 K. Wingler, M. B6cher, L. Floh6, H. Kollmus, and R. Brigelius-Floh6, Eur. J. Biochem. 259, 149 (1999).

[9] INDIVIDUAL GPxs 109

ceils (Fig. 1, lane 4), as expected. In rats, GI-GPx mRNA was present in colon (Fig. 1, lane 5) but not in spleen (Fig. 1, lane 6), as known from previous studies. 5 Surprisingly, GI-GPx mRNA was also detectable in rat liver (Fig. 1, lane 7). This is in contrast to the results from Chu et al., 5 who did not find GI-GPx mRNA in rat but only in human liver by Northern blotting. This demonstrates the preference for RT-PCR over Northern analyses if the expression levels are low.

Comments. Because of the different ranking of glutathione peroxidases in the hierarchy of selenoproteins, the amount of mRNA does not necessarily reflect the amount of protein being translated therefrom. Protein and mRNA levels can be correlated only when the selenium status of the tissues investigated is adequate. RT-PCR can, however, be used to study the actual amount of RNA in tissues and cells at different selenium states, if an appropriate method to quantify the PCR products is available.

D e t e r m i n a t i o n b y I m m u n o c h e m i c a l M e t h o d s

Western Blotting

Western blotting allows both qualitative and quantitative detection of glu- tathione peroxidase proteins. It indicates the real amount of a certain GPx that is translated into the protein from the respective mRNA. RNA and protein content do not necessarily correlate, especially when measured in cells or tissues grown in a limited supply selenium (see above).

A major problem in the detection by immunochemical methods is that specific antibodies are not commercially available for all types of GPx. Only for cGPx is a small number of antibodies or antisera available with, at least in our hands, insufficient specificity. Antisera against pig PHGPx have been raised in rabbits 29 and against rat liver PHGPx. 3° We have had the opportunity to test the former and found a high specificity. Because of the small amount of PHGPx in most cell lines and tissues, it must be concentrated before the assays. This can be done by running the samples through a small column filled with bromosulfophthalein Sepharose and eluting PHGPx with a high-salt buffer [e.g., three times through a small volume of 25 mM Tris-HC1, (pH 8.0), 0.5 M KC1, 10% (v/v) glycerol]. An exception is testis, which has the highest PHGPx levels of all mammalian tissues. For GI-GPx only two antisera have been described so far. 31'32 The first has been raised against a recombinant GI-GPx protein in which the selenocysteine was replaced by cysteine. 31 The second was raised against the 17 C-terminal amino acids of GI-GPx. 32 This peptide is identical in humans, rats, and mice. The antiserum can therefore be used for the detection of GI-GPx in a variety of species.

29 A. Roveri, M. Maiorino, and E Ursini, Methods EnzymoI. 233, 202 (1994). 3o M. Nakashima, S. Komura, N. Ohishi, and K. Yagi, Biochem. Mol. Biol. Int. 29, 1139 (1993). 31 R. S. Esworthy, K. M. Swiderek, Y.-S. Ho, and E-E Chu, Biochim. Biophys. Acta 381, 213 (1998). 32 M. Brcher, T. Brldike, M. Kiess, and U. Bilitewski, J. Immunol. Methods 208~ 191 (1997).

110 SELENOPROTEINS [9]

Procedures SAMPLING. (1) Cultured cells are homogenized and extracts prepared as

described. TM Wash harvested cells in phosphate-buffered saline (PBS) and cen- trifuge at 150g for 8 rain at 4 °. For enzymatic determinations homogenize cell pellets containing 2-4 x 107 cells in 0.7-1.5 ml of Tris buffer (100 mMTris-HC1, 300 mM KC1, pH 7.6) containing 0.1% (v/v) peroxide-free Triton X-100 by soni- fication with 10 strokes at 70% energy and 30% duty cycle. (2) Tissues are taken from anesthesized animals and immediately frozen in liquid nitrogen. Transfer small pieces of frozen tissue samples into ice-cold buffer [100 mM Tris (pH 7.6), 300 mM KC1, 0.1% (v/v) peroxide-free Triton X-100; 1 ml/500 mg tissue] and homogenize with an Ultra-Turrax (e.g., an Ultra Turrax T25 from IKA, Staufen, Germany). Clear homogenates by centrifugation (10,000g, 10 rain, 4°). Separate 100-150/zg of protein on a 12.5% (w/v) polyacrylamide gel according to standard electrophoresis protocols.

BLOTTING. Estimation of cGPx and GI-GPx requires different procedures. For cGPx determination, the nitrocellulose membrane and six blotting papers (Schleicher & Schuell, Dassel, Germany) are mounted between the cathode and anode of a semidry blotting system as follows: cathode, three blotting papers equilibrated in buffer C (see below), polyacrylamide gel, membrane wetted with buffer B, one blotting paper equilibrated in buffer B, and two blotting papers equi- librated in buffer A. For GI-GPx determination the arrangement is the same but only buffer D is needed. Protein transfer is performed at 0.8 mA/cm 2. The blotting time for cGPx is 8 hr; the blotting time for GI-GPx is 2 hr. Other glutathione peroxidases can be blotted like GI-GPx.

Buffer A: Buffer B: Buffer C:

BufferD:

300 mM Tris-HC1, 20% (v/v) methanol, pH 10.4 20 mM Tris-HC1, 20% (v/v) methanol, pH 10.4 25 mM Tris-HC1, 20% (v/v) methanol, 40 mM aminocaproic acid, pH 9.4 25 mMTris-HC1, 190 mM glycine, 15% (v/v) methanol, pH 8.5

IMMUNOCHEMICAL DETECTION

1. Blocking of nonspecific binding sites on the membrane. The nitrocellulose membrane is kept overnight in 2% (cGPx) or 5% (GI-GPx) low-fat milk powder in TTBS [50 mM Tris-HC1, 150 mM NaC1, 0.1% (v/v) Tween 20, pH 7.5] at 4 ° with gentle shaking. Excess milk powder is removed by washing with TTBS.

2. An appropriate GPx antibody must be selected and used as the first antibody. The antibody or serum, respectively, is diluted in TTBS. TTBS for dilution may contain 3% (w/v) bovine serum albumin. The reaction is performed for 3 hr at room temperature with gentle shaking. Thereafter, excess antiserum is removed by washing (four times, 10 min each) with TTBS.

[9] INDIVIDUAL G P x s 111

3. As second antibody, a horseradish peroxidase-coupled sheep anti-rabbit !gG fraction (Dako, Hamburg, Germany) is used. This is diluted 1 : 3000 with TTBS and the blot is incubated with the dilution for 1 hr at room temperature. Thereafter, the blot is washed with TTBS (four times, 10 rain each).

4. The chemiluminescence reaction is initiated by treating the blot with en- hanced chemiluminescence (ECL) solution (Amersham, Braunschweig, Germany) for 2 min. Chemiluminescence is made visible either by autoradiography (X-Omat, Kodak, Stuttgart, Germany) for various times or by densitometry (in a Fuji LAS- 1000-CCD-camera system) (Raytest, Straubenhardt, Germany).

According to the procedures described, various tissues from rat and three cul- tured cell lines were investigated for cGPx and GI-GPx expression. Two cell lines (HepG2 and CaCo-2) are known to express GI-GPx, and one (ECV, a human bladder carcinoma epithelial cell line) is known not to express GI-GPx. As first antibody for GI-GPx the antiserum described by B6cher et al. 32 was used, and for cGPx an antiserum kindly provided by Q. Shen (University of Massachusetts, Worcester, MA) was used. Anti-cGPx antiserum was diluted 1 : 1000 and anti- GI-GPx antiserum 1 : 5000, both in TTBS. As shown in Fig. 2 (middle) all cells

GI-GPx, 2 h ~ ~ ' =~

cGPx, 8 h

GI-GPx, 8 h

FIG. 2. cGPx and GI-GPx in various cell lines and rat tissues as detected by antisera against the respective enzymes. Tissues were sampled from male Wistar rats housed in individual conventional cages under controlled conditions of temperature and humidity. Animals were given free access to water and standard pelleted rat diet (C 1000; Altromin, Lage, Germany). Rats were anesthesized and perfused in situ with ice-cold sterile 0.15 M KC1. The tissues were then quickly removed and the intestinal lumen was flushed with ice-cold sterile phosphate-buffered saline. The tissues were cut into small pieces of 450-100 rag, immediately frozen in liquid nitrogen, and stored at - 8 0 °. Samples were prepared and analyzed as described in Procedures under Determination by Immunochemical Methods. Top: Blotting under conditions required for GI-GPx and detection by anti-GI-GPx serum. Middle: Blotting under conditions required for cGPx and detection by anti-cGPx serum. Bottom: Blotting under conditions required for cGPx and detection by anti-GI-GPx serum.

112 SELENOPROTEINS [91

and tissues express cGPx although in different amounts. GI-GPx was found in HepG2 and CaCo-2 cells, but not in ECV cells, as expected (Fig. 2, top). It was also found in rat colon and ileum and, surprisingly, in rat liver. This shows that GI-GPx is indeed expressed in liver from a rodent species that has previously been excluded. 5 Cross-reactivity of GI-GPx antiserum with cGPx or PHGPx was checked earlier in blots prepared for GI-GPx determination (2 hr) and excluded, z6 To rule out misinterpretations due to the different blotting times required for cGPx and GI-GPx, specificity of the antisera used was reevaluated. Figure 2 (middle) shows the blot analyzed with anti-cGPx antiserum (blotting time, 8 hr) and indi- cates cGPx is present in all cells and tissues, whereas Fig. 2, (bottom) demonstrates that anti-GI-GPx antiserum detects GI-GPx only in HepG2 and CaCo-2 cells and in rat colon, ileum, and liver. ECV cells and rat spleen did not react with GI-GPx antiserum, indicating that it does not cross-react with cGPx.

C o n c l u s i o n s

Differential determination of individual glutathione peroxidases by activity measurements is complicated by overlapping specificities. Only PHGPx can be differentiated in this way from other intracellular glutathione peroxidases by means of the specific substrate PCOOH.

The mRNAs can, of course, be estimated specifically by Northern blotting or, more sensitively, by PCR. Unfortunately, the mRNA levels do not correlate well with pertinent enzyme levels, because the different mRNAs are not transcribed with identical efficiencies and their stabilities are differentially affected by the selenium status.

Immunological assays promise the most specific measurement of GPx isotypes. However, the protein content, as is evident at least for PHGPx, does not always reflect active enzyme. Also, the immunological determination of individual GPx types is hampered by the lack of commercial availability of most of the anti- bodies required. The analysis of the diversified GPx system must still therefore employ complementary methodological approaches. A differentiation between the individual types of glutathione peroxidases appears mandatory, because they are evidently engaged in distinct biological roles.

A c k n o w l e d g m e n t s

This work was supported by the Deutsche Forschungsgemeinschaft (INK 26/B1-1 and SPP 1087, Br 778/5-1).