Human Stomach Alcohol Dehydrogenase: Isoenzyme Composition and Catalytic Properties John D. Hempel, M.S. and Regina Pietruszko, Ph.D.
Two isoenzymes of alcohol dehydrogenase have been purified from human stomach and characterized with regard to electrophoretic mobility and kinetic properties with ethanol, hexanol, and acetaldehyde. Both undergo a time-dependent formation of multiple electrophoretic bands; the total amount of alcohol dehydrogenase activity in an average human stomach is only about 0.2% of that of the liver.
LCOHOL dehydrogenase from rat stomach A differs greatly from that of rat liver in electrophoretic mobility and affinity for ethanol. Its Michaelis constant for ethanol is about 700 times greater than that of rat liver alcohol dehydrogenase.' It seemed probable that if a similar alcohol dehydrogenase were present in significant amounts in the human stomach, it might be important in metabolism of beverage ethanoL2 For this reason, the content and nature of human stomach alcohol dehydrogenase have been investigated. Previous work by Smith, Hopkinson, and H a d d provided a genetic basis for heterogeneity and polymorphism of alcohol dehydrogenase from human tissues, yet only qualitative data on the activities of these isoenzymes were reported. In the stomach, according to these authors, genetic polymorphism exists at the ADH3 locus where either the ADH: or ADH: gene may exist, coding for a y' or y2 polypeptide subunit of the dimeric isoenzymes. A heterozygote will thus possess three electrophoretically separable isoenzymes which are seen to migrate toward the cathode on starch-gel electrophoresis. The isoenzymes of alcohol dehydrogenase in the human stomach are examined quantitatively in this paper. MATERIALS
AND
METHODS
Human stomachs were obtained at autopsy, usually 8-14 hr after death, and immediately placed in cold 150 mM Tris/HCI buffer, pH 8.5, during transport to the laboratory, where they were either frozen at -7OOC immediately (without buffer) or first scraped of the mucosa. which was then frozen until use. Mucosa from 3 or 4 stomachs was homogenized in 3 volumes of 20 mM TrisfHCI, pH 8.8. containing 0.1% ' (v/v)
2-mercaptoethanol in a Virtis homogenizer, followed by more complete homogenization in a motor-driven Teflon pestle homogenizer. Particulate material was sedimented at 22.000 g for I hr. The supernatant was subjected to (NH4),S04 fractionation between 40% and 70% saturation. The 70% precipitate was ruuspended in a minimum volume of homogenization buffer addifionally containing 20% (v/v) glycerol (column buffer). followed by dialysis against the same, until the presence of (NH4)$04 could no longer be detected in the dialysis medium. The dialysate was then loaded onto a 2.2 X 35 cm column of Whatman DE-I1 cellulose equilibrated in column buffer. Fractions containing enzyme activity were combined and concentrated by vacuum dialysis according to their isoenzyme content. which was determined electrophoretically. The partially purified isoenzymes were either used as such, in which case 2-mercaptoethanol was removed by dialysis prior to use, or were subjected to additional purification by affinity chromatography on columns containing 1 g (dry weight) 5' AMP Sepharose (Pharmacia Fine Chemicals), which was swelled in 50 mM phosphate buffer, pH 7.5, containing 1 mM glutathione. Elution of alcohol dehydrogenase was effected by coenzyme in a stepwise manner. After elution, coenzyme was removed by exhaustive dialysis against 20 mM Tris/HCI, pH 8.8. Total alcohol dehydrogenase at this stage was determined fluorometrically by titration with NADH in the presence of isobutyramide as follows: To a cuvette containing 0.1 M isobutyramide in 0.1 M phosphate. pH 7, and about 100 pg enzyme, 10-pl additions of standardized: ca. 100 pN NADH, were made. The increase in fluorescence at 410 nm from excitation at 330 nm of alcohol dehydrogenase . NADH . isobutyramide was measured until no further increase occurred. Additional small increases were due to fluorescenceof free NADH. The amount of complex formed, and thus the amount of enzyme present, was calculated from the end-point of the increase in fluorescence.' Protein at all stages was determined by the Lowry procedure using bovine serum albumin as primary standard? Alcohol dehydrogenase activity was determined at 25OC in 3-ml cuvettu of I-cm light path at 340 nm in a Varian 635 spectrophotometer. Assay mixtures for the oxidation of alcohol contained 500 pm NAD and 10.8 mM ethanol in 62 mM glycine/NaOH buffer, pH 10, unless otherwise specified. Assays for the reduction of aldehydes contained 170 pM From ihe Cenier of Alcohol Studies. Rutgers University. New Brunswick. N.J. Supporied by a National Council on Alcoholism Predoctoral Fellowship (J.D.H.)and by USPHS Grant AA00186. Received for publication August 7. 1978; accepied Sepiember 28, 1978. Reprini requesis should be addressed to Regina Pieirusrko. Ph.D., Center of Alcohol Studies. Ruigers Universiiy. New Brunswick. N.J. 08903. 01979 by Grune & Straiton. Inc. 01454008/79/0301400l$0l .OO/O
Alcoholism: ClinicalandExperimentalReseiuch, Vol. 3,No. 2 (April), 1979
95
HEMPEL AND PIETRUSZKO
96
Table 1. Purification Scheme of Human Stomech Alcohol Dehydrogenase Puiflcstion stagea
40%-70% lNH,)2S0, dialysete Peak 1, DE-11 Peak 2, DE-11 Peak 1, after S'AMP Sepharose Peak 2, after S'AMP Sepharose
Total
Total
specific
Activity (IU)
Protein (mpl
Activity (IU/rngl
70.3 16.4 26.3 9.3 4.2
1140 21 64.7 1.1 0.68
0.06 0.77 0.40 8.2 6.2
Recovery Percant
Puificaion Fdd
100
-
23 37 13 6
12.8 6.6 133 103
and m-nitrobenzaldehyde 11.3 pM) at pH Activity was measured in a 3-ml cuvette of l-cm light path at 340 nm using NADH (17 0 7.0 in 0.1 M phosphate buffer at 26°C. Starting material wan 260 g mucoaa combined from 4 stomachs.
NADH in 0.1 M phosphate bufler, pH 7.0, and 1.2 mM acetaldehyde or 1.3 mM m-nitrobenzaldehyde. For inhibition studies, NAD, pyrazole, and enzyme were preincubated for 5 min before ethanol was added to initiate the reaction. Electrophoresis was carried out in horizontal starch gels (Electrostarch, Otto Hiller & Co., Madison, Wisc.) as described previously.'o
RESULTS
Column chromatography of the (NH4&304 dialysate on DE-11 cellulose resolved two peaks of alcohol dehydrogenase activity that had distinct electrophoretic mobilities. The enzyme eluting first was named peak 1, and the latter, peak 2 alcohol dehydrogenase. Fractions containing each peak were pooled, giving purifications of 8.4- and 2.1-fold for peaks I and 2, respectively. Total recovery was approximately 60%. Chromatography of resolved materials on 5'AMP Sepharose affinity columns resulted in additional purification, to at least 100-fold (Table I). This resin retained peak 1 alcohol dehydrogenase against 4 mM NAD; elution occurring with 6 mM NADH, resulting in an enzyme approximately 20% pure by criteria of fluorometric titration versus protein (using 80,000 as molecular weight5). Peak 2 alcohol dehydrogenase was retained a t 0.2 mM NAD and was eluted by 5 mM NAD, giving a preparation approximately 50% pure. The data concerning purification are summarized in Table 1. Owing to the low recoveries achieved and a time-dependent decomposition (described. below), purification beyond this stage was not attempted. The total content of alcohol dehydrogenase per human stomach was estimated from the total ethanol activity at the (NH4),S04dialysate stage in one preparation of four stomachs and found to be 1.5 IU/stomach. In Fig. 1, the electrophoretic mobilities of the two components of stomach alcohol dehydrogenase are shown in channel A. Peak 1 alcohol
dehydrogenase from the stomach migrates as fast as the fastest migrating of the human liver isoenzymes (channel G), while peak 2 alcohol dehydrogenase migration resembles that of the EE isoenzyme from horse liver (channels E and H). Channels B and C demonstrate the resolution of these two components by DE-11 chromatography. It may be noted, however, that decomposition into multiple bands of activity occurs during this procedure. That this is not an artifact of chromatography, but rather a time-dependent phenomenon is demonstrated in channel D; this is the same material as in channel A (72 hr later), which was not subjected to chromatography. Inasmuch as this chromatography was done in the presence of 2-mercaptoethanol, this multiple band formation does not appear to be the result of formation of disulfide bridges. Further-
A
B
C D E
F G H
PEAK 2
PEAK I
CATHODE Fig. 1. Starch-gel electrophoresis of human stomach ammonium sulfate dialymte (A) ahowing electrophoretic mobilities of the peek 1 and 2 iwenzymes staining for alcohol dehydrogenase activity. After separation on DE-11 cellulose, both peak 2 (B) and peak 1 (C) have formed multiple bands. Electrophoresis of the dialymte (A) after the same time interval shows similar changes (D). This is seen in another dialysate (F) after the same interval. Samples ere run against the horse liver (€1 and (HI and human liver ( 0 ) alcohol dehydrogenase isoenzymea for comperison of mobilities. The samples in (F) and ( 0 )aro from the name individual. Arrows indicate origin.
HUMAN STOMACH ALCOHOL DEHYDROGENASE Table 2.
97
Michaelis Constants and k, Values for Human Stomach Alcohol Dehydrogenase Peak 1
Substrate
Ethanol Hexanol Acetaldehyde NAD
Range Used in Determination(mMI
0.68-2 1.6 1.35-108 0.017-0.53 0.036-2.36 0.73- 1 1.8 0.016-0.5
more, preincubation with an excess of mercaptoethanol ( 1 %, v/v) also failed to reverse the multiple banding. The Michaelis constants and maximal turnover numbers obtained for peak 1 and 2 alcohol dehydrogenase are shown in Table 2. The only identities to be seen between the two isoenzymes are the K,,,values for NAD. For either ethanol or acetaldehyde, the catalytic rate constants (k-,) with peak 1 alcohol dehydrogenase are greater than those of peak 2. Conversely, the K, values for these substrates are both lower with the peak 1 enzyme. The K, values differ greatly between ethanol and hexanol for peak 1 alcohol dehydrogenase, yet the turnover numbers are the same. Competitive inhibition of ethanol activity at pH 7 by pyrazole was found for peak 1 alcohol dehydrogenase, with a Ki of 9.1 pM. DISCUSSION
Human stomach alcohol dehydrogenase was observed to consist of two kinetically and electrophoretically distinct isoenzymes seen in all preparations of the enzymes as well as in two stomachs examined individually. Peak 2 alcohol dehydrogenase has never before been observed in human liver, and although observed in the stomach by Smith, Hopkinson and Harris: it was not investigated. This enzyme is even more anodic* than, and is kinetically distinct from, the *“Anodic,” or r alcohol dehydrogenase2*”~”was also observed in one liver specimen examined by Pietruszko, Theorell, and deZalenski,” who referred to it as isoenzyme 7, having a mobility like the ES” isoenzyme of horse liver. On this basis, T alcohol dehydrogenase is seen in channel G of Fig. 1 as the slowest migrating component. Clearly, peak 2 stomach alcohol dehydrogenase has a slower electrophoretic mobility than this isoenzyme. While no special importance should be attached to the alcohol dehydrogenase isoenzyme having the slowest electrophoretic mobility per se, it is important that peak 2 stomach alcohol dehydrogenase be distinguished from r alcohol dehydrogenase.
K,
(mMI
Peak 2 k,
(rnin-’1
2.2
200
0.07 0.5
200
Kin (mMl
7.7
0.066
kca (min-’1
25
4.500
-
150
-
11.1 0.054
-
“anodic” liver isoenzyme of Li and Magnes.”*’* Both peak 1 and 2 alcohol dehydrogenases have properties in common with isoenzymes from human liver,”-I6 although peak 1 alcohol dehydrogenase is generally more similar to the known liver isoenzymes. Both peak 1 and peak 2 alcohol dehydrogenase experienced a time-dependent formation of multiple electrophoretic bands arising from single bands, as seen in Fig. 1. This occurred in nine stomachs examined; the cause of this is unknown, with the exception that disulfidebridge-mediated changes may probably be ruled out. Significantly, the large kinetic differences between rat stomach and liver alcohol dehydrogenase are not found in the human. Since the alcohol dehydrogenase content of human stomach is only 1-2 IU, in comparison with ca. 1000 IU present in the liver,13 these isoenzymes are probably quantitatively unimportant in beverage ethanol metabolism. REFERENCES 1. Cederbaum AI, Pietruszko R. Hempel J, Becker F. Rubin E: Characterization of a non-hepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach. Arch Biochem Biophys 17 1 :348-360, 1975 2. Li T-K,Bosron WF. Dafeldecker WP, Lange LG, Vallee BL: Isolation of a r alcohol dehydrogenase of human liver: Is it a determinant of alcoholism? Proc Natl Acad Sci USA 7443784381,1977 3. Smith M, Hopkinson DA. Harris H: Developmental changes and polymorphism in human alcohol dehydrogenase. Ann Hum Genet 34:251-271, 1971 4. Smith M. Hopkinson DA, Harris H: Alcohol dehydrogenase isozymes in adult human stomach and liver: Evidence for activity of the ADH3 locus. Ann Hum Genet 35:243253. 1971 5 . Smith M. Hopkinson DA, Harris H: Studies on the subunit structure and molecular size of the human alcohol dehydrogenase isozymes determined by the different loci. ADHI. ADH2, and ADH3. Ann Hum Genet 36:401-414, 1973
98
6. Smith M, Hopkinson DA. Harris H: Studies on the properties of the human alcohol dehydrogenase isozymes determined by the diferent loci ADHI, ADH2. ADH3. Ann Hum Genet 37:49-67,1973 7. Ciotti MM. Kaplan NO: Procedures for determination of pyridine nucleotides. in Colowick SP, Kaplan NO (4s): Methods in Enzymology vol. 111. New York. Academic, 1957, p 890 8. Winer A. Theorell H: Dissociation constants of ternary complexes of fatty acids and amides with horse liver alcohol dehydrogenase-coenzyme complexes. Acta Chem Scand 14:1729-1742, 1960 9. Lowry OH, Rosebrough NJ. Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275. 1951 10. Pietruszko R, Theorell H: Subunit composition of horse liver alcohol dehydrogenase. Arch Biochem Biophys I31 :288-298, 1969 1 I. Li T-K. Magnes LJ: Identification of a distinctive molecular form of alcohol dehydrogenase in human livers
HEMPEL AND PlElRUSZKO
with high activity. Biochem Biophys Res Commun 63:202208,1975 12. Bosron WF, Li T-K, Lange LG, Dafeldtcker WP. Vallee BL: Isolation and characterization of an anodic form of human liver alcohol dehydrogenase. Biochem Biophys Res Commun 74:85-91,1977 13. Mourad N, Woronick C L Crystallization of human liver alcohol dehydrogenase. Arch Biochem Biophys 121:431-439, 1967 14. Pietruszko R, Theorell H, dealenski C: Heterogeneity of alcohol dehydrogenase from human liver. Arch Biochem Biophys 153:279-293. 1972 IS. Lange LG. Sytkowski AJ, Vallee BL: Human liver alcohol dehydrogenase: Purification, composition, and catalytic features. Biochemistry 1546874693, 1976 16. Pietruszko R. Crawford K. Lester D: Comparison of substrate specificity of alcohol dehydrogenases from human liver. horse liver, and yeast towards saturated and 2-enoic alcohols and aldehydes. Arch Biochem Biophys 159:50-60, 1973
Two isoenzymes of alcohol dehydrogenase have been purified from human stomach and characterized with regard to electrophoretic mobility and kinetic properties with ethanol, hexanol, and acetaldehyde. Both undergo a time-dependent formation of multiple electrophoretic bands; the total amount of alcohol dehydrogenase activity in an average human stomach is only about 0.2% of that of the liver.
LCOHOL dehydrogenase from rat stomach A differs greatly from that of rat liver in electrophoretic mobility and affinity for ethanol. Its Michaelis constant for ethanol is about 700 times greater than that of rat liver alcohol dehydrogenase.' It seemed probable that if a similar alcohol dehydrogenase were present in significant amounts in the human stomach, it might be important in metabolism of beverage ethanoL2 For this reason, the content and nature of human stomach alcohol dehydrogenase have been investigated. Previous work by Smith, Hopkinson, and H a d d provided a genetic basis for heterogeneity and polymorphism of alcohol dehydrogenase from human tissues, yet only qualitative data on the activities of these isoenzymes were reported. In the stomach, according to these authors, genetic polymorphism exists at the ADH3 locus where either the ADH: or ADH: gene may exist, coding for a y' or y2 polypeptide subunit of the dimeric isoenzymes. A heterozygote will thus possess three electrophoretically separable isoenzymes which are seen to migrate toward the cathode on starch-gel electrophoresis. The isoenzymes of alcohol dehydrogenase in the human stomach are examined quantitatively in this paper. MATERIALS
AND
METHODS
Human stomachs were obtained at autopsy, usually 8-14 hr after death, and immediately placed in cold 150 mM Tris/HCI buffer, pH 8.5, during transport to the laboratory, where they were either frozen at -7OOC immediately (without buffer) or first scraped of the mucosa. which was then frozen until use. Mucosa from 3 or 4 stomachs was homogenized in 3 volumes of 20 mM TrisfHCI, pH 8.8. containing 0.1% ' (v/v)
2-mercaptoethanol in a Virtis homogenizer, followed by more complete homogenization in a motor-driven Teflon pestle homogenizer. Particulate material was sedimented at 22.000 g for I hr. The supernatant was subjected to (NH4),S04 fractionation between 40% and 70% saturation. The 70% precipitate was ruuspended in a minimum volume of homogenization buffer addifionally containing 20% (v/v) glycerol (column buffer). followed by dialysis against the same, until the presence of (NH4)$04 could no longer be detected in the dialysis medium. The dialysate was then loaded onto a 2.2 X 35 cm column of Whatman DE-I1 cellulose equilibrated in column buffer. Fractions containing enzyme activity were combined and concentrated by vacuum dialysis according to their isoenzyme content. which was determined electrophoretically. The partially purified isoenzymes were either used as such, in which case 2-mercaptoethanol was removed by dialysis prior to use, or were subjected to additional purification by affinity chromatography on columns containing 1 g (dry weight) 5' AMP Sepharose (Pharmacia Fine Chemicals), which was swelled in 50 mM phosphate buffer, pH 7.5, containing 1 mM glutathione. Elution of alcohol dehydrogenase was effected by coenzyme in a stepwise manner. After elution, coenzyme was removed by exhaustive dialysis against 20 mM Tris/HCI, pH 8.8. Total alcohol dehydrogenase at this stage was determined fluorometrically by titration with NADH in the presence of isobutyramide as follows: To a cuvette containing 0.1 M isobutyramide in 0.1 M phosphate. pH 7, and about 100 pg enzyme, 10-pl additions of standardized: ca. 100 pN NADH, were made. The increase in fluorescence at 410 nm from excitation at 330 nm of alcohol dehydrogenase . NADH . isobutyramide was measured until no further increase occurred. Additional small increases were due to fluorescenceof free NADH. The amount of complex formed, and thus the amount of enzyme present, was calculated from the end-point of the increase in fluorescence.' Protein at all stages was determined by the Lowry procedure using bovine serum albumin as primary standard? Alcohol dehydrogenase activity was determined at 25OC in 3-ml cuvettu of I-cm light path at 340 nm in a Varian 635 spectrophotometer. Assay mixtures for the oxidation of alcohol contained 500 pm NAD and 10.8 mM ethanol in 62 mM glycine/NaOH buffer, pH 10, unless otherwise specified. Assays for the reduction of aldehydes contained 170 pM From ihe Cenier of Alcohol Studies. Rutgers University. New Brunswick. N.J. Supporied by a National Council on Alcoholism Predoctoral Fellowship (J.D.H.)and by USPHS Grant AA00186. Received for publication August 7. 1978; accepied Sepiember 28, 1978. Reprini requesis should be addressed to Regina Pieirusrko. Ph.D., Center of Alcohol Studies. Ruigers Universiiy. New Brunswick. N.J. 08903. 01979 by Grune & Straiton. Inc. 01454008/79/0301400l$0l .OO/O
Alcoholism: ClinicalandExperimentalReseiuch, Vol. 3,No. 2 (April), 1979
95
HEMPEL AND PIETRUSZKO
96
Table 1. Purification Scheme of Human Stomech Alcohol Dehydrogenase Puiflcstion stagea
40%-70% lNH,)2S0, dialysete Peak 1, DE-11 Peak 2, DE-11 Peak 1, after S'AMP Sepharose Peak 2, after S'AMP Sepharose
Total
Total
specific
Activity (IU)
Protein (mpl
Activity (IU/rngl
70.3 16.4 26.3 9.3 4.2
1140 21 64.7 1.1 0.68
0.06 0.77 0.40 8.2 6.2
Recovery Percant
Puificaion Fdd
100
-
23 37 13 6
12.8 6.6 133 103
and m-nitrobenzaldehyde 11.3 pM) at pH Activity was measured in a 3-ml cuvette of l-cm light path at 340 nm using NADH (17 0 7.0 in 0.1 M phosphate buffer at 26°C. Starting material wan 260 g mucoaa combined from 4 stomachs.
NADH in 0.1 M phosphate bufler, pH 7.0, and 1.2 mM acetaldehyde or 1.3 mM m-nitrobenzaldehyde. For inhibition studies, NAD, pyrazole, and enzyme were preincubated for 5 min before ethanol was added to initiate the reaction. Electrophoresis was carried out in horizontal starch gels (Electrostarch, Otto Hiller & Co., Madison, Wisc.) as described previously.'o
RESULTS
Column chromatography of the (NH4&304 dialysate on DE-11 cellulose resolved two peaks of alcohol dehydrogenase activity that had distinct electrophoretic mobilities. The enzyme eluting first was named peak 1, and the latter, peak 2 alcohol dehydrogenase. Fractions containing each peak were pooled, giving purifications of 8.4- and 2.1-fold for peaks I and 2, respectively. Total recovery was approximately 60%. Chromatography of resolved materials on 5'AMP Sepharose affinity columns resulted in additional purification, to at least 100-fold (Table I). This resin retained peak 1 alcohol dehydrogenase against 4 mM NAD; elution occurring with 6 mM NADH, resulting in an enzyme approximately 20% pure by criteria of fluorometric titration versus protein (using 80,000 as molecular weight5). Peak 2 alcohol dehydrogenase was retained a t 0.2 mM NAD and was eluted by 5 mM NAD, giving a preparation approximately 50% pure. The data concerning purification are summarized in Table 1. Owing to the low recoveries achieved and a time-dependent decomposition (described. below), purification beyond this stage was not attempted. The total content of alcohol dehydrogenase per human stomach was estimated from the total ethanol activity at the (NH4),S04dialysate stage in one preparation of four stomachs and found to be 1.5 IU/stomach. In Fig. 1, the electrophoretic mobilities of the two components of stomach alcohol dehydrogenase are shown in channel A. Peak 1 alcohol
dehydrogenase from the stomach migrates as fast as the fastest migrating of the human liver isoenzymes (channel G), while peak 2 alcohol dehydrogenase migration resembles that of the EE isoenzyme from horse liver (channels E and H). Channels B and C demonstrate the resolution of these two components by DE-11 chromatography. It may be noted, however, that decomposition into multiple bands of activity occurs during this procedure. That this is not an artifact of chromatography, but rather a time-dependent phenomenon is demonstrated in channel D; this is the same material as in channel A (72 hr later), which was not subjected to chromatography. Inasmuch as this chromatography was done in the presence of 2-mercaptoethanol, this multiple band formation does not appear to be the result of formation of disulfide bridges. Further-
A
B
C D E
F G H
PEAK 2
PEAK I
CATHODE Fig. 1. Starch-gel electrophoresis of human stomach ammonium sulfate dialymte (A) ahowing electrophoretic mobilities of the peek 1 and 2 iwenzymes staining for alcohol dehydrogenase activity. After separation on DE-11 cellulose, both peak 2 (B) and peak 1 (C) have formed multiple bands. Electrophoresis of the dialymte (A) after the same time interval shows similar changes (D). This is seen in another dialysate (F) after the same interval. Samples ere run against the horse liver (€1 and (HI and human liver ( 0 ) alcohol dehydrogenase isoenzymea for comperison of mobilities. The samples in (F) and ( 0 )aro from the name individual. Arrows indicate origin.
HUMAN STOMACH ALCOHOL DEHYDROGENASE Table 2.
97
Michaelis Constants and k, Values for Human Stomach Alcohol Dehydrogenase Peak 1
Substrate
Ethanol Hexanol Acetaldehyde NAD
Range Used in Determination(mMI
0.68-2 1.6 1.35-108 0.017-0.53 0.036-2.36 0.73- 1 1.8 0.016-0.5
more, preincubation with an excess of mercaptoethanol ( 1 %, v/v) also failed to reverse the multiple banding. The Michaelis constants and maximal turnover numbers obtained for peak 1 and 2 alcohol dehydrogenase are shown in Table 2. The only identities to be seen between the two isoenzymes are the K,,,values for NAD. For either ethanol or acetaldehyde, the catalytic rate constants (k-,) with peak 1 alcohol dehydrogenase are greater than those of peak 2. Conversely, the K, values for these substrates are both lower with the peak 1 enzyme. The K, values differ greatly between ethanol and hexanol for peak 1 alcohol dehydrogenase, yet the turnover numbers are the same. Competitive inhibition of ethanol activity at pH 7 by pyrazole was found for peak 1 alcohol dehydrogenase, with a Ki of 9.1 pM. DISCUSSION
Human stomach alcohol dehydrogenase was observed to consist of two kinetically and electrophoretically distinct isoenzymes seen in all preparations of the enzymes as well as in two stomachs examined individually. Peak 2 alcohol dehydrogenase has never before been observed in human liver, and although observed in the stomach by Smith, Hopkinson and Harris: it was not investigated. This enzyme is even more anodic* than, and is kinetically distinct from, the *“Anodic,” or r alcohol dehydrogenase2*”~”was also observed in one liver specimen examined by Pietruszko, Theorell, and deZalenski,” who referred to it as isoenzyme 7, having a mobility like the ES” isoenzyme of horse liver. On this basis, T alcohol dehydrogenase is seen in channel G of Fig. 1 as the slowest migrating component. Clearly, peak 2 stomach alcohol dehydrogenase has a slower electrophoretic mobility than this isoenzyme. While no special importance should be attached to the alcohol dehydrogenase isoenzyme having the slowest electrophoretic mobility per se, it is important that peak 2 stomach alcohol dehydrogenase be distinguished from r alcohol dehydrogenase.
K,
(mMI
Peak 2 k,
(rnin-’1
2.2
200
0.07 0.5
200
Kin (mMl
7.7
0.066
kca (min-’1
25
4.500
-
150
-
11.1 0.054
-
“anodic” liver isoenzyme of Li and Magnes.”*’* Both peak 1 and 2 alcohol dehydrogenases have properties in common with isoenzymes from human liver,”-I6 although peak 1 alcohol dehydrogenase is generally more similar to the known liver isoenzymes. Both peak 1 and peak 2 alcohol dehydrogenase experienced a time-dependent formation of multiple electrophoretic bands arising from single bands, as seen in Fig. 1. This occurred in nine stomachs examined; the cause of this is unknown, with the exception that disulfidebridge-mediated changes may probably be ruled out. Significantly, the large kinetic differences between rat stomach and liver alcohol dehydrogenase are not found in the human. Since the alcohol dehydrogenase content of human stomach is only 1-2 IU, in comparison with ca. 1000 IU present in the liver,13 these isoenzymes are probably quantitatively unimportant in beverage ethanol metabolism. REFERENCES 1. Cederbaum AI, Pietruszko R. Hempel J, Becker F. Rubin E: Characterization of a non-hepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach. Arch Biochem Biophys 17 1 :348-360, 1975 2. Li T-K,Bosron WF. Dafeldecker WP, Lange LG, Vallee BL: Isolation of a r alcohol dehydrogenase of human liver: Is it a determinant of alcoholism? Proc Natl Acad Sci USA 7443784381,1977 3. Smith M, Hopkinson DA. Harris H: Developmental changes and polymorphism in human alcohol dehydrogenase. Ann Hum Genet 34:251-271, 1971 4. Smith M. Hopkinson DA, Harris H: Alcohol dehydrogenase isozymes in adult human stomach and liver: Evidence for activity of the ADH3 locus. Ann Hum Genet 35:243253. 1971 5 . Smith M. Hopkinson DA, Harris H: Studies on the subunit structure and molecular size of the human alcohol dehydrogenase isozymes determined by the different loci. ADHI. ADH2, and ADH3. Ann Hum Genet 36:401-414, 1973
98
6. Smith M, Hopkinson DA. Harris H: Studies on the properties of the human alcohol dehydrogenase isozymes determined by the diferent loci ADHI, ADH2. ADH3. Ann Hum Genet 37:49-67,1973 7. Ciotti MM. Kaplan NO: Procedures for determination of pyridine nucleotides. in Colowick SP, Kaplan NO (4s): Methods in Enzymology vol. 111. New York. Academic, 1957, p 890 8. Winer A. Theorell H: Dissociation constants of ternary complexes of fatty acids and amides with horse liver alcohol dehydrogenase-coenzyme complexes. Acta Chem Scand 14:1729-1742, 1960 9. Lowry OH, Rosebrough NJ. Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275. 1951 10. Pietruszko R, Theorell H: Subunit composition of horse liver alcohol dehydrogenase. Arch Biochem Biophys I31 :288-298, 1969 1 I. Li T-K. Magnes LJ: Identification of a distinctive molecular form of alcohol dehydrogenase in human livers
HEMPEL AND PlElRUSZKO
with high activity. Biochem Biophys Res Commun 63:202208,1975 12. Bosron WF, Li T-K, Lange LG, Dafeldtcker WP. Vallee BL: Isolation and characterization of an anodic form of human liver alcohol dehydrogenase. Biochem Biophys Res Commun 74:85-91,1977 13. Mourad N, Woronick C L Crystallization of human liver alcohol dehydrogenase. Arch Biochem Biophys 121:431-439, 1967 14. Pietruszko R, Theorell H, dealenski C: Heterogeneity of alcohol dehydrogenase from human liver. Arch Biochem Biophys 153:279-293. 1972 IS. Lange LG. Sytkowski AJ, Vallee BL: Human liver alcohol dehydrogenase: Purification, composition, and catalytic features. Biochemistry 1546874693, 1976 16. Pietruszko R. Crawford K. Lester D: Comparison of substrate specificity of alcohol dehydrogenases from human liver. horse liver, and yeast towards saturated and 2-enoic alcohols and aldehydes. Arch Biochem Biophys 159:50-60, 1973
