419
Biuchimica e~ Biophysica Acta, 1074 (1991)419-423 © 1~91 Elsevier Science Pub]isher~. B.V. 1}304.416.5/91/511J,50 ADONIS " ~ t"~ 165t~1002093
B ~ A G E N 23560
Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies Yu-Wai Ho, Wing-Ping Fong and Walter K.K. Ho Del~artment of Biochemi.~try, The Ch#tes¢ U'ml cr~iq' o]"ttong Kong. .~hatin, N.T. (Hong Ko~g)
(Rece~:ved7 Ma~:1~11 Keywords: Monoclonalantil~dy;Metalloenzyme:Dehydrogertase;(Human) The human class I alcohol dehydreganase isozyme ~218z was used a.~ the antigen to raise mouoclnnal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was ise~jme specific and all of them exhibited a similar affiniff against all isezymes oftbe human class 1 ADH. Five out @fthe seven moneclonal antibodies had no effect on PzP2 activity. Antibody G3 acted as a non-competitive inhibitor with a/~t of 3 p g / m l at pH 7.5. Increasing pH was effective in reducing the [evel of inhibition, On the other hand, antibody ID4 exhibited a pH-delgndent activation of ADH activity. In the prgsem~ of this antibody, the pH optimum of OzlB= was shifted from 9 to 8.$ and total activity was increased by 70% at this optimal pH, Kinetic analysis indicates that 11)4 probably acts as a nen-cempetiti've activator and may exert its action by interacting with the ¢oenzyme binding site,
lntreductim Human alcol-.ol dehydrogenases (ADH, alcohol: N A D * oxidoreductase. EC l J .1.1) are dimeric, metat-
Ioenzymes that catalyze the interconversion of alcohols with the corresponding aldehydes/ketones. They exist as a number of isozymes which can be differentiated into three classes according to a number of structural, phy~icochemical and catalytic properties. Such classification b consistent with and supported by immunological data. Each class of ADH is immunologically distinct and antibodies against it fail to interact interclass-wise; nevertheless, they do react with the same class of enzyme [!]. The varion~ isozymes of ADH have been purified and studied extensively with respect to their primary structure, physical properties, substrate specificity and kinetic parameters. ADH oxidizes and reduces a number o1 physiologically important metabolites; inspire of recent developments, the exact physiological role of the enzyme remains unknown. Monoclonal antibodies have been generated against ADH of different species including rat [21, horse [3] and Drosophila [4]. Polyelonal antibodies against different human isozymes have also been produced [5,6].
These antibodies can be used to measure ADH quantitatively and sensitively. The goal of the present study is ~o generate a panel of monoclonal antibodies against a human ADH isozyme, ~2P2- These monoclonal antibodies would be valuable for further physiological, enzymatic, biochemical and purification studies. Materials and Methods
Purification of ADH bozymes The human class I ADH isozyrnes a/3,, B,y., and ,8,B2 were purified from liver of homozygous ADH_, 2-2 phertotype by chromatography on DEAE-cellulose, CapGapp-Sepharose and CM-cettulose according to the procedure of long and Keung [7]. The final enz3'me preparation was freed of NAD(H) by Affi-Gel Blue affinity resin and then rechromatographed by HPLC in a SP-SPW cation exchange column (Waters Milford, MA). The homodimeri¢ isozlnne y~y~ was obtained from the betcrodimer P27~ by using a •freeze/thaw technique [81. The isoz'tmes were identified by starch gel [9] and polyacrylamide gel deetrophoresis in the presence of 7 M urea [10].
Enzyme and protein assay Conespondence:W.K.K.Ho. Departmentof Biochemistry.The Chinese Universityof HongKong,ShatinN.T+,HoneKone.
ADH activity was assayed spectrophotometrically by following the change .~n absorbance at 340 nm on a Varian 634 spectrophotometer ~hermostated at 25°C.
One unit of enzyme activity is defined as the amount of enzyme that catalyzes the appearance/disappearance of 1 tzmol of NADH/min at 25~C, using a molar absorptivity of 6220 M t cm-t for NADH. Protein concentration was determined colorimctrieally by the method of Lowry et al. [11] using bovine serum albumin as the standard.
tibodies were determined by discontinuous SDS-polyacrylamide gel electrophoresis [14] in a 12% gel
Results
Identification and characterization of monoclonal antibodies against human 13zlJ2 ADH
Produczion of monoclonal antibody 6- to 8-week-old Balb/c mice were immunized by injection with a purified preparation of human ADH //2//2 isozyme (200 /sg/animal) at 2 week intervals until a high titer serum was obtained. Animals were killed on the day of the fusion experiment and lymphoeytes from the spleens were prepared and fused with a myeloma cell line, NS-I, according to standard protocol using poly(etbylene glycol) [12]. Antibody activity of the hybridomas was determined by enzyme-linked immunosorbent assay (ELISA) using a preparation of unresolved class i ADH as antigen. Alter at least three cycles of limiting di]ution, the established hybridoma lines were either frozen for future use or injected into Balb/c mice to produce aseites. Monuclonal antibodies were isolated from the aseites fluid by 50% ammonium sulphate precipitation followed by affinity chromatography on a protein A-Sepharose column.
Characterization of monoelonal antibodies Characterization of monoclona! antibody immuneglobulin class/subclass was performed by Ouehterlony double immunodiffusion method [13}, using the mouse monoclonal typing kit from $erotec, U.K. Purity and molecular weight characteristics of the r~onoclonal an-
Altogether seven hybridoma lines secreting monoclonal antibodies against human [izflz ADH were isolated. The monoclonal antibodies purified by affinity chromatography were characterized with respect to their reactivity ao,'l immunoglobulin type (Table I). They were compared on a weight basis for their eapaeity to bind to immobilized ADH. Different human ADH isozymes, inch,ding ~2/~, Yl?t, ~2Yl and a / ~ as welt as horse liver ADH and yeast ADH were used for the binding study. The relative binding affinities for //2ff2 are listed in T~:ble i. Antibody FI2 showed the highest affinity while antibodies 1-19and (34 showed the lowest. The binding affinity of a particular monoelonal antibody toward the other human ADH isozymes and horse liver ADH were similar. In the case of yeast ADH, 11o appreciable binding could be detected even at high concentrations of the antibodies ( > 10/~g/ml). The effect of these antibodies on ADH activity was further examined by using the isozvm¢ p:dSz. As indicated in Table I, 5 out of the 7 monoelonal antibodies prepared did not have any significant effect on enzyme activity. Only anti~dy G3 showed a dramatic inhibition of ADH activity while antflxxly ID4 potentiated it by about 70%. There appears to be no correlation between the binding capacity of a monoclonal antibody and its effect on enzyme activity. Antibodies 03 and
TABLE [
Characterinicsof monoclonalantibodies raisedagainst human [92[Jz ADI-I Monoclonai antibody
Class/ subclass
MW('IQ ~) heavy chain
lisht chain
ll¢]aIiv© binding affinity '
Effcgt on A D H activity b (% control)
ID4 ]"19 (34 B8 H5 G3 Fi2
ISG1 IgG2b Ig(31 IgOI IgGI lag I lgEil
50 60 .50 50 50 50 48
25 29 25 2~ 25. 25 24
5- l(~2 2 1 ~. :.0~' 1"lOs 2' 104 5' 105
170 100 fin 100 100 20 94
a The relative binding affinity was estimated from the concentration of antibody required to give 50% of the maximum binding in an e ~ e
linked immunosorbantassay.HPLC purifiedADH isozyrnuswere used to coat micmtiterplates, h 5 p.g/n11of pSI.J2was incubatedwith 20/~g/ml an'~ibodiesin 0,] M NaPe4, 2,5 mM NAD~ (pH 7.5~'at roomtcmperalure [or I h and was the,. assayedfor activitywith 40 mM ethanol
421
/
lo
*~N
A
8
:t -
6
t~
4
300
Cone+ ol 83 [:Iglml] Fig. I. Inhibitinn of A D | I activity by the raonoclona] antibody G3.5 ~ g / m l of ADH isozyraes ~zfl2 (s). a[~2 t o ) or 7,Y, (&) was incuhaled with va~ing amounts of G3 ia ~tA M NaPe4 (pH 7.5) at room temperature for t h and then assaye, I for activity with 2.5 mM N A D ~ and 40raM ethanol.
;
2oo
1D4 were consequently selected for further characterization.
Eff¢ca of G3 on ADH actieity The inhibition of G3 ca 828: w~s time-dependent. It reached ~ maximum after the enzyme was incubated with (33 (10/.tg/ml) for 30 rain, Th0 inhibition w~s virtually tderRical in the presence ::rid absence of the coenzyme, HAD* (2.5 raM; data not sho~..n). The inhibition mediated by G3 was dose-dependent. The concentration of G3 required to inhibit 50% of ~2~2 (5 ~tg/ml) activity wes approx. 3 ~tg/ml at pH 7.5 (Fig. 1). The specificity of the inhibition was examined by charact0rizing the effect of 0 3 on other ADH isozyraes. (;3 inhibited the a,::ivities of both ~t/-/2 and 7t7,- The dose-response carves obtained were similar tO thai of g]z.62 (Fig. It.
The effect of G3 on enzyme activity was also dependent on pH. Inhibition was observed throughout the entire pH range (Fig. 2At, although the effect was less significant on the alkaline side. For example, the percent inhibition of activity decreased from 85 to 45% when the pit w~s increased from 7.5 to 10.5 (Fig. 2B).
-iJ 7
O
~
tO
I1
pH
Fig. 2. (At The pH-~etivity ptofiTes oF ~ 2 ~ in the a0senc¢ (s} and pr¢~nce of (33 (o) or tD4 (x). (B) "rh¢ rclativt: activits"of/32/92 in the presence of G3 t o ) or 11)4 (.t.) at diffet~nl pH value¢ B:fl~ (5 p g / m | ) was incubated with or without the antibody (20 #g/m]) in different buffers at ~ temperature for 30 min nod then d~aycd for activity with ~ mM NAD ÷ a~d 40 mM ethanol, The btlffers ased ~,~ere0.1 M NaPe 4 (pH 7.0-8.0) and O.i M g~cinn-NaOH (pH ~.5-1|.0).
TABLE Ii
S,~tmte ~'.~ficity of ~_,IL. ia tile presence ~ d absence of ID4 ~ Sui~trate
No antibody
Plus till.
V ~ (U/rag} K,. (raM) V.m (U/rag} Km (raM) Ethanol ~ 1-Butane| l-Hcxa~ol
!1.0 6..'i8 6.60
0.874 0.256 0.115
28.6 0.152
2-Butanol
2,03
Effect of ID4 on ADH activity
NAD + c
_
The ac'fivation of ID4 on ~2.~z activity occurred almost instanteously; maximal activation was observed within 2 mln of incubation and was independent of antibody concentration from 5 to 20 ~g/ml. The activation was the same either in the presence or absence of the coenz'yme NAD ÷ (2.5 nLM; data not shown). The effect of 1D4 on/~2~2 activity was pH dependent. In the absence of atUibo~.iy, the pH ~ptim~m for ~2~2 oxidation of ethat~ol (40 raM) was 9.~-. lncubaOs~n
Acetaldehyde d 68.2 NADH f -
0.0955 • 0.0520
t8.7 10.3 ]0.1
238 104
3.91 U.750 0.234
t45 0.320 0,110 0.104
J En:ryrfl¢acli~[y was determined in 0.l M NaPe 4, pH 7,5~ b Kinetic coastanls for the alcohols were determined at 2.~; mM NAD +. ¢ Km for NAD + was determined at 40 rnM ¢lhaool, a Kinetic eonstan~ for acetaldehyde were determined at 0.2 ram NADH. Co~ce,nhaiio~ of acetaldehyde was corrected lot t'lydra!ion [22]. r Km for NAD~'I was determined at 5 raM acetaldehyde.
422
,0
1.~
/-....
~
Di~us~on
--'~"-"""""
~~
N ~ 0.8
~
0.41
+°+ r, o,z ,IC
/
,.o
*
~,a
•
1,o
+
/
o.s o~ ?
0
P 41
10
i___ 11'
pH
Fig+ 3, T h e pH-aetivity profiles af (A) ~2+Yl, ( B ) ) q V l , and (C) horse liver A D H in the absence (4.) and presence ( A ) of 11)4. Conditions
were the sameas tho~edescribedia Fig.2.
with 1D4 shifted its pH optimum to 8.5 (Fig. 2A). The activation effect of ID4 was more significant at low pH. The activation was 2.78 times at pH 7.1), it de. creased progressively with increase in pH such that at pH 9.0-!0.0, 1D4 had negligible effect on the activity of the enzyme. At the higher pH of 10,5, inhibition of enzyme activity rather than activation was observed (Fig. 2B). The effect of 1D4 on other human ADH isoz'~es (//27,, 7W,) and horse liver ADH was studied with respect to the pH-activity profile. The effect was similar to that on /3x#2, namely ID4 caused (1) a shift of pH optimum toward the acidic fide by 0.25 to 0.50 units, (2) activation at low pH and (3) inhibRion at high pn (Fig. 3). 1D4 also affects g2Oz activity toward other substrates, includine, primary and secondary alcohols as well as acetaldehyde (Table II), In all cases Vm, was increased in the presence of 111)4. The magnitude of increase differed slightly among different substrates. It ranged from 1.17 ti,r.es for 2-butanol to 1.70 times for ethanol. The increase in Vm.:
Biuchimica e~ Biophysica Acta, 1074 (1991)419-423 © 1~91 Elsevier Science Pub]isher~. B.V. 1}304.416.5/91/511J,50 ADONIS " ~ t"~ 165t~1002093
B ~ A G E N 23560
Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies Yu-Wai Ho, Wing-Ping Fong and Walter K.K. Ho Del~artment of Biochemi.~try, The Ch#tes¢ U'ml cr~iq' o]"ttong Kong. .~hatin, N.T. (Hong Ko~g)
(Rece~:ved7 Ma~:1~11 Keywords: Monoclonalantil~dy;Metalloenzyme:Dehydrogertase;(Human) The human class I alcohol dehydreganase isozyme ~218z was used a.~ the antigen to raise mouoclnnal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was ise~jme specific and all of them exhibited a similar affiniff against all isezymes oftbe human class 1 ADH. Five out @fthe seven moneclonal antibodies had no effect on PzP2 activity. Antibody G3 acted as a non-competitive inhibitor with a/~t of 3 p g / m l at pH 7.5. Increasing pH was effective in reducing the [evel of inhibition, On the other hand, antibody ID4 exhibited a pH-delgndent activation of ADH activity. In the prgsem~ of this antibody, the pH optimum of OzlB= was shifted from 9 to 8.$ and total activity was increased by 70% at this optimal pH, Kinetic analysis indicates that 11)4 probably acts as a nen-cempetiti've activator and may exert its action by interacting with the ¢oenzyme binding site,
lntreductim Human alcol-.ol dehydrogenases (ADH, alcohol: N A D * oxidoreductase. EC l J .1.1) are dimeric, metat-
Ioenzymes that catalyze the interconversion of alcohols with the corresponding aldehydes/ketones. They exist as a number of isozymes which can be differentiated into three classes according to a number of structural, phy~icochemical and catalytic properties. Such classification b consistent with and supported by immunological data. Each class of ADH is immunologically distinct and antibodies against it fail to interact interclass-wise; nevertheless, they do react with the same class of enzyme [!]. The varion~ isozymes of ADH have been purified and studied extensively with respect to their primary structure, physical properties, substrate specificity and kinetic parameters. ADH oxidizes and reduces a number o1 physiologically important metabolites; inspire of recent developments, the exact physiological role of the enzyme remains unknown. Monoclonal antibodies have been generated against ADH of different species including rat [21, horse [3] and Drosophila [4]. Polyelonal antibodies against different human isozymes have also been produced [5,6].
These antibodies can be used to measure ADH quantitatively and sensitively. The goal of the present study is ~o generate a panel of monoclonal antibodies against a human ADH isozyme, ~2P2- These monoclonal antibodies would be valuable for further physiological, enzymatic, biochemical and purification studies. Materials and Methods
Purification of ADH bozymes The human class I ADH isozyrnes a/3,, B,y., and ,8,B2 were purified from liver of homozygous ADH_, 2-2 phertotype by chromatography on DEAE-cellulose, CapGapp-Sepharose and CM-cettulose according to the procedure of long and Keung [7]. The final enz3'me preparation was freed of NAD(H) by Affi-Gel Blue affinity resin and then rechromatographed by HPLC in a SP-SPW cation exchange column (Waters Milford, MA). The homodimeri¢ isozlnne y~y~ was obtained from the betcrodimer P27~ by using a •freeze/thaw technique [81. The isoz'tmes were identified by starch gel [9] and polyacrylamide gel deetrophoresis in the presence of 7 M urea [10].
Enzyme and protein assay Conespondence:W.K.K.Ho. Departmentof Biochemistry.The Chinese Universityof HongKong,ShatinN.T+,HoneKone.
ADH activity was assayed spectrophotometrically by following the change .~n absorbance at 340 nm on a Varian 634 spectrophotometer ~hermostated at 25°C.
One unit of enzyme activity is defined as the amount of enzyme that catalyzes the appearance/disappearance of 1 tzmol of NADH/min at 25~C, using a molar absorptivity of 6220 M t cm-t for NADH. Protein concentration was determined colorimctrieally by the method of Lowry et al. [11] using bovine serum albumin as the standard.
tibodies were determined by discontinuous SDS-polyacrylamide gel electrophoresis [14] in a 12% gel
Results
Identification and characterization of monoclonal antibodies against human 13zlJ2 ADH
Produczion of monoclonal antibody 6- to 8-week-old Balb/c mice were immunized by injection with a purified preparation of human ADH //2//2 isozyme (200 /sg/animal) at 2 week intervals until a high titer serum was obtained. Animals were killed on the day of the fusion experiment and lymphoeytes from the spleens were prepared and fused with a myeloma cell line, NS-I, according to standard protocol using poly(etbylene glycol) [12]. Antibody activity of the hybridomas was determined by enzyme-linked immunosorbent assay (ELISA) using a preparation of unresolved class i ADH as antigen. Alter at least three cycles of limiting di]ution, the established hybridoma lines were either frozen for future use or injected into Balb/c mice to produce aseites. Monuclonal antibodies were isolated from the aseites fluid by 50% ammonium sulphate precipitation followed by affinity chromatography on a protein A-Sepharose column.
Characterization of monoelonal antibodies Characterization of monoclona! antibody immuneglobulin class/subclass was performed by Ouehterlony double immunodiffusion method [13}, using the mouse monoclonal typing kit from $erotec, U.K. Purity and molecular weight characteristics of the r~onoclonal an-
Altogether seven hybridoma lines secreting monoclonal antibodies against human [izflz ADH were isolated. The monoclonal antibodies purified by affinity chromatography were characterized with respect to their reactivity ao,'l immunoglobulin type (Table I). They were compared on a weight basis for their eapaeity to bind to immobilized ADH. Different human ADH isozymes, inch,ding ~2/~, Yl?t, ~2Yl and a / ~ as welt as horse liver ADH and yeast ADH were used for the binding study. The relative binding affinities for //2ff2 are listed in T~:ble i. Antibody FI2 showed the highest affinity while antibodies 1-19and (34 showed the lowest. The binding affinity of a particular monoelonal antibody toward the other human ADH isozymes and horse liver ADH were similar. In the case of yeast ADH, 11o appreciable binding could be detected even at high concentrations of the antibodies ( > 10/~g/ml). The effect of these antibodies on ADH activity was further examined by using the isozvm¢ p:dSz. As indicated in Table I, 5 out of the 7 monoelonal antibodies prepared did not have any significant effect on enzyme activity. Only anti~dy G3 showed a dramatic inhibition of ADH activity while antflxxly ID4 potentiated it by about 70%. There appears to be no correlation between the binding capacity of a monoclonal antibody and its effect on enzyme activity. Antibodies 03 and
TABLE [
Characterinicsof monoclonalantibodies raisedagainst human [92[Jz ADI-I Monoclonai antibody
Class/ subclass
MW('IQ ~) heavy chain
lisht chain
ll¢]aIiv© binding affinity '
Effcgt on A D H activity b (% control)
ID4 ]"19 (34 B8 H5 G3 Fi2
ISG1 IgG2b Ig(31 IgOI IgGI lag I lgEil
50 60 .50 50 50 50 48
25 29 25 2~ 25. 25 24
5- l(~2 2 1 ~. :.0~' 1"lOs 2' 104 5' 105
170 100 fin 100 100 20 94
a The relative binding affinity was estimated from the concentration of antibody required to give 50% of the maximum binding in an e ~ e
linked immunosorbantassay.HPLC purifiedADH isozyrnuswere used to coat micmtiterplates, h 5 p.g/n11of pSI.J2was incubatedwith 20/~g/ml an'~ibodiesin 0,] M NaPe4, 2,5 mM NAD~ (pH 7.5~'at roomtcmperalure [or I h and was the,. assayedfor activitywith 40 mM ethanol
421
/
lo
*~N
A
8
:t -
6
t~
4
300
Cone+ ol 83 [:Iglml] Fig. I. Inhibitinn of A D | I activity by the raonoclona] antibody G3.5 ~ g / m l of ADH isozyraes ~zfl2 (s). a[~2 t o ) or 7,Y, (&) was incuhaled with va~ing amounts of G3 ia ~tA M NaPe4 (pH 7.5) at room temperature for t h and then assaye, I for activity with 2.5 mM N A D ~ and 40raM ethanol.
;
2oo
1D4 were consequently selected for further characterization.
Eff¢ca of G3 on ADH actieity The inhibition of G3 ca 828: w~s time-dependent. It reached ~ maximum after the enzyme was incubated with (33 (10/.tg/ml) for 30 rain, Th0 inhibition w~s virtually tderRical in the presence ::rid absence of the coenzyme, HAD* (2.5 raM; data not sho~..n). The inhibition mediated by G3 was dose-dependent. The concentration of G3 required to inhibit 50% of ~2~2 (5 ~tg/ml) activity wes approx. 3 ~tg/ml at pH 7.5 (Fig. 1). The specificity of the inhibition was examined by charact0rizing the effect of 0 3 on other ADH isozyraes. (;3 inhibited the a,::ivities of both ~t/-/2 and 7t7,- The dose-response carves obtained were similar tO thai of g]z.62 (Fig. It.
The effect of G3 on enzyme activity was also dependent on pH. Inhibition was observed throughout the entire pH range (Fig. 2At, although the effect was less significant on the alkaline side. For example, the percent inhibition of activity decreased from 85 to 45% when the pit w~s increased from 7.5 to 10.5 (Fig. 2B).
-iJ 7
O
~
tO
I1
pH
Fig. 2. (At The pH-~etivity ptofiTes oF ~ 2 ~ in the a0senc¢ (s} and pr¢~nce of (33 (o) or tD4 (x). (B) "rh¢ rclativt: activits"of/32/92 in the presence of G3 t o ) or 11)4 (.t.) at diffet~nl pH value¢ B:fl~ (5 p g / m | ) was incubated with or without the antibody (20 #g/m]) in different buffers at ~ temperature for 30 min nod then d~aycd for activity with ~ mM NAD ÷ a~d 40 mM ethanol, The btlffers ased ~,~ere0.1 M NaPe 4 (pH 7.0-8.0) and O.i M g~cinn-NaOH (pH ~.5-1|.0).
TABLE Ii
S,~tmte ~'.~ficity of ~_,IL. ia tile presence ~ d absence of ID4 ~ Sui~trate
No antibody
Plus till.
V ~ (U/rag} K,. (raM) V.m (U/rag} Km (raM) Ethanol ~ 1-Butane| l-Hcxa~ol
!1.0 6..'i8 6.60
0.874 0.256 0.115
28.6 0.152
2-Butanol
2,03
Effect of ID4 on ADH activity
NAD + c
_
The ac'fivation of ID4 on ~2.~z activity occurred almost instanteously; maximal activation was observed within 2 mln of incubation and was independent of antibody concentration from 5 to 20 ~g/ml. The activation was the same either in the presence or absence of the coenz'yme NAD ÷ (2.5 nLM; data not shown). The effect of 1D4 on/~2~2 activity was pH dependent. In the absence of atUibo~.iy, the pH ~ptim~m for ~2~2 oxidation of ethat~ol (40 raM) was 9.~-. lncubaOs~n
Acetaldehyde d 68.2 NADH f -
0.0955 • 0.0520
t8.7 10.3 ]0.1
238 104
3.91 U.750 0.234
t45 0.320 0,110 0.104
J En:ryrfl¢acli~[y was determined in 0.l M NaPe 4, pH 7,5~ b Kinetic coastanls for the alcohols were determined at 2.~; mM NAD +. ¢ Km for NAD + was determined at 40 rnM ¢lhaool, a Kinetic eonstan~ for acetaldehyde were determined at 0.2 ram NADH. Co~ce,nhaiio~ of acetaldehyde was corrected lot t'lydra!ion [22]. r Km for NAD~'I was determined at 5 raM acetaldehyde.
422
,0
1.~
/-....
~
Di~us~on
--'~"-"""""
~~
N ~ 0.8
~
0.41
+°+ r, o,z ,IC
/
,.o
*
~,a
•
1,o
+
/
o.s o~ ?
0
P 41
10
i___ 11'
pH
Fig+ 3, T h e pH-aetivity profiles af (A) ~2+Yl, ( B ) ) q V l , and (C) horse liver A D H in the absence (4.) and presence ( A ) of 11)4. Conditions
were the sameas tho~edescribedia Fig.2.
with 1D4 shifted its pH optimum to 8.5 (Fig. 2A). The activation effect of ID4 was more significant at low pH. The activation was 2.78 times at pH 7.1), it de. creased progressively with increase in pH such that at pH 9.0-!0.0, 1D4 had negligible effect on the activity of the enzyme. At the higher pH of 10,5, inhibition of enzyme activity rather than activation was observed (Fig. 2B). The effect of 1D4 on other human ADH isoz'~es (//27,, 7W,) and horse liver ADH was studied with respect to the pH-activity profile. The effect was similar to that on /3x#2, namely ID4 caused (1) a shift of pH optimum toward the acidic fide by 0.25 to 0.50 units, (2) activation at low pH and (3) inhibRion at high pn (Fig. 3). 1D4 also affects g2Oz activity toward other substrates, includine, primary and secondary alcohols as well as acetaldehyde (Table II), In all cases Vm, was increased in the presence of 111)4. The magnitude of increase differed slightly among different substrates. It ranged from 1.17 ti,r.es for 2-butanol to 1.70 times for ethanol. The increase in Vm.:
