Vol. August
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
HUMAN
COMMUNICATIONS
Pages
15, 1991
LIVER CLASS III ALCOHOL
FORMALDEHYDE
AND GLUTATHIONE
DEHYDRGGENASE
1371-1377
DEPENDENT
ARE THE SAME ENZYME
Barton Holmquist and Bert L. Vallee+ The Center for Biochemical and BiophysicaI Sciences and Medicine Harvard Medical School, 250 Longwood Ave., Boston, MA 02115
Received
July
8, 1991
Human liver class III alcohol dehydrogenase kx-ADH) and glutathione dependent formaldehyde dehydrogenase are the same enzyme. The enzyme, xx-ADH, exhibits a k, of 200 mid’ and a K, of 4 PM for the oxidation of formaldehyde, but only in the presence of GSH. In the absence of GSH the enzyme is essentially inactive toward formaldehyde but very active toward long chain alcohols. Thus, as in the rat (Koivusalo, M., Baumann, M., and Uotila, L. (1989) FEBS Letters 257, 105-109), the class III alcohol dehydrogenase and the GSH dependent formaldehyde dehydrogenase are identical enzymes. S-Hydroxymethyl derivatives of 8-thiooctanoate and lipoate are also very active substrates. The activity is specific for class III alcohol dehydrogenase; neither the class I and II nor the horse EE, ES, and SS isozymes oxidize hemithiolacetals. o-Phenanthroline competitively inhibits both activities and the two substrate types compete with each other. B 1991Academic Press, Inc.
Class III alcohol dehydrogenase (EC 1.1.1.1) was first detected in human placenta (1) and has been identified in liver and most other mammalian
tissues (2,3,4,5).
It is the only ADH detectable in
the human brain, placenta, and testis (6), and its sequence identity with class I ADH is >60% (7). The sequences of the rat and horse class III ADHs (8,9), in turn, are very similar to that of the human enzyme; out of a total of 373 residues only 21 and 19 residues, respectively, differ. The enzyme has been implicated
in ethanol (l), w-hydroxy fatty acid (lo), and leukotriene
Its activity toward ethanol is low due to an extremely high K,,, (> 3 M). ADHs, however, in oxidizing long chain primary alcohols (10,12). other ADHs by its insensitivity to 4-methylpyrazole,
(11) metabolism.
It is as effective as other
It is further distinguished from
a classical inhibitor
of ADHs (13), and by its
anodic migration on starch gel electrophoresis.
‘To whom correspondence should be addressed. Abbreviations: ADH, alcohol dehydrogenase; FDH, glutathione dependent formaldehyde dehydrogenase; GSH, glutathione; HM-GSH, S-hydroxymethyl glutathione; D’IT, dithiothreitol; DTNB, 5,5’dithiobis-(2-nitrobenzoic) acid; lZHDA, 12-hydroxydodecanoate acid. 0006-291X/91
1371
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The amino acid sequence of the X-ADH subunit is identical to that of FDH (14), based on the composition and sequence of rat liver FDH peptides. The rat liver FDH exhibits ADH activity toward primary alcohols characteristic of XX-ADH.
This activity, however, does not require GSH.
The GSH dependent enzyme catalyzed oxidation of formaldehyde proceeds through the spontaneously formed S-hydroxymethyl
glutathione and results in the oxidative acylation of GSH to form the thiol
ester, S-formylglutathione.
This GSH dependent activity is widely distributed in many animals, plants,
bacteria, and yeasts and thought to function as a scavenger of formaldehyde,
either formed
endogenously through several metabolic pathways or when taken up as a xenobiotic either directly or formed from exogenous precursors. We here show that the class III ADH from human liver isolated as XX-ADH is the same enzyme as human liver FDH, that this activity is not observed with class I or II ADHs, and that a number of additional thiols can replace GSH. MATERIALS
AND METHODS
Materials. Glutathione, 2-mercaptoethanol, lipoic acid, coenzyme A, glycolaldehyde, N-acetyl-L-cysteine, L-cysteine ethyl ester, DTNB, NAD+, and 2,3glyceraldehyde, dimercaptopropanesulfonic acid sodium salt were purchased from Sigma Chemical Co., St. Louis, MO. Formaldehyde (methanol free) was from Ladd Research Inc., Burlington, VT. The peptide VACENGLPVH was a gift from Dr. D. S. Auld, and captopril was a gift from Dr. M. Ondetti. Other reagents were the best grade commercially available. Human class I ADH @I,&, 01yi, yryi) isozymes (15) and class II ADH (16) were prepared by published procedures and the horse EE isozyme by an improved HPLC procedure (Futer, O., personal communication). 7-Thiooctanoic acid was prepared from 7-bromooctanoate by reaction with thiourea followed by hydrolysis to the free thiol acid (17) and glutathione ethyl ester was prepared as described (18). Methods. Enzyme activities were determined spectrophotometrically at 25°C on either a Cary 219 or a Gilford 2600 spectrophotometer. Standard assays for FDH activity contained 50 mM sodium phosphate, pH 8.0, 1.25 mM NAD+ (grade III), 1 mM formaldehyde, and 1 mM glutathione and were followed by monitoring the increase in absorbance due to NADH at 340 nm, E = 6220 M-km-‘. Measurements were usually started by addition of enzyme following incubation of the mixture for at least 1 min to allow hemithiolacetal formation. One unit (U) is defined as the amount of ADH required to produce 1 pmol of NADH/min at 25°C. Aldehyde reduction assays employed 0.16 mM NADH, 50 mM sodium phosphate, pH 7.5. Starch gel electrophoresis gels (10) were strained for activity using pentanol and GSH/formaldehyde (19) as substrates. Free sulphydryl groups were determined using DTN’B (18).
RESULTS Human liver class III ADH, purified to homogeneity by ion exchange HPLC (12), exhibits high activity in the oxidation of formaldehyde but only in the presence of glutathione.
With 1 mM GSH and
formaldehyde at pH 8.0, conditions previously established as near optimal for FDH activity, XX-ADH has a specific activity of 3 U/mg, comparable to that of 3.2 U/mg for the human liver glutathione dependent FDH (20). The K,,, is 4 PM, close to the value of 8 PM determined previously. Co-purijkation
of FDH and xx-ADH
activities.
A standard preparation of xx-ADH (12) served to
examine whether the enzyme retained the ethanol and FDH activities throughout the isolation process 1372
Vol.
178,
No.
3, 1991
BIOCHEMICAL
r 1.5
AND
A I -\\
BIOPHYSICAL
RESEARCH
B
i II It
.I.5
\
\
COMMUNICATIONS
0.6
\ \
ll F&TION
\
.I
\ \
0.1 .
\
20 TIME,
N”fBER
40 min
Figure 1. A. Chromatography of humanliver extract after elution from DEAE-cellulose on an AMP-Sepharoseaffinity columnshowingthe coelutionof the HM-GSH activity, U/mL, (-) measured underthe standardassayconditionsand the activity toward 0.5 M ethanol,pH 10, 0.1 M glycine, 2.4 mM NAD+ (*... ). The absorbance at 280 nm (- - -) is alsoshown. B. Ion exchange HPLC of the active fractionsof A on a WatersDEAE 5PW column(12) in 10 mM Tris, pH 8.0, 0.1 mM dithiothretol. Elution is with linearsaltgradientsto 0.02 M NaCl in 10 minthen to 0.06 M NaCl in 140min.
(Figure 1). The results obtained by ion exchangeHPLC chromatography of the pooled active fractions from the adenosinemonophosphateaffinity chromatography indicate that the two activities reside on the sameenzyme and are not due to contamination of one by another.
Specificity toward other thiols. In addition to GSH, numerousother thiols elicit high FDH activity some of which approach that of GSH (Table 1). No other aldehydes were found to substitute for formaldehyde.
Table 1. HemithiolacetalDehydrogenase Activity of xx-ADH: RelativeActivity of VariousThiolg andEthanolb % Activity
Substrate
100 70 50 25 32 12 10 4 23
GSH Cys-Gly 7-ThiooctanoicAcid Captopril Lipoic Acid Dithiothreitol Cysteine Decapeptide(VACENGLPVH) Ethanolb
Inactive: 2-mercaptoethanol, 3-mercaptopropionic acid, dodecane thiol, 2,3dimercaptopropanesulfonic acid, N-acetylcysteine,thioglycolate,thiolhistidine,thiolactate, thiomalate,D,L-homocysteine,coenzymeA, cysteineethyl ester, GSSG, acetaldehyde, glycolaldehyde,glyceraldehyde,propionaldehyde, isobutyraldehyde,glutaraldehyde, 3-phosphoglyceraldehyde, benzaldehyde,phenylacetaldehyde, glyoxylic acid. ‘50 mM Na phosphate buffer, pH 8.0, 1.2 mM NAD+, 1 mM formaldehyde,1 mM thiol. bO.1 M Glycine, pH 10, 2.5 mM NAD+, 0.5 M ethanol. 1373
Vol.
178,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100 80 -4
80 .E 40
i 220 :s
0
0
t
5
IWI.
I
I
0.1
0.2
10
,,,,,,I
0.5
15
mM I
1 [OPf
1
mM
5
,.,I
I
lo
2o
Figure Inhibition of the class III ADH catalyzed oxidation of the hemithiolacetal of glutathione ethyl ester (25, 0 and 50, A PM) by o-phenanthroliie at pH 8.0, 1 mM formaldehyde, 1.2 mM NAD+. Inset: Dixon plot showing competitive nature of the inhibition.
Substrate competirion.
The competition between HM-GSH and n-octanol and 12-HDA was examined
to assess whether or not these substrates undergo oxidation at the same active center of the enzyme. In a system where a single enzyme acts on two substrates leading to a common product that is detected, NADH in this case, the equation describing the system has been derived (21). Employing
constant
ratios of substrates, plots of l/v, vs l/[S] at various ratios of substrates provides a family of lines with a common point of intersection.
Using the equilibrium
constant for HM-GSH
formation, determined
to be 0.38 mM (20) under the conditions used, theoretical plots calculated for various substrate ratios were constructed and compared to actual rate data obtained. In the competition between HM-GSH and 12-HDA, the experimental points coincide with the rates predicted for a purely competitive interaction. Similar results were obtained in similar competition experiments performed between n-octanol and HMGSH and between n-octanol and 12-HDA. Inhibition
by o-phenanthroline
1 mM GSH/formaldehyde
(OP) and 4-pentylpyrazole.
Under the standard assay conditions of
(S/K,,, of 250), pH 8.0, OP up to 15 mM does not inhibit.
However,
inhibition is observed when glutathione ethyl ester, K, = 200 PM, is used at substrate concentrations considerably below the k,
25 and 50 PM.
(Figure 2) indicate a competitive
Dixon plots at two substrate and five OP concentrations
& of 3 mM.
Similarly,
4-pentylpyrazole
inhibits
oxidation
competitively with a & of 30 PM. Effect of fatty acids.
Fatty acids do not activate the rate of oxidation of HM-GSH.
With
formaldehyde, 1 mM, and for concentrations of GSH from 0.01 to 1 mM, propionate (1 to 50 mM), pentanoate (0.1 to 50 mM),
and octanoate (0.1 to 2 mM) had no effect on reaction velocity.
Pentanoate, 50 mM, a concentration that activates methylcrotyl alcohol oxidation 1Zfold (12) had no effect on the oxidation of 1 mM formaldehyde in the presence of 0.05 to 1 mM Cys-Gly nor 60 pM glutathione ethyl ester. 1374
Vol.
178, No. 3, 1991
BIOCHEMICAL
Activity with other ADHs. GSH and the hemithiolacetal
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The class III ADH from rabbit liver exhibits high activity toward HMof 8-thiooctanoate (Kunze, L., unpublished) with K, values of 4.3 and 5.7
PM, respectively, as does the class III ADH from E. coli, whose n-terminal sequence (47 residues) is over 60% identical to human liver class III ADH. &/3r, ‘y&,
However, the human liver class I isozymes (q,,
class II ADH (xx), and the horse EE and SS isozymes exhibit less than 0.1% of the activity
toward HM-GSH
when compared to their respective standard activity toward 33 mM ethanol, pH 10.
Neither the class I isozymes nor the horse EE isozyme are active toward 7-thioheptanoate or GSH ethyl ester in combination with either formaldehyde or propanal (1 mM).
Further, when examined with 1
mM formaldehyde, the following thiols, all 1 mM, failed to exhibit activity toward class I ADH (i.e.
300 mM, which greatly exceeds its
solubility limits.
Therefore, no inhibition would be expected for OP and none was found (20). When
as with glutathione ethyl ester, [S]
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
HUMAN
COMMUNICATIONS
Pages
15, 1991
LIVER CLASS III ALCOHOL
FORMALDEHYDE
AND GLUTATHIONE
DEHYDRGGENASE
1371-1377
DEPENDENT
ARE THE SAME ENZYME
Barton Holmquist and Bert L. Vallee+ The Center for Biochemical and BiophysicaI Sciences and Medicine Harvard Medical School, 250 Longwood Ave., Boston, MA 02115
Received
July
8, 1991
Human liver class III alcohol dehydrogenase kx-ADH) and glutathione dependent formaldehyde dehydrogenase are the same enzyme. The enzyme, xx-ADH, exhibits a k, of 200 mid’ and a K, of 4 PM for the oxidation of formaldehyde, but only in the presence of GSH. In the absence of GSH the enzyme is essentially inactive toward formaldehyde but very active toward long chain alcohols. Thus, as in the rat (Koivusalo, M., Baumann, M., and Uotila, L. (1989) FEBS Letters 257, 105-109), the class III alcohol dehydrogenase and the GSH dependent formaldehyde dehydrogenase are identical enzymes. S-Hydroxymethyl derivatives of 8-thiooctanoate and lipoate are also very active substrates. The activity is specific for class III alcohol dehydrogenase; neither the class I and II nor the horse EE, ES, and SS isozymes oxidize hemithiolacetals. o-Phenanthroline competitively inhibits both activities and the two substrate types compete with each other. B 1991Academic Press, Inc.
Class III alcohol dehydrogenase (EC 1.1.1.1) was first detected in human placenta (1) and has been identified in liver and most other mammalian
tissues (2,3,4,5).
It is the only ADH detectable in
the human brain, placenta, and testis (6), and its sequence identity with class I ADH is >60% (7). The sequences of the rat and horse class III ADHs (8,9), in turn, are very similar to that of the human enzyme; out of a total of 373 residues only 21 and 19 residues, respectively, differ. The enzyme has been implicated
in ethanol (l), w-hydroxy fatty acid (lo), and leukotriene
Its activity toward ethanol is low due to an extremely high K,,, (> 3 M). ADHs, however, in oxidizing long chain primary alcohols (10,12). other ADHs by its insensitivity to 4-methylpyrazole,
(11) metabolism.
It is as effective as other
It is further distinguished from
a classical inhibitor
of ADHs (13), and by its
anodic migration on starch gel electrophoresis.
‘To whom correspondence should be addressed. Abbreviations: ADH, alcohol dehydrogenase; FDH, glutathione dependent formaldehyde dehydrogenase; GSH, glutathione; HM-GSH, S-hydroxymethyl glutathione; D’IT, dithiothreitol; DTNB, 5,5’dithiobis-(2-nitrobenzoic) acid; lZHDA, 12-hydroxydodecanoate acid. 0006-291X/91
1371
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The amino acid sequence of the X-ADH subunit is identical to that of FDH (14), based on the composition and sequence of rat liver FDH peptides. The rat liver FDH exhibits ADH activity toward primary alcohols characteristic of XX-ADH.
This activity, however, does not require GSH.
The GSH dependent enzyme catalyzed oxidation of formaldehyde proceeds through the spontaneously formed S-hydroxymethyl
glutathione and results in the oxidative acylation of GSH to form the thiol
ester, S-formylglutathione.
This GSH dependent activity is widely distributed in many animals, plants,
bacteria, and yeasts and thought to function as a scavenger of formaldehyde,
either formed
endogenously through several metabolic pathways or when taken up as a xenobiotic either directly or formed from exogenous precursors. We here show that the class III ADH from human liver isolated as XX-ADH is the same enzyme as human liver FDH, that this activity is not observed with class I or II ADHs, and that a number of additional thiols can replace GSH. MATERIALS
AND METHODS
Materials. Glutathione, 2-mercaptoethanol, lipoic acid, coenzyme A, glycolaldehyde, N-acetyl-L-cysteine, L-cysteine ethyl ester, DTNB, NAD+, and 2,3glyceraldehyde, dimercaptopropanesulfonic acid sodium salt were purchased from Sigma Chemical Co., St. Louis, MO. Formaldehyde (methanol free) was from Ladd Research Inc., Burlington, VT. The peptide VACENGLPVH was a gift from Dr. D. S. Auld, and captopril was a gift from Dr. M. Ondetti. Other reagents were the best grade commercially available. Human class I ADH @I,&, 01yi, yryi) isozymes (15) and class II ADH (16) were prepared by published procedures and the horse EE isozyme by an improved HPLC procedure (Futer, O., personal communication). 7-Thiooctanoic acid was prepared from 7-bromooctanoate by reaction with thiourea followed by hydrolysis to the free thiol acid (17) and glutathione ethyl ester was prepared as described (18). Methods. Enzyme activities were determined spectrophotometrically at 25°C on either a Cary 219 or a Gilford 2600 spectrophotometer. Standard assays for FDH activity contained 50 mM sodium phosphate, pH 8.0, 1.25 mM NAD+ (grade III), 1 mM formaldehyde, and 1 mM glutathione and were followed by monitoring the increase in absorbance due to NADH at 340 nm, E = 6220 M-km-‘. Measurements were usually started by addition of enzyme following incubation of the mixture for at least 1 min to allow hemithiolacetal formation. One unit (U) is defined as the amount of ADH required to produce 1 pmol of NADH/min at 25°C. Aldehyde reduction assays employed 0.16 mM NADH, 50 mM sodium phosphate, pH 7.5. Starch gel electrophoresis gels (10) were strained for activity using pentanol and GSH/formaldehyde (19) as substrates. Free sulphydryl groups were determined using DTN’B (18).
RESULTS Human liver class III ADH, purified to homogeneity by ion exchange HPLC (12), exhibits high activity in the oxidation of formaldehyde but only in the presence of glutathione.
With 1 mM GSH and
formaldehyde at pH 8.0, conditions previously established as near optimal for FDH activity, XX-ADH has a specific activity of 3 U/mg, comparable to that of 3.2 U/mg for the human liver glutathione dependent FDH (20). The K,,, is 4 PM, close to the value of 8 PM determined previously. Co-purijkation
of FDH and xx-ADH
activities.
A standard preparation of xx-ADH (12) served to
examine whether the enzyme retained the ethanol and FDH activities throughout the isolation process 1372
Vol.
178,
No.
3, 1991
BIOCHEMICAL
r 1.5
AND
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BIOPHYSICAL
RESEARCH
B
i II It
.I.5
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COMMUNICATIONS
0.6
\ \
ll F&TION
\
.I
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0.1 .
\
20 TIME,
N”fBER
40 min
Figure 1. A. Chromatography of humanliver extract after elution from DEAE-cellulose on an AMP-Sepharoseaffinity columnshowingthe coelutionof the HM-GSH activity, U/mL, (-) measured underthe standardassayconditionsand the activity toward 0.5 M ethanol,pH 10, 0.1 M glycine, 2.4 mM NAD+ (*... ). The absorbance at 280 nm (- - -) is alsoshown. B. Ion exchange HPLC of the active fractionsof A on a WatersDEAE 5PW column(12) in 10 mM Tris, pH 8.0, 0.1 mM dithiothretol. Elution is with linearsaltgradientsto 0.02 M NaCl in 10 minthen to 0.06 M NaCl in 140min.
(Figure 1). The results obtained by ion exchangeHPLC chromatography of the pooled active fractions from the adenosinemonophosphateaffinity chromatography indicate that the two activities reside on the sameenzyme and are not due to contamination of one by another.
Specificity toward other thiols. In addition to GSH, numerousother thiols elicit high FDH activity some of which approach that of GSH (Table 1). No other aldehydes were found to substitute for formaldehyde.
Table 1. HemithiolacetalDehydrogenase Activity of xx-ADH: RelativeActivity of VariousThiolg andEthanolb % Activity
Substrate
100 70 50 25 32 12 10 4 23
GSH Cys-Gly 7-ThiooctanoicAcid Captopril Lipoic Acid Dithiothreitol Cysteine Decapeptide(VACENGLPVH) Ethanolb
Inactive: 2-mercaptoethanol, 3-mercaptopropionic acid, dodecane thiol, 2,3dimercaptopropanesulfonic acid, N-acetylcysteine,thioglycolate,thiolhistidine,thiolactate, thiomalate,D,L-homocysteine,coenzymeA, cysteineethyl ester, GSSG, acetaldehyde, glycolaldehyde,glyceraldehyde,propionaldehyde, isobutyraldehyde,glutaraldehyde, 3-phosphoglyceraldehyde, benzaldehyde,phenylacetaldehyde, glyoxylic acid. ‘50 mM Na phosphate buffer, pH 8.0, 1.2 mM NAD+, 1 mM formaldehyde,1 mM thiol. bO.1 M Glycine, pH 10, 2.5 mM NAD+, 0.5 M ethanol. 1373
Vol.
178,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
100 80 -4
80 .E 40
i 220 :s
0
0
t
5
IWI.
I
I
0.1
0.2
10
,,,,,,I
0.5
15
mM I
1 [OPf
1
mM
5
,.,I
I
lo
2o
Figure Inhibition of the class III ADH catalyzed oxidation of the hemithiolacetal of glutathione ethyl ester (25, 0 and 50, A PM) by o-phenanthroliie at pH 8.0, 1 mM formaldehyde, 1.2 mM NAD+. Inset: Dixon plot showing competitive nature of the inhibition.
Substrate competirion.
The competition between HM-GSH and n-octanol and 12-HDA was examined
to assess whether or not these substrates undergo oxidation at the same active center of the enzyme. In a system where a single enzyme acts on two substrates leading to a common product that is detected, NADH in this case, the equation describing the system has been derived (21). Employing
constant
ratios of substrates, plots of l/v, vs l/[S] at various ratios of substrates provides a family of lines with a common point of intersection.
Using the equilibrium
constant for HM-GSH
formation, determined
to be 0.38 mM (20) under the conditions used, theoretical plots calculated for various substrate ratios were constructed and compared to actual rate data obtained. In the competition between HM-GSH and 12-HDA, the experimental points coincide with the rates predicted for a purely competitive interaction. Similar results were obtained in similar competition experiments performed between n-octanol and HMGSH and between n-octanol and 12-HDA. Inhibition
by o-phenanthroline
1 mM GSH/formaldehyde
(OP) and 4-pentylpyrazole.
Under the standard assay conditions of
(S/K,,, of 250), pH 8.0, OP up to 15 mM does not inhibit.
However,
inhibition is observed when glutathione ethyl ester, K, = 200 PM, is used at substrate concentrations considerably below the k,
25 and 50 PM.
(Figure 2) indicate a competitive
Dixon plots at two substrate and five OP concentrations
& of 3 mM.
Similarly,
4-pentylpyrazole
inhibits
oxidation
competitively with a & of 30 PM. Effect of fatty acids.
Fatty acids do not activate the rate of oxidation of HM-GSH.
With
formaldehyde, 1 mM, and for concentrations of GSH from 0.01 to 1 mM, propionate (1 to 50 mM), pentanoate (0.1 to 50 mM),
and octanoate (0.1 to 2 mM) had no effect on reaction velocity.
Pentanoate, 50 mM, a concentration that activates methylcrotyl alcohol oxidation 1Zfold (12) had no effect on the oxidation of 1 mM formaldehyde in the presence of 0.05 to 1 mM Cys-Gly nor 60 pM glutathione ethyl ester. 1374
Vol.
178, No. 3, 1991
BIOCHEMICAL
Activity with other ADHs. GSH and the hemithiolacetal
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The class III ADH from rabbit liver exhibits high activity toward HMof 8-thiooctanoate (Kunze, L., unpublished) with K, values of 4.3 and 5.7
PM, respectively, as does the class III ADH from E. coli, whose n-terminal sequence (47 residues) is over 60% identical to human liver class III ADH. &/3r, ‘y&,
However, the human liver class I isozymes (q,,
class II ADH (xx), and the horse EE and SS isozymes exhibit less than 0.1% of the activity
toward HM-GSH
when compared to their respective standard activity toward 33 mM ethanol, pH 10.
Neither the class I isozymes nor the horse EE isozyme are active toward 7-thioheptanoate or GSH ethyl ester in combination with either formaldehyde or propanal (1 mM).
Further, when examined with 1
mM formaldehyde, the following thiols, all 1 mM, failed to exhibit activity toward class I ADH (i.e.
300 mM, which greatly exceeds its
solubility limits.
Therefore, no inhibition would be expected for OP and none was found (20). When
as with glutathione ethyl ester, [S]
