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Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase
ARCHIVES
Vol.
OF BIOCHEMISTRY
235, No. 2, December,
AND
BIOPHYSICS
pp. 589-595,
1984
Inhibitory Monoclonal Antibodies against Rat Liver Alcohol Dehydrogenase’ PUSHKARAJ Department
J. LAD,2 DALE of Medicine, Received
B. SCHENK,
AND
HYAM
L. LEFFERT3
Division of Pharmaco logy, M-013 H, University San Diego, La Jolla, California 9.2095 May
30, 1934, and in revised
form
August
of Cal&rnia,
1’7, 1984
Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-l and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-ll>R-9>R-4>R-6>R-lO>R-&R-2=R-3=R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH ‘7.7-7.8, a condition thought to protonate the enzyme’s active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase. 0 1984 Academic
Press, Inc.
Mammalian liver alcohol dehydrogenase (alcohol:NAD+ oxido-reductase, EC 1.1.1.1) is a zinc-containing dimeric protein which exhibits various isozymes (l-3). Studies of catalytic mechanisms usually have relied upon kinetic analyses with the horse enzyme, which is commercially available. By contrast, similar studies with rat alcohol dehydrogenase, the species most often used to analyze ethanol metabolism, have been limited due to the rat enzyme’s unstable nature. Although rapid purification procedures for the rat liver enzyme have recently facilitated kinetic analyses (4, 5), a controversy remains regarding the extent to which alcohol dehydrogenase contributes to liver alcohol metabolism.
Pharmacological agents have been employed to resolve this controversy. For example, pyrazole and its derivatives (e.g., 4-methylpyrazole) that inhibit alcohol dehydrogenase have been used to determine the contribution of alcohol dehydrogenase in total alcohol metabolism (6, 7). These agents have limited utility since, as competitive inhibitors, they are needed in high amounts to inhibit enzyme activity at high ethanol concentrations (8). Consequently, these agents nonspecifically inhibit catalase and microsomal ethanoloxidizing system activities as well (9). Therefore, potent specific inhibitors of rat alcohol dehydrogenase might help to distinguish its role from other enzymes involved in hepatic alcohol metabolism. One way to obtain such inhibitors is to produce specific neutralizing antibodies against alcohol dehydrogenase. Although two groups have developed polyclonal antibodies against the horse enzyme, these
’ This work was supported by USPHS Grants AM 28392, AA 03504, AM 28215 and GM 07752. a Present address: Genencor, Inc. 130 Kimball Way, South San Francisco, Calif. 94080. 3 To whom correspondence should be addressed. 589
0003-9861/&I Copyright All rights
$3.00
0 1984 by Academic Press. Inc. of reproduction in any form reserved.
590
LAD,
SCHENK,
antisera cross-reacted poorly with the rat enzyme (10-13). Inhibitory polyclonal rabbit antiserum against the rat liver enzyme has also been obtained, but it inhibited only 60% of enzyme activity (14). In contrast to these earlier findings, we report here the development of specific monoclonal antibodies against rat liver alcohol dehydrogenase with potent and full neutralizing activity. Because these antibodies can be obtained in unlimited amounts and in pure form, this antibody panel should prove valuable for further physiological, enzymatic, biochemical, and purification studies of rat liver alcohol dehydrogenase. EXPERIMENTAL
PROCEDURES
Materials. Adult male Fischer-344 rats (200-250 g) and male Balb/c mice (lo-20g) were obtained from Charles River Breeding Laboratories (Wilmington, Mass.). Fetal bovine calf serum (Rehtumin FS) was obtained from Armour Pharmaceutical Company (Kankakee, Ill.). DEAE-Affi-Gel Blue, AfhGel Blue, and Coomassie brilliant blue R-250 were purchased from Bio-Rad (Richmond, Calif.). Enzymegrade ammonium sulfate was supplied by Schwa& Mann (Orangeburg, N. Y.). Acrylamide (enzyme grade), N,N,N:N’-tetramethylethylene diamine, and ammonium persulfate were from Eastman-Kodak (Rochester, N. Y.). SDS” was from BDH Chemicals, Ltd. (Poole, England). Goat anti-mouse (IgG + IgM) antibody-peroxidase conjugate was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, Ind.). Rabbit anti-mouse (IgG + IgM + IgA) antibodies were from Cappel Laboratories (Westchester, Pa.). Goat antibodies against mouse IgG,, IgG%, IgGab, and IgM were obtained from Meloy (Springfield, Va.). Polyethylene glycol (Avg M, 1500) and DMSO were from Aldrich Chemical Company (Milwaukee, Wise.) and Pierce Chemical Company (Rockford, Ill.), respectively. Porcine glucagon-free insulin was a gift from Dr. W. Bromer (Eli Lilly Research Labs, Indianapolis, Ind.). Sephadex G-100, cyanogen bromide-activated Sepharose-4B, AMPagarose, NADf (Grade III), and other routinely used chemicals were from Sigma Chemical Company (St. Louis, MO.). Plastic microtiter plates (96 wells) and 24-well culture plates were from Flow Laboratories (Inglewood, Calif.). ’ Abbreviations used: DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunoadsorbent assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
AND
LEFFERT
Purifkatim and assay of rat liver alcohol dehydm gcnase. Rat liver alcohol dehydrogenase was purified to homogeneity as described previously (5). The standard procedure included differential centrifugation, ammonium sulfate precipitation, and Sephadex G-100, DEAE-A&Gel Blue, Affi-Gel Blue, and AMP-agarose chromatography. The purity of the enzyme preparation was checked by SDS-polyacrylamide gel electrophoresis and isoelectrofocusing (5). In order to study enzyme inhibition, partially purified enzyme preparations were used. These were prepared routinely as follows: 20 g frozen (-20°C) liver tissue was thawed and homogenized in 106 ml 10 mM Tris-HCl buffer (pH 7.0). A 100,OOOg supernatant was obtained from the homogenate, and was applied to a 50-ml Affi-Gel Blue column. The column was washed with 150 ml 10 mM Tris-HCI (pH 8.0) and 300 ml 10 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCI. The activity was eluted with a solution of 10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, and 1.0 mM NAD+. Alcohol dehydrogenase activity was measured at 37°C in a Beckman DU-8 spectrophotometer with a kinetics accessory unit, by monitoring the rate of NAD+ conversion to NADH at 340 nm. The standard reaction mixture (2 ml final volume) contained 0.5 M Tris-HCl (pH 7.2), 2.5 mM NAD+, and 8.0 mM ethanol (15,16). Proteins were determined according to Lowry et al. (17), with bovine serum albumin as the standard. Hybrids p-oductim Male Balb/c mice were immunized intramuscularly in the hind leg with lo15 pg of purified rat liver alcohol dehydrogenase in complete Freund’s adjuvant. Twenty five days later, booster injections of 7-10 pg pure enzyme in incomplete Freund’s adjuvant were made in the hind leg. One week later, antisera titers were determined with an enzyme-linked immunoadsorbent assay (ELLSA) (18). Mice which showed detectable antibodies (antisera dilution al:3600 in ELISA) were injected intraperitoneally and intravenously (tail vein) with 5 pg pure enzyme. Four days later the spleens were used for cell fusions. Cell fusions were performed by standard procedures (19-22). The parental mouse myeloma NS-l/ I’Ag 4.1 cell line was obtained from Dr. S. Sell (University of Texas, Houston, Tex.). Hybridoma colonies producing antibodies against rat liver alcohol dehydrogenase were detected by ELISA (18). These colonies were propagated and cloned in soft agar or by limited dilution techniques (21). Clones producing antibodies against rat liver alcohol dehydrogenase (detected by ELISA) were further propagated and stored in 95% (v/v) fetal bovine serum, 5% (v/v) DMSO at -70°C (21). Thawed clones continued to produce antibodies without detectable changes for more than 4 months in continuous culture.
MONOCLONAL
ANTIBODIES
AGAINST
Antibody purGfication and characterization Monoclonal antibodies were purified by immunoaffinity chromatography over rabbit anti-mouse (IgG + IgM + IgA) antibodies coupled to cyanogen bromideactivated Sepharose-4B (3 mg antibody/ml packed gel). Bound immunoglobulins were eluted with 0.1 M glycine (pH 2.8), neutralized with 1.0 M Tris base to pH 7.0, dialysed against distilled water or phosphatebuffered saline (20 mM sodium phosphate, pH 7.4, 0.15 M NaCl) for 72 h at 4°C and concentrated (by filtration at 4°C or by lyophilization). Protein concentrations were determined (17) after precipitation with silicotungstic acid (23). Characterization of monoclonal antibody immunoglobulin class/subclass was performed by Ouchterlony double immunodiffusion (24). About 100 gg goat IgG against specific mouse immunoglobulins (I&I, kGzw k’&ts, or IgM) from outer wells and 10 kg of monoclonal antibody from the central well were allowed to diffuse at 21°C until precipitin bands appeared. SDS-polyacryhmide gel electrophoresis. Discontinuous SDS-PAGE was performed under denaturing conditions (25). The stacking and running gels were 2 and 10% (w/v) acrylamide, respectively. Before each run, the samples (monoclonal antibodies, anti-
TABLE
ALCOHOL
591
DEHYDROGENASE
gen, or standards) were denatured at 100°C for 1.5 min in 50 mM Tris-HCI, pH 7.0, containing 2% (w/ v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.01% (w/ v) bromophenol blue, and were centrifuged in an Eppendorf microfuge for 5 min at 4°C. Electrophoresis was performed at 4°C for 3.5-4 h under a constant current of 13-14 mA. Proteins in the gels were stained with 0.03% (w/v) Coomassie brilliant blue R-250 in 10% (v/v) acetic acid and 25% (v/v) isopropanol. Gels were destained with a solution of 10% (v/v) acetic acid and 10% (v/v) isopropanol. RESULTS
Identi$cation and mono&ma1 antibodies cohol dehydrogenase.
characterization of against rat liver al-
Hybridomas that secrete monoclonal antibodies to rat liver alcohol dehydrogenase were obtained from two cell fusion experiments. Eleven stable clones were established (Table I). Monoclonal antibodies (R-1-R-11, Table I) secreted by these hybridomas were purified by immunoaffinity chromatography, and were compared on a weight basis, by I
MONOCLONALANTIBODIESAGAINSTRATLIVERALCOHOLDEHYDROGENASE
Antibody
iIf, (x10-3)
required
for
50% bd Monoclonal antibody R-l R-2 R-3 R-4 R-5 R-6 R-7 R-8 R-S R-10 R-11
Hybridoma clone 18C7 18GlO 18H8 18HlO 16E6 21F4 22D2 22FlO 24ElO 25Fl 26F7
Class/subclass of antibody IgM I@.% I@ I&, W I@ IN I&,” IgM IgGP I&P
Heavy chain 70 56 70 56 70 70 69 56 69 56 56
Light chain 30, 28 29 28 30 30 30 29 28 30 30 30
Binding”
Inhibitionb
0.71 2.15 2.70 0.36 0.63 0.84 4.70 3.50 0.11 1.40 0.64
>lOOd >lOOd >lOOd 35 >lOOd 98 >lOOd >lOOd 28 98 20
Note. Hybridoma clones secreting antibodies R-l to R-11 were obtained from fusion experiments. Immunoglobulin class/subclasses of antibodies were determined by Ouchterlony double diffusion. Molecular weights were determined by SDS-polyacrylamide gel electrophoresis as described under Experimental Procedures. a 50% binding represents A,% = 0.55 in Fig. 1. * 50% inhibition represents 50% inhibition of enzyme activity in Fig. 2. ‘Tentative, see Results. d Antibodies were not tested at concentrations higher than 100 pg.
592
LAD,
SCHENK,
AND
ELISA, for their capacity to bind to immobilized pure alcohol dehydrogenase (Fig. 1). Antibody R-9 showed the highest titer whereas antibodies R-7 and R-8 showed the lowest titers (Fig. 1). Relative binding titers, defined by the amount of antibody giving 50% of the maximum absorbance in the ELISA, are listed in Table I. Since the peroxidase-conjugated second antibody used was an undefined mixture of antibodies with potentially different avidities toward mouse IgG subclasses and IgM, absolute antibody affinities to alcohol dehydrogenase cannot be calculated from these studies. Affinity-purified antibodies were further characterized for their heavy- and lightchain molecular weights and immunoglobulin class/subclasses (Table I). Antibodies R-8, R-10, and R-11 did not form precipitin lines in double-diffusion tests with antisera against mouse IgGl, IgGz,, or IgM. However, these three Id&b, monoclonal antibodies contained heavy chains of molecular weight 54,000-57,000 (Table I). This suggests that they are either IgG, or IgA (more probably IgG3, because rabbit antimouse antibody used
122 IDE 0.8 r P 2 0.6. 0.4 0.2 -
II O.O 001
h
0.1
1.0
I loo
4
I 100
PG ANTIBODY /ICO~L
FIG. 1. Monoclonal antibody titration curves by ELISA. Microtiter plates were coated with a solution of 3 pg pure rat liver alcohol dehydrogenase/ml. After washing and blocking with a solution of 10 mg BSA/ml, the wells were incubated with varying amounts (0.01-40 @g/100 ~1) of antibodies [R-l (0), R-2 (A), R-3 (O), R-4 (V), R-5 (O), R-6 (A), R-7 (H), R-8 (W), R-9 (0), R-10 (+), and R-11 (X)]. Bound antibodies were detected with rabbit anti-mouse (IgG + IgM) IgG-peroxidase conjugate as described previously (18).
LEFFERT
i
FIG. 2. Inhibition of rat liver alcohol dehydrogenase activity by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 gg proteins) was incubated with varying amounts (O-100 pg) of antibodies [R-l (0), R-2 (A), R-3 (Cl), R-4 (V), R-5 (a), R-6 (A), R-7 (m), R-8 (v), R-9 (V), R-10 (+), and R-11 (X)] for 4 h at 21°C (pH 7.2), and was assayed for enzymatic activity. Control activity (100%) was 0.04 flM NADH formed min-’ assay mix-‘.
in screening by ELISA was against mouse
[hi& + IgMl).
Efict of rrumoc kmal antibodies on alcohol dehydrogenase activity. Partially purified
enzyme (100 pg proteins) was incubated with different concentrations of antibodies (O-100 pg/2 ml reaction volume) at 21°C for 4 h, and was assayed for enzymatic activity. As seen in Fig. 2, antibodies R11, R-9, and R-4 inhibited activity maximally (90-92s). Antibodies R-l and R-7 did not inhibit enzyme activity. Inhibitory antibodies caused a concentration-dependent inhibition. The concentrations of antibody required to inhibit 50% activity are given in Table I. Due to their higher potency for enzyme inhibition and/or binding to pure enzyme (Fig. l), antibodies R-4, R-9, and R-11 were used for further inhibition studies. The specificity of inhibition was examined by characterizing the effects of these three antibodies on alcohol dehydrogenases from other sources. All failed to inhibit horse enzyme activity (data not shown). However, partially purified enzyme from outbred Sprague-Dawley rats was inhibited by each of them in a dosedependent manner (data not shown). The inhibition curves obtained were identical
MONOCLONAL
ANTIBODIES
AGAINST
to those seen for enzyme from inbred Fischer-344 rats. Time and pH dependence of enzyme inhibition by mono&ma1 antibodies. Partially purified rat liver alcohol dehydrogenase was incubated with varying amounts (0,20,50, and 100 pg) of antibody R-11 at 21°C for different times, and was assayed for enzymatic activity. As seen in Fig. 3, the inhibition by antibody R-9 was time-dependent at each concentration tested; 100 pg antibody caused the highest inhibition at all time points examined (27, 81, 95, 100, and 100% after 0, 30, 60, 150, and 270 min of incubation, respectively). Since both 50 and 100 pg antibody inhibited enzyme maximally after 150 min, these conditions were used routinely in later experiments. Figure 4 shows the effect of pH on inhibition of enzyme activity by antibodies R-4, R-9, and R-11. Partially purified enzyme was incubated with or without antibodies in 0.5 M Tris-HCl buffer of different pH values for 2.5 h at 21”C, and was assayed for activity in the same buffers. The percentage inhibition values were calculated from control incubation samples at each pH value set equal to 100% activity (0% inhibition). All three anti-
loo t
5 F
so-
560z l-40z P :ZO0
d I 0
1 I2
I TIME
I 3
I 4
I 5
(HOURS)
FIG. 3. Effect of time on enzyme inhibition by monoclonal antibody R-11. Partially purified rat liver alcohol dehydrogenase (60 fig proteins) was incubated with 20 (0), 50 (A), or 106 pg (Cl) antibody R-11 at 21’C (pH 7.2) for varying times (O-270 min), and was assayed for enzymatic activity as described under Experimental Procedures. Control activity (0% inhibition) was 0.02 CM NADH formed min-’ assay mix-‘.
ALCOHOL
DEHYDROGENASE
593
FIG. 4. Effect of pH on enzyme inhibition by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 pg proteins) was incubated with water (0), or 100 ng antibody R-4 (A), 70 pg antibody R-9 (w), or 90 wg antibody R-11 (V) in 0.5 M Tris-HCI buffer of varying pH values for 2.5 h at 21°C, and was assayed in the same buffers as described under Experimental Procedures. Activity in the absence of monoclonal antibodies (0) was set equal to 100% (0% inhibition) at each pH value.
bodies inhibited activity at pH ~7.8. The inhibitory effects obtained were significantly enhanced at pH ~7.0, conditions in which enzyme activity was roughly halfmaximal. By contrast, under conditions of maximal enzymatic activity, pH values >7.8, all three antibodies caused less than 15% inhibition. Similar results also were seen with pure enzyme (data not shown). Eflect of enzyme substrates on enzyme inhibition by mcmoclcmal antibodies. Partially purified enzyme was incubated with water, or 50 pg R-9 or R-11 for 2.5 h at 21°C and then assayed for enzymatic activity at varying ethanol concentrations (0.1-50.0 mM). The percentage inhibition at different ethanol concentrations was calculated from control incubation samples set equal to 100% activity. Figure 5 shows that both antibodies caused 52% inhibition of activity at all ethanol concentrations. Table II shows the effects of antibodies R-9 and R-11 on enzyme activity when other alcohols were used as substrates. Both antibodies (50 pg) inhibited isopropanol oxidation -45%, comparable to that of ethanol (52-55%; Table 11, Fig. 5). However, the same antibodies inhibited enzyme activity only 11-16 and 22% when butanol or propanol were used as substrates, respectively (Table II). These studies were
594
LAD,
SCHENK,
AND
YJo; , , , , , , ,1”03
07 I.0 30 [ETHANOL]
7oGo mM
300
Too
FIG. 5. Effect of ethanol on enzyme inhibition by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 pg proteins) was incubated at 21°C (pH 7.2) with water (0), 50 pg antibody R-9 (A), or 50 pg antibody R-11 (M) for 2.5 h, and was assayed for enzymatic activity at varying ethanol concentrations (0.1-50.0 ml@
performed at pH 7.2 (as they were for ethanol), so it is unclear as yet whether or not the pH-dependent inhibitory effects (Fig. 4) are substrate-specific. DISCUSSION
The present study describes the development and characterization of 11 stable hybridoma clones which synthesize and secrete monoclonal antibodies to rat liver alcohol dehydrogenase from inbred and outbred strains. Within this panel, three antibodies (R-4, R-9, and R-11) showed potent anticatalytic effects. Although mammalian alcohol dehydrogenase is a TABLE EFFECT
OF MONOCLONAL
ANTIBODIES
ON ALCOHOL
LEFFERT
highly conserved protein, rat liver alcohol dehydrogenase must differ sufficiently from the mouse enzyme to have permitted the mouse immune system to produce anticatalytic antibodies. This is further indicated by findings that these antibodies did not inhibit the horse enzyme. Thus, these antibodies are species- but not strain-specific. Although both enzymes show 80% primary sequence homology (26), peptide maps of the horse enzyme differ from those of the rat (27). Therefore, it is unclear whether the antigenic sites recognized by the current antibody panel are generated by nonhomologous sequences or, alternatively, as in the case of influenza virus (28), whether a variation of as little as one amino acid between enzymes is responsible for the properties of the monoclonal antibodies obtained. An invariant inhibition of the initial velocities of enzyme reactions by antibodies in Fig. 5 indicates a noncompetitive nature of the inhibition (29) for ethanol. When this was examined by plotting a Lineweaver-Burk double reciprocal plot, it was evident that antibodies decreased the V,,, without changing the Km for ethanol (not shown). Results from kinetic experiments (Fig. 5) and Table I indicate that 20-50 pg of antibodies R-9 and R-11 inhibited activity about 50%, suggesting that the Ki value for these noncompetitively inhibiting antibodies is about 2050 pg/2 ml assay mix (lo-20 nM for antibody R-11 [IgM] or 30-80 nM for antibody II
DEHYDROGENASE
Activities Substrate Ethanol
Butanol Propanol Isopropanol
Concentration (mM)
No antibody
ACTIVITY (FM NADH
+Antibody
USING
formed R-9
DIFFERENT
min-’
assay
SUBSTRATES
mix-‘)
+Antibody
R-11
1 5 10
0.0156 0.0205 0.0210
0.0074 0.0098 0.0101
(53) (52) (52)
0.0072 0.0092 0.0096
(54) (55) (54)
5
0.0222
0.0198
(11)
0.0187
(16)
15
0.0187
0.0145
(22)
0.0145
(22)
5
0.0201
0.0116
(42)
0.0110
(45)
Note Partially purified rat liver alcohol dehydrogenase (100 pg/200 ~1) was incubated with water or 50 #g R-9 or R-11 for 2.5 h, and was assayed for enzymatic activity at pH 7.2 in the presence of one of the following substrates: ethanol, butanol, propanol, and isopropanol. Numbers in parentheses indicate percentage inhibition by antibody.
MONOCLONAL
ANTIBODIES
AGAINST
R-9 [IgG]) at pH 7.2. This value may be overestimated because partially purified enzyme was used. In the case of NAD+, inhibitory effects of antibody R-9 were invariant with respect to NAD+ concentrations (0.1-10 mM) at a constant ethanol concentration (8.0 mM), again indicating a noncompetitive nature of the inhibition (data not shown). The reasons why the antibodies inhibited alcohol dehydrogenase (when ethanol was used as substrate) only under conditions of pH ~‘7.7-7.8 are unclear. Elevated pH levels (>7.7) did not seem to affect the antibodies directly, because the inhibitory effects of antibodies upon enzyme activity (assayed at pH 7.2) remained unaltered following antibody incubations in high pH buffers (data not shown). Previous kinetic studies of the pure rat liver enzyme have suggested that three characteristic ionization constants exist with pK’s 7.4-7.5, 8.0-8.1, and 9.1 (5). The enzyme * NAD+ complex has been assigned pK 7.4-7.5, determined by plotting the log of V,,, (at high substrate concentration) against pH (5). When results from Fig. 4 are replotted accordingly, the enzyme in the presence or absence of antibodies exhibits a molecular ionization constant of 7.5 and inhibition (evident by an altered slope) only at lowered pH values. This suggests that alcohol dehydrogenase is inhibited by the antibodies R-4, R-9, and R-11 when the enzyme * NAD+ complex is protonated. Further work is needed to determine the precise antibody binding sites on the enzyme in order to explain their anticatalytic effects. REFERENCES 1. VALEE, B. L., AND BAZZONE, T. J. (1983) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalios, J. G., and Whitt, G. S., eds.), Vol. 8, pp. 219244, Alan R. Liss, New York. 2. LI, T. K. (1977) A&J. Enzymol. 45, 427-483. 3. PIETRUSZKO, R. (1980) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalious, J. G., and Whitt, G. S., eds.), Vol. 4, pp. 107130, Alan R. Liss, New York. 4. CRABB, D. W., BOSRON, W. F., AND LI, T.-K. (1983) Arch. Biochem. Biophys. 224,299-309.
ALCOHOL
DEHYDROGENASE
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5. LAD, P. J., AND LEFFERT, H. L. (1983) And B&hem. 133,350-361. 6. THEORELL, H., AND YONETANI, T. (1969) Acta Chem Scud 23,255-260. 7. REYNIER, M. (1969) Acta Chem Scan& 23,11191129. 8. GOLDBERG, L., AND RYDBERG, II. (1969) Biochem. Pharmacol 18.1749-1762. 9. LIEBER, C. S., RUBIN, E., DECARLI, L. M., MISRA, P., AND GANG, H. (1970) Lab. Invest. 22, 615621. 10. PIETRUSZKO, R., AND RINGOLD, H. J. (1968) Biochem Biophys. Res. Commun 33,497-502. 11. PIETRUSZKO, R., AND RINGOLD, H. J. (1968) B&hem. Biophys. Res. Commun 33,503-507. 12. FULLER, T. C., AND MARUCCI, A. A. (1971) J. Zmmunol. 106, 110-119. 13. FULLER, T. C., ANDMARUCCI, A. A. (1972) Enzymologia 42, 139-152. 14. DUNCAN, R. J. S., KLINE, J. E., AND SOKOLOFF, L. (1976) B&hem. J. 153, 561-566. 15. CROW, K. E., CORNELL, N. W., AND VEECH, R. L. (197’7) Alcoholism Clin Exp. Res. 1,43-47. 16. LUMENG, L., BOSRON, W. F., AND LI, T. K. (1979) B&hem. Phurmnco~ 28,1547-1551. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem 193, 265-275. 18. LAD, P. J., AND LEFFERT, H. L. (1983) Ad Biochem. 133,362-373. 19. GALFRE, G., AND MILSTEIN, C. (1981) in Methods in Enzymology (Langone, J. L., and Van Vunakis, H., eds.), Vol. 73, pp. 3-46, Academic Press, New York. 20. GODING, J. W. (1980) J. Zmmunol. Methods 39, 285-308. 21. KENNETT, R. H., MCKEARN, T. J., AND BECHTOL, K. B. (eds.), (1980) Monoclonal Antibodies: Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York/London. 22. SCHENK, D. B., AND LEFFERT, H. L. (1983) Proc Natl. Acad. Sci USA 80, 5281-5285. 23. WHITE, A. A., NORTHUP, S. J., AND ZENSER, T. V. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp. 125-167, Dekker, New York. 24. MANCINI, G., CARBONARA, A. O., AND HEREMMANS, J. K. (1965) Zmmunochxmistry 2,235-243. 25. LAEMMLI, U. K. (19’70) Nature &m&m) 227,680685. 26. JORNVALL, H., AND MARKOVIC, 0. (1972) Eur. J. Biochem. 25,283-290. 27. LAD, P. J., HUBERT, J. J., SHOEMAKER, W. J., AND LEFFERT, H. L. (1983) Camp. Biochem. Physiol B 75,373-378. 28. LAVER, W. G., GERHARD, W., WEBSTER, R. G., FRANKEL, M. E., AND AIR, G. M. (1979) Proc. Natl. Acad Sci. USA 76,1425-1429. 29. SEGEL, I. H. (1975) Enzyme Kinetics, Wiley, New York.
Vol.
OF BIOCHEMISTRY
235, No. 2, December,
AND
BIOPHYSICS
pp. 589-595,
1984
Inhibitory Monoclonal Antibodies against Rat Liver Alcohol Dehydrogenase’ PUSHKARAJ Department
J. LAD,2 DALE of Medicine, Received
B. SCHENK,
AND
HYAM
L. LEFFERT3
Division of Pharmaco logy, M-013 H, University San Diego, La Jolla, California 9.2095 May
30, 1934, and in revised
form
August
of Cal&rnia,
1’7, 1984
Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-l and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-ll>R-9>R-4>R-6>R-lO>R-&R-2=R-3=R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH ‘7.7-7.8, a condition thought to protonate the enzyme’s active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase. 0 1984 Academic
Press, Inc.
Mammalian liver alcohol dehydrogenase (alcohol:NAD+ oxido-reductase, EC 1.1.1.1) is a zinc-containing dimeric protein which exhibits various isozymes (l-3). Studies of catalytic mechanisms usually have relied upon kinetic analyses with the horse enzyme, which is commercially available. By contrast, similar studies with rat alcohol dehydrogenase, the species most often used to analyze ethanol metabolism, have been limited due to the rat enzyme’s unstable nature. Although rapid purification procedures for the rat liver enzyme have recently facilitated kinetic analyses (4, 5), a controversy remains regarding the extent to which alcohol dehydrogenase contributes to liver alcohol metabolism.
Pharmacological agents have been employed to resolve this controversy. For example, pyrazole and its derivatives (e.g., 4-methylpyrazole) that inhibit alcohol dehydrogenase have been used to determine the contribution of alcohol dehydrogenase in total alcohol metabolism (6, 7). These agents have limited utility since, as competitive inhibitors, they are needed in high amounts to inhibit enzyme activity at high ethanol concentrations (8). Consequently, these agents nonspecifically inhibit catalase and microsomal ethanoloxidizing system activities as well (9). Therefore, potent specific inhibitors of rat alcohol dehydrogenase might help to distinguish its role from other enzymes involved in hepatic alcohol metabolism. One way to obtain such inhibitors is to produce specific neutralizing antibodies against alcohol dehydrogenase. Although two groups have developed polyclonal antibodies against the horse enzyme, these
’ This work was supported by USPHS Grants AM 28392, AA 03504, AM 28215 and GM 07752. a Present address: Genencor, Inc. 130 Kimball Way, South San Francisco, Calif. 94080. 3 To whom correspondence should be addressed. 589
0003-9861/&I Copyright All rights
$3.00
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590
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antisera cross-reacted poorly with the rat enzyme (10-13). Inhibitory polyclonal rabbit antiserum against the rat liver enzyme has also been obtained, but it inhibited only 60% of enzyme activity (14). In contrast to these earlier findings, we report here the development of specific monoclonal antibodies against rat liver alcohol dehydrogenase with potent and full neutralizing activity. Because these antibodies can be obtained in unlimited amounts and in pure form, this antibody panel should prove valuable for further physiological, enzymatic, biochemical, and purification studies of rat liver alcohol dehydrogenase. EXPERIMENTAL
PROCEDURES
Materials. Adult male Fischer-344 rats (200-250 g) and male Balb/c mice (lo-20g) were obtained from Charles River Breeding Laboratories (Wilmington, Mass.). Fetal bovine calf serum (Rehtumin FS) was obtained from Armour Pharmaceutical Company (Kankakee, Ill.). DEAE-Affi-Gel Blue, AfhGel Blue, and Coomassie brilliant blue R-250 were purchased from Bio-Rad (Richmond, Calif.). Enzymegrade ammonium sulfate was supplied by Schwa& Mann (Orangeburg, N. Y.). Acrylamide (enzyme grade), N,N,N:N’-tetramethylethylene diamine, and ammonium persulfate were from Eastman-Kodak (Rochester, N. Y.). SDS” was from BDH Chemicals, Ltd. (Poole, England). Goat anti-mouse (IgG + IgM) antibody-peroxidase conjugate was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, Ind.). Rabbit anti-mouse (IgG + IgM + IgA) antibodies were from Cappel Laboratories (Westchester, Pa.). Goat antibodies against mouse IgG,, IgG%, IgGab, and IgM were obtained from Meloy (Springfield, Va.). Polyethylene glycol (Avg M, 1500) and DMSO were from Aldrich Chemical Company (Milwaukee, Wise.) and Pierce Chemical Company (Rockford, Ill.), respectively. Porcine glucagon-free insulin was a gift from Dr. W. Bromer (Eli Lilly Research Labs, Indianapolis, Ind.). Sephadex G-100, cyanogen bromide-activated Sepharose-4B, AMPagarose, NADf (Grade III), and other routinely used chemicals were from Sigma Chemical Company (St. Louis, MO.). Plastic microtiter plates (96 wells) and 24-well culture plates were from Flow Laboratories (Inglewood, Calif.). ’ Abbreviations used: DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunoadsorbent assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
AND
LEFFERT
Purifkatim and assay of rat liver alcohol dehydm gcnase. Rat liver alcohol dehydrogenase was purified to homogeneity as described previously (5). The standard procedure included differential centrifugation, ammonium sulfate precipitation, and Sephadex G-100, DEAE-A&Gel Blue, Affi-Gel Blue, and AMP-agarose chromatography. The purity of the enzyme preparation was checked by SDS-polyacrylamide gel electrophoresis and isoelectrofocusing (5). In order to study enzyme inhibition, partially purified enzyme preparations were used. These were prepared routinely as follows: 20 g frozen (-20°C) liver tissue was thawed and homogenized in 106 ml 10 mM Tris-HCl buffer (pH 7.0). A 100,OOOg supernatant was obtained from the homogenate, and was applied to a 50-ml Affi-Gel Blue column. The column was washed with 150 ml 10 mM Tris-HCI (pH 8.0) and 300 ml 10 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCI. The activity was eluted with a solution of 10 mM Tris-HCl (pH 8.0), 0.15 M NaCl, and 1.0 mM NAD+. Alcohol dehydrogenase activity was measured at 37°C in a Beckman DU-8 spectrophotometer with a kinetics accessory unit, by monitoring the rate of NAD+ conversion to NADH at 340 nm. The standard reaction mixture (2 ml final volume) contained 0.5 M Tris-HCl (pH 7.2), 2.5 mM NAD+, and 8.0 mM ethanol (15,16). Proteins were determined according to Lowry et al. (17), with bovine serum albumin as the standard. Hybrids p-oductim Male Balb/c mice were immunized intramuscularly in the hind leg with lo15 pg of purified rat liver alcohol dehydrogenase in complete Freund’s adjuvant. Twenty five days later, booster injections of 7-10 pg pure enzyme in incomplete Freund’s adjuvant were made in the hind leg. One week later, antisera titers were determined with an enzyme-linked immunoadsorbent assay (ELLSA) (18). Mice which showed detectable antibodies (antisera dilution al:3600 in ELISA) were injected intraperitoneally and intravenously (tail vein) with 5 pg pure enzyme. Four days later the spleens were used for cell fusions. Cell fusions were performed by standard procedures (19-22). The parental mouse myeloma NS-l/ I’Ag 4.1 cell line was obtained from Dr. S. Sell (University of Texas, Houston, Tex.). Hybridoma colonies producing antibodies against rat liver alcohol dehydrogenase were detected by ELISA (18). These colonies were propagated and cloned in soft agar or by limited dilution techniques (21). Clones producing antibodies against rat liver alcohol dehydrogenase (detected by ELISA) were further propagated and stored in 95% (v/v) fetal bovine serum, 5% (v/v) DMSO at -70°C (21). Thawed clones continued to produce antibodies without detectable changes for more than 4 months in continuous culture.
MONOCLONAL
ANTIBODIES
AGAINST
Antibody purGfication and characterization Monoclonal antibodies were purified by immunoaffinity chromatography over rabbit anti-mouse (IgG + IgM + IgA) antibodies coupled to cyanogen bromideactivated Sepharose-4B (3 mg antibody/ml packed gel). Bound immunoglobulins were eluted with 0.1 M glycine (pH 2.8), neutralized with 1.0 M Tris base to pH 7.0, dialysed against distilled water or phosphatebuffered saline (20 mM sodium phosphate, pH 7.4, 0.15 M NaCl) for 72 h at 4°C and concentrated (by filtration at 4°C or by lyophilization). Protein concentrations were determined (17) after precipitation with silicotungstic acid (23). Characterization of monoclonal antibody immunoglobulin class/subclass was performed by Ouchterlony double immunodiffusion (24). About 100 gg goat IgG against specific mouse immunoglobulins (I&I, kGzw k’&ts, or IgM) from outer wells and 10 kg of monoclonal antibody from the central well were allowed to diffuse at 21°C until precipitin bands appeared. SDS-polyacryhmide gel electrophoresis. Discontinuous SDS-PAGE was performed under denaturing conditions (25). The stacking and running gels were 2 and 10% (w/v) acrylamide, respectively. Before each run, the samples (monoclonal antibodies, anti-
TABLE
ALCOHOL
591
DEHYDROGENASE
gen, or standards) were denatured at 100°C for 1.5 min in 50 mM Tris-HCI, pH 7.0, containing 2% (w/ v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.01% (w/ v) bromophenol blue, and were centrifuged in an Eppendorf microfuge for 5 min at 4°C. Electrophoresis was performed at 4°C for 3.5-4 h under a constant current of 13-14 mA. Proteins in the gels were stained with 0.03% (w/v) Coomassie brilliant blue R-250 in 10% (v/v) acetic acid and 25% (v/v) isopropanol. Gels were destained with a solution of 10% (v/v) acetic acid and 10% (v/v) isopropanol. RESULTS
Identi$cation and mono&ma1 antibodies cohol dehydrogenase.
characterization of against rat liver al-
Hybridomas that secrete monoclonal antibodies to rat liver alcohol dehydrogenase were obtained from two cell fusion experiments. Eleven stable clones were established (Table I). Monoclonal antibodies (R-1-R-11, Table I) secreted by these hybridomas were purified by immunoaffinity chromatography, and were compared on a weight basis, by I
MONOCLONALANTIBODIESAGAINSTRATLIVERALCOHOLDEHYDROGENASE
Antibody
iIf, (x10-3)
required
for
50% bd Monoclonal antibody R-l R-2 R-3 R-4 R-5 R-6 R-7 R-8 R-S R-10 R-11
Hybridoma clone 18C7 18GlO 18H8 18HlO 16E6 21F4 22D2 22FlO 24ElO 25Fl 26F7
Class/subclass of antibody IgM I@.% I@ I&, W I@ IN I&,” IgM IgGP I&P
Heavy chain 70 56 70 56 70 70 69 56 69 56 56
Light chain 30, 28 29 28 30 30 30 29 28 30 30 30
Binding”
Inhibitionb
0.71 2.15 2.70 0.36 0.63 0.84 4.70 3.50 0.11 1.40 0.64
>lOOd >lOOd >lOOd 35 >lOOd 98 >lOOd >lOOd 28 98 20
Note. Hybridoma clones secreting antibodies R-l to R-11 were obtained from fusion experiments. Immunoglobulin class/subclasses of antibodies were determined by Ouchterlony double diffusion. Molecular weights were determined by SDS-polyacrylamide gel electrophoresis as described under Experimental Procedures. a 50% binding represents A,% = 0.55 in Fig. 1. * 50% inhibition represents 50% inhibition of enzyme activity in Fig. 2. ‘Tentative, see Results. d Antibodies were not tested at concentrations higher than 100 pg.
592
LAD,
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AND
ELISA, for their capacity to bind to immobilized pure alcohol dehydrogenase (Fig. 1). Antibody R-9 showed the highest titer whereas antibodies R-7 and R-8 showed the lowest titers (Fig. 1). Relative binding titers, defined by the amount of antibody giving 50% of the maximum absorbance in the ELISA, are listed in Table I. Since the peroxidase-conjugated second antibody used was an undefined mixture of antibodies with potentially different avidities toward mouse IgG subclasses and IgM, absolute antibody affinities to alcohol dehydrogenase cannot be calculated from these studies. Affinity-purified antibodies were further characterized for their heavy- and lightchain molecular weights and immunoglobulin class/subclasses (Table I). Antibodies R-8, R-10, and R-11 did not form precipitin lines in double-diffusion tests with antisera against mouse IgGl, IgGz,, or IgM. However, these three Id&b, monoclonal antibodies contained heavy chains of molecular weight 54,000-57,000 (Table I). This suggests that they are either IgG, or IgA (more probably IgG3, because rabbit antimouse antibody used
122 IDE 0.8 r P 2 0.6. 0.4 0.2 -
II O.O 001
h
0.1
1.0
I loo
4
I 100
PG ANTIBODY /ICO~L
FIG. 1. Monoclonal antibody titration curves by ELISA. Microtiter plates were coated with a solution of 3 pg pure rat liver alcohol dehydrogenase/ml. After washing and blocking with a solution of 10 mg BSA/ml, the wells were incubated with varying amounts (0.01-40 @g/100 ~1) of antibodies [R-l (0), R-2 (A), R-3 (O), R-4 (V), R-5 (O), R-6 (A), R-7 (H), R-8 (W), R-9 (0), R-10 (+), and R-11 (X)]. Bound antibodies were detected with rabbit anti-mouse (IgG + IgM) IgG-peroxidase conjugate as described previously (18).
LEFFERT
i
FIG. 2. Inhibition of rat liver alcohol dehydrogenase activity by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 gg proteins) was incubated with varying amounts (O-100 pg) of antibodies [R-l (0), R-2 (A), R-3 (Cl), R-4 (V), R-5 (a), R-6 (A), R-7 (m), R-8 (v), R-9 (V), R-10 (+), and R-11 (X)] for 4 h at 21°C (pH 7.2), and was assayed for enzymatic activity. Control activity (100%) was 0.04 flM NADH formed min-’ assay mix-‘.
in screening by ELISA was against mouse
[hi& + IgMl).
Efict of rrumoc kmal antibodies on alcohol dehydrogenase activity. Partially purified
enzyme (100 pg proteins) was incubated with different concentrations of antibodies (O-100 pg/2 ml reaction volume) at 21°C for 4 h, and was assayed for enzymatic activity. As seen in Fig. 2, antibodies R11, R-9, and R-4 inhibited activity maximally (90-92s). Antibodies R-l and R-7 did not inhibit enzyme activity. Inhibitory antibodies caused a concentration-dependent inhibition. The concentrations of antibody required to inhibit 50% activity are given in Table I. Due to their higher potency for enzyme inhibition and/or binding to pure enzyme (Fig. l), antibodies R-4, R-9, and R-11 were used for further inhibition studies. The specificity of inhibition was examined by characterizing the effects of these three antibodies on alcohol dehydrogenases from other sources. All failed to inhibit horse enzyme activity (data not shown). However, partially purified enzyme from outbred Sprague-Dawley rats was inhibited by each of them in a dosedependent manner (data not shown). The inhibition curves obtained were identical
MONOCLONAL
ANTIBODIES
AGAINST
to those seen for enzyme from inbred Fischer-344 rats. Time and pH dependence of enzyme inhibition by mono&ma1 antibodies. Partially purified rat liver alcohol dehydrogenase was incubated with varying amounts (0,20,50, and 100 pg) of antibody R-11 at 21°C for different times, and was assayed for enzymatic activity. As seen in Fig. 3, the inhibition by antibody R-9 was time-dependent at each concentration tested; 100 pg antibody caused the highest inhibition at all time points examined (27, 81, 95, 100, and 100% after 0, 30, 60, 150, and 270 min of incubation, respectively). Since both 50 and 100 pg antibody inhibited enzyme maximally after 150 min, these conditions were used routinely in later experiments. Figure 4 shows the effect of pH on inhibition of enzyme activity by antibodies R-4, R-9, and R-11. Partially purified enzyme was incubated with or without antibodies in 0.5 M Tris-HCl buffer of different pH values for 2.5 h at 21”C, and was assayed for activity in the same buffers. The percentage inhibition values were calculated from control incubation samples at each pH value set equal to 100% activity (0% inhibition). All three anti-
loo t
5 F
so-
560z l-40z P :ZO0
d I 0
1 I2
I TIME
I 3
I 4
I 5
(HOURS)
FIG. 3. Effect of time on enzyme inhibition by monoclonal antibody R-11. Partially purified rat liver alcohol dehydrogenase (60 fig proteins) was incubated with 20 (0), 50 (A), or 106 pg (Cl) antibody R-11 at 21’C (pH 7.2) for varying times (O-270 min), and was assayed for enzymatic activity as described under Experimental Procedures. Control activity (0% inhibition) was 0.02 CM NADH formed min-’ assay mix-‘.
ALCOHOL
DEHYDROGENASE
593
FIG. 4. Effect of pH on enzyme inhibition by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 pg proteins) was incubated with water (0), or 100 ng antibody R-4 (A), 70 pg antibody R-9 (w), or 90 wg antibody R-11 (V) in 0.5 M Tris-HCI buffer of varying pH values for 2.5 h at 21°C, and was assayed in the same buffers as described under Experimental Procedures. Activity in the absence of monoclonal antibodies (0) was set equal to 100% (0% inhibition) at each pH value.
bodies inhibited activity at pH ~7.8. The inhibitory effects obtained were significantly enhanced at pH ~7.0, conditions in which enzyme activity was roughly halfmaximal. By contrast, under conditions of maximal enzymatic activity, pH values >7.8, all three antibodies caused less than 15% inhibition. Similar results also were seen with pure enzyme (data not shown). Eflect of enzyme substrates on enzyme inhibition by mcmoclcmal antibodies. Partially purified enzyme was incubated with water, or 50 pg R-9 or R-11 for 2.5 h at 21°C and then assayed for enzymatic activity at varying ethanol concentrations (0.1-50.0 mM). The percentage inhibition at different ethanol concentrations was calculated from control incubation samples set equal to 100% activity. Figure 5 shows that both antibodies caused 52% inhibition of activity at all ethanol concentrations. Table II shows the effects of antibodies R-9 and R-11 on enzyme activity when other alcohols were used as substrates. Both antibodies (50 pg) inhibited isopropanol oxidation -45%, comparable to that of ethanol (52-55%; Table 11, Fig. 5). However, the same antibodies inhibited enzyme activity only 11-16 and 22% when butanol or propanol were used as substrates, respectively (Table II). These studies were
594
LAD,
SCHENK,
AND
YJo; , , , , , , ,1”03
07 I.0 30 [ETHANOL]
7oGo mM
300
Too
FIG. 5. Effect of ethanol on enzyme inhibition by monoclonal antibodies. Partially purified rat liver alcohol dehydrogenase (100 pg proteins) was incubated at 21°C (pH 7.2) with water (0), 50 pg antibody R-9 (A), or 50 pg antibody R-11 (M) for 2.5 h, and was assayed for enzymatic activity at varying ethanol concentrations (0.1-50.0 ml@
performed at pH 7.2 (as they were for ethanol), so it is unclear as yet whether or not the pH-dependent inhibitory effects (Fig. 4) are substrate-specific. DISCUSSION
The present study describes the development and characterization of 11 stable hybridoma clones which synthesize and secrete monoclonal antibodies to rat liver alcohol dehydrogenase from inbred and outbred strains. Within this panel, three antibodies (R-4, R-9, and R-11) showed potent anticatalytic effects. Although mammalian alcohol dehydrogenase is a TABLE EFFECT
OF MONOCLONAL
ANTIBODIES
ON ALCOHOL
LEFFERT
highly conserved protein, rat liver alcohol dehydrogenase must differ sufficiently from the mouse enzyme to have permitted the mouse immune system to produce anticatalytic antibodies. This is further indicated by findings that these antibodies did not inhibit the horse enzyme. Thus, these antibodies are species- but not strain-specific. Although both enzymes show 80% primary sequence homology (26), peptide maps of the horse enzyme differ from those of the rat (27). Therefore, it is unclear whether the antigenic sites recognized by the current antibody panel are generated by nonhomologous sequences or, alternatively, as in the case of influenza virus (28), whether a variation of as little as one amino acid between enzymes is responsible for the properties of the monoclonal antibodies obtained. An invariant inhibition of the initial velocities of enzyme reactions by antibodies in Fig. 5 indicates a noncompetitive nature of the inhibition (29) for ethanol. When this was examined by plotting a Lineweaver-Burk double reciprocal plot, it was evident that antibodies decreased the V,,, without changing the Km for ethanol (not shown). Results from kinetic experiments (Fig. 5) and Table I indicate that 20-50 pg of antibodies R-9 and R-11 inhibited activity about 50%, suggesting that the Ki value for these noncompetitively inhibiting antibodies is about 2050 pg/2 ml assay mix (lo-20 nM for antibody R-11 [IgM] or 30-80 nM for antibody II
DEHYDROGENASE
Activities Substrate Ethanol
Butanol Propanol Isopropanol
Concentration (mM)
No antibody
ACTIVITY (FM NADH
+Antibody
USING
formed R-9
DIFFERENT
min-’
assay
SUBSTRATES
mix-‘)
+Antibody
R-11
1 5 10
0.0156 0.0205 0.0210
0.0074 0.0098 0.0101
(53) (52) (52)
0.0072 0.0092 0.0096
(54) (55) (54)
5
0.0222
0.0198
(11)
0.0187
(16)
15
0.0187
0.0145
(22)
0.0145
(22)
5
0.0201
0.0116
(42)
0.0110
(45)
Note Partially purified rat liver alcohol dehydrogenase (100 pg/200 ~1) was incubated with water or 50 #g R-9 or R-11 for 2.5 h, and was assayed for enzymatic activity at pH 7.2 in the presence of one of the following substrates: ethanol, butanol, propanol, and isopropanol. Numbers in parentheses indicate percentage inhibition by antibody.
MONOCLONAL
ANTIBODIES
AGAINST
R-9 [IgG]) at pH 7.2. This value may be overestimated because partially purified enzyme was used. In the case of NAD+, inhibitory effects of antibody R-9 were invariant with respect to NAD+ concentrations (0.1-10 mM) at a constant ethanol concentration (8.0 mM), again indicating a noncompetitive nature of the inhibition (data not shown). The reasons why the antibodies inhibited alcohol dehydrogenase (when ethanol was used as substrate) only under conditions of pH ~‘7.7-7.8 are unclear. Elevated pH levels (>7.7) did not seem to affect the antibodies directly, because the inhibitory effects of antibodies upon enzyme activity (assayed at pH 7.2) remained unaltered following antibody incubations in high pH buffers (data not shown). Previous kinetic studies of the pure rat liver enzyme have suggested that three characteristic ionization constants exist with pK’s 7.4-7.5, 8.0-8.1, and 9.1 (5). The enzyme * NAD+ complex has been assigned pK 7.4-7.5, determined by plotting the log of V,,, (at high substrate concentration) against pH (5). When results from Fig. 4 are replotted accordingly, the enzyme in the presence or absence of antibodies exhibits a molecular ionization constant of 7.5 and inhibition (evident by an altered slope) only at lowered pH values. This suggests that alcohol dehydrogenase is inhibited by the antibodies R-4, R-9, and R-11 when the enzyme * NAD+ complex is protonated. Further work is needed to determine the precise antibody binding sites on the enzyme in order to explain their anticatalytic effects. REFERENCES 1. VALEE, B. L., AND BAZZONE, T. J. (1983) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalios, J. G., and Whitt, G. S., eds.), Vol. 8, pp. 219244, Alan R. Liss, New York. 2. LI, T. K. (1977) A&J. Enzymol. 45, 427-483. 3. PIETRUSZKO, R. (1980) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalious, J. G., and Whitt, G. S., eds.), Vol. 4, pp. 107130, Alan R. Liss, New York. 4. CRABB, D. W., BOSRON, W. F., AND LI, T.-K. (1983) Arch. Biochem. Biophys. 224,299-309.
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DEHYDROGENASE
595
5. LAD, P. J., AND LEFFERT, H. L. (1983) And B&hem. 133,350-361. 6. THEORELL, H., AND YONETANI, T. (1969) Acta Chem Scud 23,255-260. 7. REYNIER, M. (1969) Acta Chem Scan& 23,11191129. 8. GOLDBERG, L., AND RYDBERG, II. (1969) Biochem. Pharmacol 18.1749-1762. 9. LIEBER, C. S., RUBIN, E., DECARLI, L. M., MISRA, P., AND GANG, H. (1970) Lab. Invest. 22, 615621. 10. PIETRUSZKO, R., AND RINGOLD, H. J. (1968) Biochem Biophys. Res. Commun 33,497-502. 11. PIETRUSZKO, R., AND RINGOLD, H. J. (1968) B&hem. Biophys. Res. Commun 33,503-507. 12. FULLER, T. C., AND MARUCCI, A. A. (1971) J. Zmmunol. 106, 110-119. 13. FULLER, T. C., ANDMARUCCI, A. A. (1972) Enzymologia 42, 139-152. 14. DUNCAN, R. J. S., KLINE, J. E., AND SOKOLOFF, L. (1976) B&hem. J. 153, 561-566. 15. CROW, K. E., CORNELL, N. W., AND VEECH, R. L. (197’7) Alcoholism Clin Exp. Res. 1,43-47. 16. LUMENG, L., BOSRON, W. F., AND LI, T. K. (1979) B&hem. Phurmnco~ 28,1547-1551. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem 193, 265-275. 18. LAD, P. J., AND LEFFERT, H. L. (1983) Ad Biochem. 133,362-373. 19. GALFRE, G., AND MILSTEIN, C. (1981) in Methods in Enzymology (Langone, J. L., and Van Vunakis, H., eds.), Vol. 73, pp. 3-46, Academic Press, New York. 20. GODING, J. W. (1980) J. Zmmunol. Methods 39, 285-308. 21. KENNETT, R. H., MCKEARN, T. J., AND BECHTOL, K. B. (eds.), (1980) Monoclonal Antibodies: Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York/London. 22. SCHENK, D. B., AND LEFFERT, H. L. (1983) Proc Natl. Acad. Sci USA 80, 5281-5285. 23. WHITE, A. A., NORTHUP, S. J., AND ZENSER, T. V. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp. 125-167, Dekker, New York. 24. MANCINI, G., CARBONARA, A. O., AND HEREMMANS, J. K. (1965) Zmmunochxmistry 2,235-243. 25. LAEMMLI, U. K. (19’70) Nature &m&m) 227,680685. 26. JORNVALL, H., AND MARKOVIC, 0. (1972) Eur. J. Biochem. 25,283-290. 27. LAD, P. J., HUBERT, J. J., SHOEMAKER, W. J., AND LEFFERT, H. L. (1983) Camp. Biochem. Physiol B 75,373-378. 28. LAVER, W. G., GERHARD, W., WEBSTER, R. G., FRANKEL, M. E., AND AIR, G. M. (1979) Proc. Natl. Acad Sci. USA 76,1425-1429. 29. SEGEL, I. H. (1975) Enzyme Kinetics, Wiley, New York.