ARCHIVES

Vol.

OF BIOCHEMISTRY

286, No. 2, May

AND

BIOPHYSICS

1, pp. 330-336,199l

Characterization and Partial Amino Acid Sequence of Human Plasma Glutathione Peroxidase’ R. Steven

Esworthy,

Department Immunology,

of Medical Beckman

Received

August

Fong-Fong

Chu, Raymond

J. Paxton,*

17, 1990, and in revised

form

December

Academic

Press,

Akman,

and James H. Doroshow2 Center, and *Division

of

17, 1990

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82-l 13 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157-178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 2 1 -kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation. 0 1991

Steven

Oncology and Therapeutics Research, City of Hope National Medical Research Institute of the City of Hope, Duarte, California 91010

Inc.

The existence of a selenium-dependent glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreduc-

tase, EC 1.11.1.9) (GSHPX)~ activity in plasma has been clearly demonstrated (1, 2). The plasma form of the enzyme has many properties in common with the classical cytosolic enzyme (GSHPx-1) including its native form, which is a tetramer of 21-kDa isomonomers with one molecule of Se per subunit; both enzymes are active toward a similar range of substrates (1, 2). However, the two enzymes differ in many properties including immunological reactivity with antisera generated against the cytosolic enzyme, kinetic parameters with respect to both hydroperoxides and GSH, sensitivity to metals, glycosylation, and kinetics of depletion and repletion of activity in response to selenium (l-4). The question remains whether or not these differences reflect modifications to the cytosolic enzyme or whether they reflect the properties of a distinct GSHPx protein. We purified and sequenced plasma GSHPx to determine the relationship of the two proteins by comparing the amino acid sequences. To distinguish the plasma GSHPx from the cellular, monomeric, selenium-dependent, phospholipid hydroperoxide glutathione peroxidase (PHGPX), we have tested the plasma GSHPx for phospholipid hydroperoxide-reducing activity (5). The level of glutathione (GSH) in the plasma is about lO,OOO-fold lessthan that found in cells (6). It is not clear why a distinct plasma form of GSHPx with kinetic properties similar to the cytosolic form should be found in the plasma. Since plasma GSHPx appears to originate from the liver, we also examined bile as a potential alternative extracellular site for the expression of this plasma enzyme. MATERIALS

i This work was supported by NIH Grants CA-31788 and CA-33572 and BRSG Grant 2SO7 RR-05471 from the National Cancer Institute, and American Heart Association Grant 901 GI-2. x To whom correspondence should be addressed at the Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010.

Materials. The enzyme

AND

METHODS

Human erythrocyte GSHPx was enriched by fractionation

was purchased over Sephadex

from Sigma. G-200 to re-

’ Abbreviations used: EGTA, ethylene glycol bis(@aminoethyl ether) N,N,N’,N’-tetraacetic acid; GSHPx, glutathione peroxidase; PHGPX, phospholipid hydroperoxide glutathione peroxidase; RBC, erythrocyte; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

330 All

Copyright 0 1991 rights of reproduction

0003-9861/91 $3.00 by Academic Press, Inc. in any form reserved.

HUMAN

PLASMA

GLUTATHIONE

move alOO-kDa contaminants. Human plasma GSHPx was isolated from frozen plasma stored at -20°C for 6-12 months that was obtained from the Blood Bank of the City of Hope National Medical Center. After approval by the Institutional Review Board of the City of Hope, fresh human bile and plasma were obtained from a single subject who had a T-tube in the biliary tree that drained through an externalized plastic catheter. The subject was receiving total parenteral nutrition with Na selenite supplementation at 32.7 pg/liter and had normal liver function (7). Phosphatidylcholine hydroperoxide was prepared by M. Maiorino (University of Padua, Padua, Italy) by the method described in Zhang et al. (5). The preparation was free of significant free fatty acid hydroperoxide contamination as human erythrocyte GSHPx did not detectably reduce any component. Plasma GSHPxpurifiation. Three batches of plasma each containing 1.5 liters were purified according to a recently described procedure with minor modifications (1). Batches of enzyme containing 16 wg of protein were chromatographed by reversed-phase HPLC with a 4.6 X 30mm Brownlee RP300 column. The column was eluted with a gradient of 95% solvent A (0.1% trifluoroacetic acid, 99.9% water) and 5% solvent B (0.1% trifluoroacetic acid, 90% acetonitrile, 9.9% water) to 100% solvent B in 60 min. One major peak (70-75%) of protein flanked by two to four minor peaks eluted at 50-60% solvent B as monitored at 214 nm. Between 6 and 8 pg of protein was recovered from the HPLC step as determined by amino acid analysis. Quantitation was performed with a Perkin-Elmer LIMS 2000 Chromatography Data System. Amino acid sequencing. The major peak from the HPLC step was concentrated to a volume of 50 pl by vacuum centrifugation, and reduced by 71 mM &mercaptoethanol in 6 M guanidine-HCl, 1 mM EDTA, and 0.25 M Tris-HCl at pH 8.5 for 14-2 h under nitrogen. The reduced protein was alkylated with 0.37 M I-vinyl pyridine for 14-2 hours under nitrogen. It was then desalted using a TSK-250 column equilibrated with 5% solvent B, 95% solvent A. The first peak eluting, as monitored at 214 nm, was collected. The volume of the desalted material was decreased to 50 ~1 and prepared for proteolysis with about 1 pg of trypsin, endopeptidase Lys-C, or endopeptidase Asp-N (Boehringer-Mannheim, Indianapolis, IN). Trypsin digestions were performed in 0.2 M ammonium bicarbonate, pH 8.0, at room temperature overnight. Asp-N digestions were performed in 25 mM sodium phosphate, pH 8.0, at 30°C overnight. Lys-C digestions were performed in 50 mM Tris-HCI, pH 8.5, at room temperature overnight. The resulting peptides were applied to a 2.1 X 250-mm Vydac Cl8 reversed-phase HPLC column and eluted with a linear gradient of 98% solvent A:2% solvent B to 30% solvent A:70% solvent B in 60 min at a flow rate of 0.15 ml/h. Peaks were monitored at 214 nm and collected manually. The fractions were concentrated to a volume of lo-20 ~1, mixed with an equal volume of acetonitrile, and spotted onto a polyvinylidene difluoride membrane (Millipore) pretreated with polybrene. Sequence analyses were performed on the polyvinylidene difluoride filters using gas-phase sequencers built at the City of Hope and equipped with a continuous-flow reactor (8, 9). Enzyme and protein assays. GSHPx activity was measured spectrophotometrically at 340 nm by following the oxidation rate of NADPH as described (10). Substrates used included H202 (lo), tert.-butylhydroperoxide (ll), and phosphatidylcholine hydroperoxide (5). Assays for GSHPx activities were performed on bile concentrated in a Centricon microconcentrator with a 30-kDa-cutoff membrane (Amicon Division, W. R. Grace & Co.) or by ammonium sulfate fractionation. The 35% ammonium sulfate precipitates were resuspended in 0.1 vol of 0.05 M sodium phosphate, 0.5 mM GSH, pH 7.0, and then dialyzed against the same buffer. For protein determinations, the BCA protein assay kit (Pierce) was used throughout the plasma GSHPx purification except at the Sephadex G-200 step, where the fluorescamine assay was performed (12). Bovine serum albumin was used as a protein concentration standard throughout. Sodium dodecyl sulfate-polyacrylamide gel electrophoresti (SDSPAGE). Sample preparation and SDS-PAGE were performed as de-

331

PEROXIDASE

scribed (13). The gels were composed of 12% acrylamide and 0.32% piperazine diacrylamide (Bio-Rad) and electrophoresis was performed at a constant current of 5-7 mA per gel slab overnight. Gels were fixed with 10% acetic acid, 40% methanol, and 50% H20, and then were processed with the Bio-Rad silver stain kit (Bio-Rad Laboratories, Richmond, CA).

RESULTS

Plasma GSHPx purification and characterization. In the final step of plasma GSHPx purification, gel filtration on Sephadex G-200, the plasma GSHPx activity eluted just before bovine serum albumin and coincidentally with cytosolic GSHPx; this is consistent with the expected 88kDa molecular mass (data not shown). The plasma GSHPx eluted from the column had a specific activity of =40 U/mg when assayed with 150 I.IM H202, 27”C, pH 7.0. The fractions containing the peak of plasma GSHPx activity were pooled, and the composition of the pooled fractions was analyzed by SDS-PAGE (12% acrylamide). One band of 21.5 kDa was detected by silver staining (Fig. 1). The plasma GSHPx and human cellular GSHPx from erythrocytes could not reduce phosphatidylcholine hydroperoxide under conditions where both are active toward HzOz (Fig. 2).

M kDa

1 2

FE

66 -

29 -

14.2

r*

-

FIG. 1. Plasma GSHPx after Sephadex G-200 gel filtration (lane 1) and after reversed-phase HPLC fractionation on a C8 column (lane 2). The samples (-2 pg in lane 1, ~1 pg in lane 2) were fractionated by SDS-PAGE and the gel was silver stained. The markers (lane M) were bovine serum albumin, carbonic anhydrase, and RNase A.

332

ESWORTHY

ET

AL.

PHGPX

WC

TIME (MINUTES)

TIME (MN)

TIME (MINUTES)

TIME (MINUTES)

FIG. 2. GSHPx assays of plasma GSHPx and RBC GSHPx-1. The left two panels are assays on enriched plasma GSHPx and human RBC GSHPx-1 using HzOz as the substrate. The plasma GSHPx activity was calculated to be 1 mu/al and the RBC GSHPx-1 activity was 0.25 mU/ ~1. The right two panels are assays on twice the amount of plasma GSHPx or RBC GSHPx-1 using phosphatidylcholine hydroperoxide (PCOOH) as the substrate. Porcine PHGPX was added to the reactions after a few minutes to confirm that the reaction mixtures were complete. The apparent deflection of the baseline in the third panel after PCOOH addition was an artifact probably caused by cuvette movement. The amount of PCOOH available to PHGPX in the third panel was not altered from the reaction in the last panel.

Analysis of bile for extracellular expression of the plasma GSHPx. The amount of GSHPx activity (0.002 U/ml) in the fresh bile obtained from a patient receiving Sesupplemented nutrients was close to the lower limit of detection in the assay system (Fig. 3). The plasma GSHPx activity expressed in the same patient was at the level of 0.20 U/ml. The GSHPx assay was also performed on three types of concentrated bile: Centricon-concentrated bile, dialyzed bile after the Centricon concentration step, and ammonium sulfate-precipitated and dialyzed material. The ammonium sulfate fractionation and dialysis steps

were performed to reduce the concentration of bile salts in the assays, since bile salts have been reported to inhibit PHGPX activity (5). However, when the purified cellular GSHPx-1 or plasma GSHPx was added to the bile sample in the GSHPx assays, the enzymatic activity was not inhibited (Fig. 3). Again, only a trace of GSHPx activity was detected in the concentrated, dialyzed material prepared by either ammonium sulfate fractionation or Centricon spin concentration (results not shown). Amino acid analysis and sequencing of plasma GSHPx. The plasma GSHPx isolated from a Sephadex G-200 siz-

/

RBC GSHPX +hli-PLASMA GSHPX 4G

0

5

Time (mid FIG. 3. GSHPx activity in bile using tert.-butylhydroperoxide (t-BOOH) as the substrate. The first panel shows that the fresh plasma sample of the subject had about 0.2 U/ml GSHPx activity. The second panel shows the association of GSHPx activity with the plasma fraction which is retained by a membrane filter with 30-kDa cutoff. No GSHPx activity is detected in the filtrate. The third panel shows that 380% of the GSHPx activity detected in the plasma can be removed by the antiplasma GSHPx IgG, whereas only 12-15% of the activity is removed by the anti-RBC GSHPx-1 IgG. The fourth panel is the control reaction demonstrating the reactivity of the anti-RBC GSHPx-1 and antiplasma GSHPx IgG preparations against RBC GSHPx-1. The fifth panel shows that 30 ~1 of bile has almost no detectable GSHPx activity. The sixth panel demonstrates that the preincubation of plasma and bile (1:l) for 2.5 h at room temperature prior to assay did not result in the loss of plasma GSHPx activity.

HUMAN

PLASMA

GLUTATHIONE

333

PEROXIDASE

TABLE cl?-

Amino Acid Composition of PlasmaGSHPx and Cellular GSHPx

0.6 -

Mole

a5! x -2

Plasma”

Q4-

CYS

0.3 Q20.1 -

r’

ot? 0

FIG. 4.

I

5

K)

15

20 25 TIME (mid

30

35

40

45

Reversed-phase HPLC fractionation of the human plasma GSHPx preparation. Sixteen micrograms of plasma GSHPx eluting from the peak activity fraction from Sephadex G-200 was applied to a Cl8 reversed-phase column for fractionation. Peak 4 was isolated and used for amino acid composition analysis and amino acid sequencing.

Asx Thr Ser Glx Pro GUY Ala Val Met Ile JAI Tyr

Phe His LYS Ax Trp

1.4c 8.6 3z 0.6 4.2 zk 0.4 4.2 2 0.7 13.7 f. 0.9 6.2 f 0.4 13.9 + 2.6 4.0 t- 0.3 6.2 f 0.5 1.5 2 0.1 5.5 f 0.1 9.4 2 0.4 4.3 2 0.9 6.6 + 0.2 2.2 * 0.2 6.3 2 0.1 3.4 + 0.1 NDd

percentage Cellular* 2.5 9.0 3.5 5.5 10.0 7.5 8.5 11.0 6.5 2.0 3.0 11.6 2.0 5.5 1.0 3.0 7.0 1.0

ing column was chromatographed further with reversedphase HPLC. The major peak (peak 4), which constituted ’ Three analyses on 0.5-1.5 pg of purified protein. * Mullenbach et al. (14). ~75% of total protein as determined by absorbance at ’ Cysteine was determined as cysteic acid in a separate analysis after 214 nm, was the 21.5kDa polypeptide resolved by SDSperformic acid oxidation. PAGE (Figs. 1 and 4). The peak 4 material from three d Not determined. independent preparations was analyzed for amino acid composition (Table I) and was used for amino acid sequencing. The amino acid composition of plasma GSHPx from pooled plasma and one allelic variant of cellular veals that the correct position of peptide T20 is at residues 129-141 or 129-145. The latter alternative, which includes GSHPx shows that there are significant differences for the glutamine/glutamic acid (Glx), glycine, alanine, leu- a four-residue gap, is displayed (Fig. 6). Additionally, we can assign peptide L7/LlO/T26 to a position next to the tine, lysine, and arginine content of the two proteins. amino-terminal end of peptide T20 (position 121-128). The amino terminus of the protein was blocked. The protein was digested with either endopeptidase trypsin, Asp-N, or Lys-C. The tryptic peptide map is shown in 0.16~ Fig. 5. The sequences of the peptide fragments from all T32 three peptide maps are listed in Table II. 0.14 Peptides T21, T30, LB, A14, and A19 represent overlapping fragments that combine to produce a single se0.12 quence of 23 amino acids; and peptides T22, T32, L17, AM, and A206 combine to yield a single sequence of 32 0.10 E amino acids. These two larger sequences can be aligned 0 -50 s ooswith confidence to the amino acid sequence of cellular .40 ” a .30 GSHPx-1 (Fig. 6). 0.06 Ji Peptide T20 was tentatively placed at the carboxyl ter.lO z minus based on the amino acid identity in three positions - 0 0!34with GSHPx-1 and the failure to detect any amino acid 002in the cycle following the serine residue. Additionally, peptide T12 was tentatively assigned to position 49-55 of 0’ I A ’ ’ 1 0 ’ . 8 5 x) 20 x) 40 50 GSHPx. We have recently isolated a cDNA clone of huTime (minutes) man plasma GSHPx from a placental library (F. F. Chu, unpublished data, 1990). The deduced amino acid se- FIG. 5. Tryptic peptide map of plasma GSHPx. The sequences of the quence that we have obtained from the cDNA clone re- labeled peptides are shown in Table II.

334

ESWORTHY TABLE

List

of Sequenced

Peptide

II

Fragments

Endopeptidase All Al4 Al6 A18 A19 A20-6

Asp-N

Lys-C

’ Residues in parentheses identified. ’ The single dash denotes to this position.

GSHPx

peptide

peptide

YTFLK -DIRWNFEK -Q-FYTFL YVRPGGGFVPNFQL MDILSYMRRQAALG

Endopeptidase T12 T20 T21 T22 T26 T28 T29 T30,31 T32

Plasma

DRLFWEPMKVQ EKFLVGPDGIPIMRRQQ(RR-VV)“*b DILSYMRRQAALG EILPTLKYVRPGGGFVPNFQLFEKG DIRWNFEKFLVGPDGIPIMRR EILPTLKYVRPGGGFVPNFQLF

Endopeptidase L7 L8 LlO L17 L24

from

Trypsin

peptide

TTVSNVK NSCPPTSELLGTS (W)NFEK QEPGENSEILPTLK FYTFLK LF-EPMK MDILSYMR FLVGPDGIPIM YVRPGGGFVPNFQLFEK were

tentatively,

that no amino

but

acid residue

not

unequivocally,

could be assigned

Peptide T12 may not be located at position 49-55 and cannot be properly placed at this time. The linear polymerase chain reaction method (17) was used to sequence the cDNA clone. The reaction was primed with a unique oligonucleotide based on the amino acid sequence of peptide All to partially sequence the clone in the direction of the amino terminus. Based on the position of peptide sequence represented by peptides T20, T26, L7, and LlO in the deduced amino acid sequence of the cDNA clone as adjacent to the primer site on the amino-terminal side, the peptide All/T28 can be placed at position 146-156. The correctly aligned partial amino acid sequence of the plasma GSHPx peptides represents 84 amino acid residues or 42% of GSHPx-1. The amino acid identity between plasma GSHPx and GSHPx-1 is 46/84 or 55% in the portions aligned in Fig. 6. The alignment between plasma GSHPx and GSHPx-2, a GSHPx-l-like cDNA isolated from liver and HepG2 cDNA libraries, reveals slightly less identity (39 identical residues out of 84). DISCUSSION

Plasma GSHPx can be distinguished from the two wellcharacterized, cellular selenium-dependent GSHPx ac-

ET

AL.

tivities (GSHPx-1 and PHGPX) and an uncharacterized GSHPx-l-like cDNA clone, GSHPx-2 (10, 15, 16). Do the data support the hypothesis that plasma GSHPx is the product of a gene different from genes for any of the characterized cellular activities? The major alternative hypotheses are that (1) the sequence data we have presented represent the sequence of a GSHPx-1 allelic variant represented in the plasma due to sampling bias or bias due to preferential leakage into the plasma, and (2) the sequence is that of some other cellular GSHPx activity which could be leaked into plasma such as PHGPX. Sampling bias of any sort is unlikely. Three separate pools of plasma consisting of eight individual plasma samples each were analyzed for the amino acid composition results shown in Table I. We detected no major differences among the pools for amino acid composition except for glycine, suggesting that we were determining the composition of the predominant form(s) of plasma enzyme which is (are) significantly different from the GSHPx-1 allelic variant used to generate the composition results used in Table I (14). This is further substantiated by the fact that the amino acid sequence deduced for the placental cDNA clone is identical to the plasma amino acid sequences aligned in Fig. 6. How much sequence variation is present in allelic variants of GSHPx-l? So far, only two obvious allelic variants of classical GSHPx-1 have been detected and sequenced (14, 18, 19). These two variants differ by one amino acid residue at position 91 (Gln-Leu). The sequence of plasma GSHPx is also quite different than the deduced amino acid sequence of a GSHPx-l-like cDNA clone, GSHPx2, which was reported by us (10, 15), and the complete sequence presented by Akasaka et al. (16). But, the existence of the GSHPx-2 sequence raises the question of whether very dissimilar GSHPx-1 variants exist. Since the cellular distribution of the GSHPx-2 mRNA is restricted to liver and liver-derived cell lines, we can tentatively discount the idea that GSHPx-2 represents an allelic structural variant of GSHPx-1 (unpublished data). The plasma GSHPx also appears to differ from the ubiquitously expressed PHGPX by virtue of the tetramerit subunit composition of the plasma enzyme versus the monomeric subunit structure of PHGPX and the lack of activity of plasma GSHPx toward phosphatidylcholine hydroperoxide, which represents one of the two major classes of substrates of PHGPX (5, 20). Since PHGPX does not exhibit significant levels of activity toward hydrogen peroxide or tert.-butyl hydroperoxide under optimal conditions or after partial denaturation in deoxycholate it is difficult to conceive of circumstances where the activity of PHGPX could give rise to the plasma GSHPx activity. Thus, based on the observations described above, plasma GSHPx activity is not derived from known ubiquitous cellular activities. In summary, our results demonstrate that plasma GSHPx is a unique mem-

HUMAN

PLASMA

mAfiAkSfYdlSAisLd

GLUTATHIONE

Px2

1

PXl

1 mcaarlaAaaAqSvYafSArpLagGEpVsIgslRGkvlLIENVaSLCS’GTTvRDyTQRNE~rRLgPRG

Px2

58 LWLGFPCNQFGHQENcqNEEIlNSLKYVRPGGGyqPtFtLvqKCE~GqneH~FAy~dkLPyPyD

Pxl

69 LWLGFPCNQFGHQENalcNEEIqNSIl(WRPCGGGFePN~LFEKC~GagaHPlFAFLreaLPaPSD

I I I I II

GEkVdfntfRGravLIKNVISLCs*GTTtRDfTQINEI.QcRf

I II I

Illll

III

PRr

III II II IIII I II

III lllllllllll I I I llllll II II I II I I Ill I II IIIIlIIIII III Ill1 III II II LViLGFPCNQFG~EPGENSEILPTLKWRPGGGFVPNFQLFEd~GekeqkFYTFLKNSCPPTSE IIIIIIIIIIIIIIIIIIIIlIlIlIIIIIII IIIlIlIIIIIIlI

Pla

QEPGENSEILPTLKWRPGGGFVPNFQLFEKG

Px2

126 DpfsLMTDPKLIiWSPVrRsDVAWNFEKFLiGPeGePf

Pxl

137

Pla

II

lIIIIIlIIlIIIIlI II IIIIIIIIl

PX3

Px3

335

PEROXIDASE

FYTFLKNSCPPTSE

RRYSRtFpTmnIEPDIkrLLhai.

I

IIIIIIIIIIlII I IIIIIIlIII II I I IIIII I I IIIII II DataLMTDPKLItWSPVcRnDVAWNFEKFLVGPDGvPl RKYSRRFqTidIEPDIeaLLsqgpsca. I I I LLGTSDR FWEPMKV 1IIIIII III/III I IlllllllllII I II LLGTSDRIFWEPMKVqDirWNFEKFLVGPDGiPimRRqq

FIG. 6.

Amino acid sequence alignment of GSHPx-1 (Pxl), GSHPx-2 (Px2) and plasma GSHPx obtained from cDNA (Px3) or protein sequence analysis (PLA). The GSHPx-1 sequence is the form presented by Mullenbach et al. (14). GSHPx-2 is the deduced amino acid of a cDNA isolated as described in Chu et al. (15; Chu, unpublished data, 1990) also reported by Akasaka et al. (16). GSHPx-3 is the deduced amino acid sequence of a partial sequence of a placental plasma GSHPx cDNA clone (Chu, unpublished data, 1990).

ber of what appears to be a GSHPx gene family. The genes of the family may be dispersed in the genome, accounting for detection of more than one structural locus for GSHPx in the study of McBride et al. (21). We have screened the media of a panel of human cell lines for a 75Se-labeledsecretory protein consisting of 21kDa monomers (data not shown). Only two hepatocellular carcinoma cell lines, HepG2 and Hep3B cells, were positive. Others, including kidney carcinoma A-498 cells, breast carcinoma MCF-7 cells, promyelocytic leukemia HL60 cells, chronic myelogenous leukemia K562 cells, and both lipopolysaccharide-activated and nontreated histiocytic lymphoma U-937 cells, were negative. The 21kDa 75Se-peptide secreted by the hepatoma cells can be immunoprecipitated by anti-human plasma GSHPx antisera but not by anti-human RBC GSHPx-1 antisera, and plasma GSHPx was not detected within HepGP cells (data not shown). GSHPx enzymatic activity was detected in the conditioned media by HepG2 cells grown in defined medium (22). These observations are in agreement with those of Avissar et al., indicating that plasma GSHPx is most likely synthesized and secreted by liver-derived cells (23). Several investigators have questioned the ability of plasma GSHPx to participate in the detoxification of plasma lipid hydroperoxides in view of the steady-state plasma level of GSH, which is lO,OOO-fold below the concentration of GSH in the cell (1,24). Bile was considered as an alternative site of expression, because (a) liver appears to be the source of both bile and plasma GSHPx, and (b) bile has millimolar concentrations of GSH. We could detect virtually no GSHPx activity in the bile. Since a near-normal level of GSHPx activity was found in the

plasma of the patient from whom we obtained bile, our results show that bile is not a site for the extracellular expression of plasma GSHPx (25). The fate and function of the plasma GSHPx remain a mystery. ACKNOWLEDGMENTS We thank Mitzi Broussard for her excellent secretarial work in the preparation of this manuscript, Matilide Maiorino for expert assistance in the assay of PHGPX and the gift of porcine PHGPX, and Lawrence Wagman, M.D., of the Department of General and Oncologic Surgery for obtaining bile and plasma samples.

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14. Mullenbach, R. A. (1987)

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15. Chu, F.-F., Akman, Cancer Res. 29,440a.

S., and Doroshow,

16. Akasaka, M., Mizoguchi, Res. IS, 4619. 17. Murray,

P., Dairman, 178, 871.

V. (1989)

J., and Takahashi,

Nucl.

18. Sukenaga, Y., Ishida, Acids Res. 15, 7178.

J. (1988)

Acids

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K., Takeda,

Proc.

K. (1990)

Am. Assoc. Nucl.

Acids

K. (1987)

Nucl.

17.8889. T., and Takagi,

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19. Chada, S., Le Beau, M. M., Casey, L., and Newburger, P. E. (1990) Gerwmics 6,268-271. 20. Thomas, J. P., Maiorino, M., Ursini, F., and Girotti, A. W. (1990) J. Biol. Chem. 266, 454-461. 21. McBride, 0. W., Mitchell, A., Lee, B. J., Mullenbach, G., and Hartfeeld. D. (1988) Biofactors 1, 285-292. 22. Darlington, G. J., Kelly, J. H., and Buffone, G. J. (1987) In Vitro Cell. Dev. Biol. 23, 349-354. 23. Avissar, N., Whitin, J. C., Allen, P. Z., Wagneu, D. D., Liegey, P., and Cohen, H. J. (1989) J. Biol. Chem. 264,15850-15855. 24. Frei, B., Stocker, R., and Ames, B. N. (1988) Proc. Natl. Acad. Sci. USA 86,9748-9752. 25. Cohen, H. J., Brown, M. R., Hamilton, D., Lyons-Patterson, J., Avissar, N., and Liegey, P. (1989) Am. J. Clin. Nub. 49,132-139.