Life Sciences Vol. 16, pp . 1563-1570 Printed in the D .S .A .
Pergamon Press
SUBCELLULAR DLSTRIBUTION AND CHARACTERIZATION OF HUMAN LIVER ALDEHYDE DEHYDROGENASE FRACTIONS Timo Koivula
Department of Medical Chemistry, University of Helsinki, SF-00170 Helsinki 17, Finland (Received in final form April 30, 1975) Summary
The eubcellular distribution of human liver aldehyde dehydrogenases (E .C . 1 .2 .1 .3) have been studied and the different types have been separated by ion exchange chromatography . The cytoplasmic fraction contained at least two chromatographically separable aldehyde dehydrogenases, which accounted for about 30°0 of the total activity . Oae of the cytoplasmic aldehyde dehydrogenases had a high Km for aldehydea (in the millimolar range) . A considerable part of the activity found is this fraction was due to an enzyme with a low Km for aldehydea (in the micromolar range) . It had properties similar to those of the mitochondrial main enzyme fraction, from where it may have originated as a contamination during eubcellular fractionation . Specific betaine aldehyde and formaldehyde dehydrogenases were separated from these unspeciûc activities in the cytoplaemic fraction . In mitochondria, where more than 50°l~0 of the total aldehyde dehydrogenaee activity was found, there was also evidence for alight high-Km activity . The microsomal fraction contained only a high-K m aldehyde dehydrogenaae, which accounted for about 10% of the total activity . In h ++~+a*, liver only one nonspecific NAD-dependent aldehyde dehydrogenase (EC 1 . 2 . 1 . 3) fraction has previously been partially purified and characterized by Kraemer & Dietrich (1) and by Blair & Bodley (2); the latter authors found indications of the presence of another minor aldehyde dehydrogeaase fraction . In rat liver, according to recent reports, there are several aldehyde dehydrogenases which catalyze NAD-dependent aldehyde oxidation (Refs. 3-5) . However, the subcellular distribution and properties of the different aldehyde dehydrogenase fractions have not been studied in human liver . Materiale and Methode NAD+, NADP+ and glucose-6-phosphate were purchased from Boehringer, pyrazole and 2,2-diethoxyethyl trimethylammonium iodide from Aldrich-Europe, and the aldehydea from Flukes AG . DEAE-cellulose was obtained from Whatmaa Biochemicale Ltd. Betaine aldehyde was synthesized from 2, 2-diethoocyethyl trimethylammonium iodide according to the principle of Bergel et al . (6) .
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Human Liver Aldehyde Dehydrogenases
Vol . 16, No . 10
A hiatologically normal biopsy specimen of human liver (weight 3 .0 g) was obtained at a stomach operation .. The specimen was immediately homogenized in 0 .25 M sucrose solution containing 10 mM sodium phosphate pH 7 .4 and 5 mM 2-mercaptoethanol . Subcellular fractionation was performed according to the principle of de Duve et al . (7) . Another fractionation was made, starting with 70 g of human liver obtained at autopsy about 1 .5 h after accidental death . The aubcellular fractions isolated from this latter specimen then served as material for partial purification by chromatography . Except for the normal premedication with phenobarbital one day before ~e operatioano drugs had been used by the persons from whom the liver samples were taken . Aldehyde dehydrogenase activity was measured at 25 °C speetrophotometrically at 340 nm or fluorometrically . The reaction mixture contained 1 .33 mM NAD, 1 .67 mM pyrazole, 0 . 070 M sodium pyrophosphate pH 8 . 0 and aldehyde . Formaldehyde dehydrogenase, glucose-b-phoaphatase, succinate-INT-reductase and glutamate dehydrogenase activities were measured as described previously (8-11) .Triton X-100 at 1 .0% concentration was added to the isolated aubcellular fractions before activity measurements in the distribution studies .
(ww)
For purification and characterization the mitochondrial and microsomal suspensions were treated with 1 .0% Triton X-100 and centrifuged for 60 min at 106000 g . Cytoplasmic and mitochondrial preparations were chromatographed in DEAF-cellulose columns equilibrated with 10 mM Tria-HCI, pH 7 . b, containing 5 mM 2-mercaptoethanol . The bound fractions were eluted with a linear salt gradient to 0 .15 M KCl concentration .
(ww)
Results The distribution of aldehyde dehydrogenase is the different aubcellular fractions of human liver was measured with propionaldehyde and acetaldehyde at two concentrations and also with glycolaldehyde as substrates (Table 1) . The distribution of the marker enzymes succinate-INT-reductase, glutamate dehydrogenase and glucose-6-phosphatase indicates that the activity found in the nuclear fraction is mostly of mitochondria) origin . The activity found in the lysosomal fraction was evidently caused by contamination with mitochondria sad microsomes . A considerable part of the activity was found in the soluble cell fraction, about 30% when measured with either the lower (60 WM) or the higher (9 and 18 mM respectively), propionaldehyde or acetaldehyde concentrations . The soluble fraction contained only 15% of the total glycolaldehyde dehydrogenase activity found . The mitochondria) matrix enzyme glutamate dehydrogenase showed moderate leakage into the cytoplasm . When allowance ie made for contamination in the nuclear and lysosomal fractions, mitochondria are found to contain at least 50% of the total activity measured at millimolar propionaldehyde or acetaldehyde concentrations, and more than 60% when measured at 60 WM concentration . Microsomal activity is about 10% of the total at higher and negligible at lower aldehyde concentrations . DEAE-cellulose chromatography of the cytoplasmic fraction yielded two peaks of aldehyde dehydrogenase activity with propionaldehyde (Fig . 1) . The first of these (cytoplaemic fraction I), the one moat loosely bound to the cellulose, was not active with glycolaldehyde . The second peak (cytoplaemic fraction II), where a small shoulder was found had activity also with glycolaldehyde . Betaine aldehyde dehydrogenase was bound most firmly to the DEAE-cellulose and was eluted separately from the other aldehyde dehydrogenase fractions . The same was true for formaldehyde dehydrogenase, which eluted between soluble fractions I and II .
Vol . 16, No . 10
1565
Human Liver Aldehyde Dehydrogenasea
TABLE 1 SUBCELLULAR DISTRIBUTION OF HUMAN LNER ALDEHYDE DEHYDROGENASE The enzyme assays are described in materials and methods . The distributions of activities are calculated as percentages of the summed activities of all fractions . The enzyme activities are expressed ae IU~g liver wet weight . Values are means of results of 2 fractionations . N = nuclear fraction, E = cytoplasmic extract, M = mitochondrial fraction, L = lysosomal fraction, P = microsomal fraction, S = final supernatant . Enzyme
Absolute values (N+E)
Aldehyde dehydrogenaae + propionaldehyde 9 mM 3 .86 60 WM 2 .38 18 mM + acetaldehyde 3 .63 3 .32 60 W M + glycolaldehyde 2 mM 3 .48 Glucose-6-phosphatase 5 .0 Succinate-INT-reductase 3 .1 Glutamate dehydrogenaae 183
Percentage values
Recovery
N+E
N
M
L
P
S
100 100 100 100 100 100 100 100
26 29 25 34 35 18 31 28
25 32 26 32 35 11 35 31
8 7 9 8 12 16 19 12
9 3 8 1 3 44 15 11
32 29 32 25 IS 7 3 18
97 77 97 73 82 95 103 97
Fig . 1 . DEAF-cellulose chromatography of human liver cytoplasmic fraction . The dialyzed sample was applied to the column (2 x 30 cm) which had been equilibrated with lOmM Tria-HC1, pH 7 .6, + 5 mM 2-mercaptoethanoL Elution was performed with a linear KC1 gradient up to 0 .2 M concentration. The fraction volume was 15 ml . Fractions were analyzed for formaldehyde dehydrogenaae (~) and aldehyde dehydro enase with propioaaldehyde ( " ), glycolaldehyde (d ) and betaine aldehyde ~O ) as substrates . Absorbance at 280 nm (- - -) . DEAE-cellulose chromatography of the mitochondrial fraction (Fig .2) gave two main peaks of activity (mitochondria) fractions I sad II) not very far from each other with both propionaldehyde sad glycolaldehyde . A minor activity was seen in the fractions eluted with the starting buffer sad these
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Human Liver Aldehyde Dehydrogenasea
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fractions also had some activity with NADP ae coenzyme . Substrate specificitiee were measured separately for soluble DEAF fractions I and II, for mitochondrial fraction I sad for the microsomal supernatant (Table 2) . Cytoplasmic fraction I seemed to be the most reall stricted in substrate specificity . It was not active with formaldehyde at and had low activity with glycolaldehyde sad with aromatic aldehydes . Cytoplasmic fraction II waa more active with formaldehyde and glycolaldehyde,I but was even lees active with aromatic aldehydes . Mitochoadrial fraction had a rather similar substrate specificity . Microsomal aldehyde dehydrogenase was active with aromatic aldehydes, but its activity with formaldehyde and glycolaldehyde was low .
Fig. 2 . DEAE-cellulose chromatography of human liver mitochondria) fraction . After treatment with detergent sad dialysis, the sample was applied to the column (1 .5 x 30 cm) which had been equilibrated with 10 mM Tris-HCl pH 7 . 6, + 5 mM 2-mercaptoethanol . Elution was performed with a linear KCl gradient up to 0 .2 M concentration . Fractions were analyzed for aldehyde dehydrogenase activity with propioaaldehyde ( " ) sad glycolaldehyde (Q ) ae substrates and for protein (- - -, absorbante at 280 nm) . All enzyme fractions had Km values for NAD between 2 ' 10 -5 and 2 " 10 -4 M (Table 3) . Mitochondria) and micro~oma,l fractions also had same activity with NADP, having a high Km (1 " 10 - M) for this coenzyme . The cytoplasmic fraction was not active with NADP . Cytoplaemic enzyme I had a very high Km value for propioaaldehyde and acetaldehyde . For the microsomal fraction the corresponding values were somewhat lower but still in the millimolar range . Cytoplasmic enzyme II and mitochondria) enzyme I had similar very low Km values for propioaaldehyde and acetaldehyde (below 10-6 M) . Discussion Evidence for the hetero eneity of human liver aldehyde dehydrogenase has been re orted earlier ~2, 12) . Neither Kraemer & Deitrich (1) nor Blair & Bodley ~2) identified the subcellular fraction from which the partial ly purified enzyme was derived. The present study was undertaken to inreatigate the aubcellular distribution of h ++*+,a*, liver aldehyde dehydrogenaee sad to characterize the main types of aldehyde dehydrogeaases by partial purification . Previous studies with rat liver have shown a considerable aldehyde dehydrogenase activity with a high Km for propioaaldehyde and acetaldeto the pres hyde in the mitochondria) and microsomal fractions . According . ent results, high Km activities are less pronounced in h +++++ an liver . The
Vol . 16, No . 10
Human Liver Aldehyde Dehydrogenases
1567
TABLE 2 SUBSTRATE SPECIFICTTY OF HUMAN LNER ALDEHYDE DEHYDROGENASE FRACTIONS The autopsy liver sample was used for isolation of the fractions . Maximal velocities were determined from Lineweaver-Burk plots . The reaults are expressed as relative activities, the activity with propionaldehyde being taken as 1 .0, and used as the standard for each fraction . ALDEH~DE
SOLUBLE
Fraction I
Formaldehyde Acetaldehyde
SOLUBLE
Fraction II
0 0 .4
Propionaldehyde
0 .4 0 .9
1 .0 0 .1
0 .2
Glycolaldehyde
Fraction I
0 .4 0 .8
1 .0 0 .1
Benzaldehyde Anisaldehyde
MITOCHONDRIAL
Fraction 0 .1
0 .8 1 .0
1 .0 0 .3
0 .2 0 .4
0 .2
MICROSOMAL
0.8 0.6
0 .2 0 .5
0 .1
TABLE 3 APPARENT MICHAELLS CONSTANTS OF HUMAN LNER ALDEHYDE DEHYDROGENASE FRACTIONS
The autopsy liver sample was used for isolation of the fractions . In the determinations with variable aldehyde concentration NAD+ waa kept at 1 .33 mM and in those with variable NAD(P)+ concentration the propionaldehyde concentration was 9 mM . I = cytoplasmic enzyme I, II = cytoplaemic enzyme II, III = betaine aldehyde dehydrogenase, N = mitochondrial enzyme I, V = microsomal eazyme . Substrate
Km (WM) I
II
Propionaldehyde 8000 10000 Acetaldehyde Betaiae aldehyde 20 NAD+ NADP+
-
III
0 .5 0 .5 100
-
-
10 80
(with 1 mM betaine aldehyde)
N
V
0 .4 0 .4 200
-
600
3000 -
70 1000
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Htmian Liver Aldehyde Dehydrogenasea
Vol. 16, No . 10
subcellular distribution of aldehyde dehydrogenase activity in the human liver is not unlike that reported for sheep liver by Crow et al . (4) . In comparison with rat liver, microsomal activity is low . The cytoplasmic fraction accounts for about 25-35% of the total activity when measured with propionaldehyde (60 WM and 9 mM) or acetaldehyde (60 ~M and 18 mM) as substrates .
The aldehyde dehydrogenase activity of the human liver cytoplasmic fraction could be fractionated into two separate peaks by DEAE-cellulose chromatography . Formaldehyde dehydrogenase and betaine aldehyde dehyd rogenase separated differently from these nonspecific activities . Cytoplasmic fraction I has very high Km values for propionaldehyde and acetaldehyde . Even at high aldehyde substrate concentrations, however, its contribution to the total aldehyde dehydrogenase activity in human liver is very small . Cytoplasmic fraction II accounts for moat of the cytoplasmic aldehyde dehydrogenase activity . Fraction II has Km values for propionaldehyde and acetaldehyde below 1 WM, as waa also found by Kraemer & Deitrich (1) sad Blair & Bodley (2) with their partially purified enzymes . A similarity also exists in substrate specificity and pH optimum (between 9 and 10) . Disulfiram caused 50% inhibition of soluble fraction II at 2 ~M concentration when 9 mM propionaldehyde was the aubatrate .
DEAE-cellulose chromatography of the mitochondrial fraction yielded two peaks which eluted very close to each other . Analyses with propionaldehyde, acetaldehyde and glycolaldehyde gave the same relative activities of the two peaks, sad the same was true for soluble fractions II and III. The substrate specificity sad kinetic constants of mitochondrial fraction I were very similar to those of soluble fraction II . The pH optimum was between 9 sad 10, and 50% inhibition was caused by 1 .5 WM disulfiram concentration when 9 mM propionaldehyde was the substrate . In rat liver the low-Km aldehyde dehydrogenase has been suggested to be located in the mitochondrial matrix compartment (3) . The moderate leakage of glutamate dehydrogenase into the cytoplasm suggests that the enzymatically similar activities of cytoplasmic fraction II and mitochoadrial fraction I may be due to the same enzyme which has leaked from the mitochondria during subcellular fractionation. 1n human liver mitochondrial suspension 20-30% higher values for aldehyde dehydrogenase activity were measured with 9 mM propionaldehyde and 18 mM acetaldehyde concentrations, respectively, than with 60 ~M con centrations . This can not be attributed to a contaminating microsomal activity nor to aubatrate activation, as shown by kinetic measurements with the partially purified enzyme fractions . Human liver mitochondria evidently do contain a high-Km aldehyde dehydrogenase, although its activity seems to be relatively low. In the fractions eluted from DEAE-cellulose, however, such high-K m activity was not detected . The properties of the microsomal aldehyde dehydrogenase of human liver are very similar to those described in rat liver ( Refa . 3 and 5) . Formaldehyde and glycolaldehyde are not good substrates sad the Km values for propionaldehyde and acetaldehyde are in the millimolar range . Previous results with rat strains selected for their ethanol preference have suggested that there may be significant strain differences in the microsomal and mitochondrial high-K m aldehyde dehydrogenasee (13) . The connection between these high-Km activities and acetaldehyde oxidation _in vivo is still, however, obscure .
Vol . 16, No . 10
Human Liver Aldehyde Dehydrogenases
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Acknowledg ements I am grateful to Associate Professor Martti Koivuealo for valuable diacussioas, to Dr . Matti Lempinen for providing the liver biopsy specimen and to Mrs . Eija Haasaaen and Mra . Ritva Leponiemi for the skillful technical aesietaace . This work waa supported by grants from the Sigrid Jusélius Foundation, the National Research Council for Medical Sciences sad the Foundation for Studies on Alcohol, Finland . Referenten 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
R . J . RRAEMER and R . A . DEITRICH, _J . Biol . Chem . _243 6402-6408 (1968) . A . H . BLAIR and F . H . BODLEY, Can . _J . Biochem . 47 265-272 (1968) . S . 0 . C . TOTTMAR, A . PETTERSSON and R .-H . KIESSLING, Biochem . _J . 135 577-586 (1973) . R . E . CROW, T . M . KITSON, A . R . H . MacGIBBON and R . D . BATT, Biochim . Biophys . Acta 350 121-128 (1974) . T . KOIVULA and M . ROIWSALO, Biochim . Biophys , Acta , in press . F . BERGEL, A . COHEN and N . C . HINDLEY, _J . Chem . _Sot . 1439-1443 (1950) . C . de DDVE, B . C . PRESSMAN, R . GIANETTO, R . WATTIAUS and F . APPELMANS, Biochem. _J . 60 604-617 (1955) . M . KOIVUSALOand L . IIOTILA, Anal . Biochem . 59 34-45 (1974) . A . E . HARPER, in Methode in Enzymatic Analysis (Bergmeyer, H .-U ., ed .) pp . 788-792, Verlag Chemie, Weinheim (1963) . R . J . PENNINGTON, Biochem . _J . 80 649-654 (1961) . E . SCHMITH, in Methoden der enzymatischen Analyse , Band I (Bergmeyer, A .-U ., ed .) pp . 607-613, Verlag Chemie, Weinheim (1970) . J . P . von WARTBIIRG, in The Biology of Alcoholism (Riesin, B . and Begleiter, H ., ede .) p . 86, Plenum Press, New York - London (1971) . T . KOIVULA, M . ROIVUSALO and K . 0 . LINDROS, Biochem . Pharmacol . in press (1975) .
Pergamon Press
SUBCELLULAR DLSTRIBUTION AND CHARACTERIZATION OF HUMAN LIVER ALDEHYDE DEHYDROGENASE FRACTIONS Timo Koivula
Department of Medical Chemistry, University of Helsinki, SF-00170 Helsinki 17, Finland (Received in final form April 30, 1975) Summary
The eubcellular distribution of human liver aldehyde dehydrogenases (E .C . 1 .2 .1 .3) have been studied and the different types have been separated by ion exchange chromatography . The cytoplasmic fraction contained at least two chromatographically separable aldehyde dehydrogenases, which accounted for about 30°0 of the total activity . Oae of the cytoplasmic aldehyde dehydrogenases had a high Km for aldehydea (in the millimolar range) . A considerable part of the activity found is this fraction was due to an enzyme with a low Km for aldehydea (in the micromolar range) . It had properties similar to those of the mitochondrial main enzyme fraction, from where it may have originated as a contamination during eubcellular fractionation . Specific betaine aldehyde and formaldehyde dehydrogenases were separated from these unspeciûc activities in the cytoplaemic fraction . In mitochondria, where more than 50°l~0 of the total aldehyde dehydrogenaee activity was found, there was also evidence for alight high-Km activity . The microsomal fraction contained only a high-K m aldehyde dehydrogenaae, which accounted for about 10% of the total activity . In h ++~+a*, liver only one nonspecific NAD-dependent aldehyde dehydrogenase (EC 1 . 2 . 1 . 3) fraction has previously been partially purified and characterized by Kraemer & Dietrich (1) and by Blair & Bodley (2); the latter authors found indications of the presence of another minor aldehyde dehydrogeaase fraction . In rat liver, according to recent reports, there are several aldehyde dehydrogenases which catalyze NAD-dependent aldehyde oxidation (Refs. 3-5) . However, the subcellular distribution and properties of the different aldehyde dehydrogenase fractions have not been studied in human liver . Materiale and Methode NAD+, NADP+ and glucose-6-phosphate were purchased from Boehringer, pyrazole and 2,2-diethoxyethyl trimethylammonium iodide from Aldrich-Europe, and the aldehydea from Flukes AG . DEAE-cellulose was obtained from Whatmaa Biochemicale Ltd. Betaine aldehyde was synthesized from 2, 2-diethoocyethyl trimethylammonium iodide according to the principle of Bergel et al . (6) .
1563
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Human Liver Aldehyde Dehydrogenases
Vol . 16, No . 10
A hiatologically normal biopsy specimen of human liver (weight 3 .0 g) was obtained at a stomach operation .. The specimen was immediately homogenized in 0 .25 M sucrose solution containing 10 mM sodium phosphate pH 7 .4 and 5 mM 2-mercaptoethanol . Subcellular fractionation was performed according to the principle of de Duve et al . (7) . Another fractionation was made, starting with 70 g of human liver obtained at autopsy about 1 .5 h after accidental death . The aubcellular fractions isolated from this latter specimen then served as material for partial purification by chromatography . Except for the normal premedication with phenobarbital one day before ~e operatioano drugs had been used by the persons from whom the liver samples were taken . Aldehyde dehydrogenase activity was measured at 25 °C speetrophotometrically at 340 nm or fluorometrically . The reaction mixture contained 1 .33 mM NAD, 1 .67 mM pyrazole, 0 . 070 M sodium pyrophosphate pH 8 . 0 and aldehyde . Formaldehyde dehydrogenase, glucose-b-phoaphatase, succinate-INT-reductase and glutamate dehydrogenase activities were measured as described previously (8-11) .Triton X-100 at 1 .0% concentration was added to the isolated aubcellular fractions before activity measurements in the distribution studies .
(ww)
For purification and characterization the mitochondrial and microsomal suspensions were treated with 1 .0% Triton X-100 and centrifuged for 60 min at 106000 g . Cytoplasmic and mitochondrial preparations were chromatographed in DEAF-cellulose columns equilibrated with 10 mM Tria-HCI, pH 7 . b, containing 5 mM 2-mercaptoethanol . The bound fractions were eluted with a linear salt gradient to 0 .15 M KCl concentration .
(ww)
Results The distribution of aldehyde dehydrogenase is the different aubcellular fractions of human liver was measured with propionaldehyde and acetaldehyde at two concentrations and also with glycolaldehyde as substrates (Table 1) . The distribution of the marker enzymes succinate-INT-reductase, glutamate dehydrogenase and glucose-6-phosphatase indicates that the activity found in the nuclear fraction is mostly of mitochondria) origin . The activity found in the lysosomal fraction was evidently caused by contamination with mitochondria sad microsomes . A considerable part of the activity was found in the soluble cell fraction, about 30% when measured with either the lower (60 WM) or the higher (9 and 18 mM respectively), propionaldehyde or acetaldehyde concentrations . The soluble fraction contained only 15% of the total glycolaldehyde dehydrogenase activity found . The mitochondria) matrix enzyme glutamate dehydrogenase showed moderate leakage into the cytoplasm . When allowance ie made for contamination in the nuclear and lysosomal fractions, mitochondria are found to contain at least 50% of the total activity measured at millimolar propionaldehyde or acetaldehyde concentrations, and more than 60% when measured at 60 WM concentration . Microsomal activity is about 10% of the total at higher and negligible at lower aldehyde concentrations . DEAE-cellulose chromatography of the cytoplasmic fraction yielded two peaks of aldehyde dehydrogenase activity with propionaldehyde (Fig . 1) . The first of these (cytoplaemic fraction I), the one moat loosely bound to the cellulose, was not active with glycolaldehyde . The second peak (cytoplaemic fraction II), where a small shoulder was found had activity also with glycolaldehyde . Betaine aldehyde dehydrogenase was bound most firmly to the DEAE-cellulose and was eluted separately from the other aldehyde dehydrogenase fractions . The same was true for formaldehyde dehydrogenase, which eluted between soluble fractions I and II .
Vol . 16, No . 10
1565
Human Liver Aldehyde Dehydrogenasea
TABLE 1 SUBCELLULAR DISTRIBUTION OF HUMAN LNER ALDEHYDE DEHYDROGENASE The enzyme assays are described in materials and methods . The distributions of activities are calculated as percentages of the summed activities of all fractions . The enzyme activities are expressed ae IU~g liver wet weight . Values are means of results of 2 fractionations . N = nuclear fraction, E = cytoplasmic extract, M = mitochondrial fraction, L = lysosomal fraction, P = microsomal fraction, S = final supernatant . Enzyme
Absolute values (N+E)
Aldehyde dehydrogenaae + propionaldehyde 9 mM 3 .86 60 WM 2 .38 18 mM + acetaldehyde 3 .63 3 .32 60 W M + glycolaldehyde 2 mM 3 .48 Glucose-6-phosphatase 5 .0 Succinate-INT-reductase 3 .1 Glutamate dehydrogenaae 183
Percentage values
Recovery
N+E
N
M
L
P
S
100 100 100 100 100 100 100 100
26 29 25 34 35 18 31 28
25 32 26 32 35 11 35 31
8 7 9 8 12 16 19 12
9 3 8 1 3 44 15 11
32 29 32 25 IS 7 3 18
97 77 97 73 82 95 103 97
Fig . 1 . DEAF-cellulose chromatography of human liver cytoplasmic fraction . The dialyzed sample was applied to the column (2 x 30 cm) which had been equilibrated with lOmM Tria-HC1, pH 7 .6, + 5 mM 2-mercaptoethanoL Elution was performed with a linear KC1 gradient up to 0 .2 M concentration. The fraction volume was 15 ml . Fractions were analyzed for formaldehyde dehydrogenaae (~) and aldehyde dehydro enase with propioaaldehyde ( " ), glycolaldehyde (d ) and betaine aldehyde ~O ) as substrates . Absorbance at 280 nm (- - -) . DEAE-cellulose chromatography of the mitochondrial fraction (Fig .2) gave two main peaks of activity (mitochondria) fractions I sad II) not very far from each other with both propionaldehyde sad glycolaldehyde . A minor activity was seen in the fractions eluted with the starting buffer sad these
1566
Human Liver Aldehyde Dehydrogenasea
Vol . 16, No . 10
fractions also had some activity with NADP ae coenzyme . Substrate specificitiee were measured separately for soluble DEAF fractions I and II, for mitochondrial fraction I sad for the microsomal supernatant (Table 2) . Cytoplasmic fraction I seemed to be the most reall stricted in substrate specificity . It was not active with formaldehyde at and had low activity with glycolaldehyde sad with aromatic aldehydes . Cytoplasmic fraction II waa more active with formaldehyde and glycolaldehyde,I but was even lees active with aromatic aldehydes . Mitochoadrial fraction had a rather similar substrate specificity . Microsomal aldehyde dehydrogenase was active with aromatic aldehydes, but its activity with formaldehyde and glycolaldehyde was low .
Fig. 2 . DEAE-cellulose chromatography of human liver mitochondria) fraction . After treatment with detergent sad dialysis, the sample was applied to the column (1 .5 x 30 cm) which had been equilibrated with 10 mM Tris-HCl pH 7 . 6, + 5 mM 2-mercaptoethanol . Elution was performed with a linear KCl gradient up to 0 .2 M concentration . Fractions were analyzed for aldehyde dehydrogenase activity with propioaaldehyde ( " ) sad glycolaldehyde (Q ) ae substrates and for protein (- - -, absorbante at 280 nm) . All enzyme fractions had Km values for NAD between 2 ' 10 -5 and 2 " 10 -4 M (Table 3) . Mitochondria) and micro~oma,l fractions also had same activity with NADP, having a high Km (1 " 10 - M) for this coenzyme . The cytoplasmic fraction was not active with NADP . Cytoplaemic enzyme I had a very high Km value for propioaaldehyde and acetaldehyde . For the microsomal fraction the corresponding values were somewhat lower but still in the millimolar range . Cytoplasmic enzyme II and mitochondria) enzyme I had similar very low Km values for propioaaldehyde and acetaldehyde (below 10-6 M) . Discussion Evidence for the hetero eneity of human liver aldehyde dehydrogenase has been re orted earlier ~2, 12) . Neither Kraemer & Deitrich (1) nor Blair & Bodley ~2) identified the subcellular fraction from which the partial ly purified enzyme was derived. The present study was undertaken to inreatigate the aubcellular distribution of h ++*+,a*, liver aldehyde dehydrogenaee sad to characterize the main types of aldehyde dehydrogeaases by partial purification . Previous studies with rat liver have shown a considerable aldehyde dehydrogenase activity with a high Km for propioaaldehyde and acetaldeto the pres hyde in the mitochondria) and microsomal fractions . According . ent results, high Km activities are less pronounced in h +++++ an liver . The
Vol . 16, No . 10
Human Liver Aldehyde Dehydrogenases
1567
TABLE 2 SUBSTRATE SPECIFICTTY OF HUMAN LNER ALDEHYDE DEHYDROGENASE FRACTIONS The autopsy liver sample was used for isolation of the fractions . Maximal velocities were determined from Lineweaver-Burk plots . The reaults are expressed as relative activities, the activity with propionaldehyde being taken as 1 .0, and used as the standard for each fraction . ALDEH~DE
SOLUBLE
Fraction I
Formaldehyde Acetaldehyde
SOLUBLE
Fraction II
0 0 .4
Propionaldehyde
0 .4 0 .9
1 .0 0 .1
0 .2
Glycolaldehyde
Fraction I
0 .4 0 .8
1 .0 0 .1
Benzaldehyde Anisaldehyde
MITOCHONDRIAL
Fraction 0 .1
0 .8 1 .0
1 .0 0 .3
0 .2 0 .4
0 .2
MICROSOMAL
0.8 0.6
0 .2 0 .5
0 .1
TABLE 3 APPARENT MICHAELLS CONSTANTS OF HUMAN LNER ALDEHYDE DEHYDROGENASE FRACTIONS
The autopsy liver sample was used for isolation of the fractions . In the determinations with variable aldehyde concentration NAD+ waa kept at 1 .33 mM and in those with variable NAD(P)+ concentration the propionaldehyde concentration was 9 mM . I = cytoplasmic enzyme I, II = cytoplaemic enzyme II, III = betaine aldehyde dehydrogenase, N = mitochondrial enzyme I, V = microsomal eazyme . Substrate
Km (WM) I
II
Propionaldehyde 8000 10000 Acetaldehyde Betaiae aldehyde 20 NAD+ NADP+
-
III
0 .5 0 .5 100
-
-
10 80
(with 1 mM betaine aldehyde)
N
V
0 .4 0 .4 200
-
600
3000 -
70 1000
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Htmian Liver Aldehyde Dehydrogenasea
Vol. 16, No . 10
subcellular distribution of aldehyde dehydrogenase activity in the human liver is not unlike that reported for sheep liver by Crow et al . (4) . In comparison with rat liver, microsomal activity is low . The cytoplasmic fraction accounts for about 25-35% of the total activity when measured with propionaldehyde (60 WM and 9 mM) or acetaldehyde (60 ~M and 18 mM) as substrates .
The aldehyde dehydrogenase activity of the human liver cytoplasmic fraction could be fractionated into two separate peaks by DEAE-cellulose chromatography . Formaldehyde dehydrogenase and betaine aldehyde dehyd rogenase separated differently from these nonspecific activities . Cytoplasmic fraction I has very high Km values for propionaldehyde and acetaldehyde . Even at high aldehyde substrate concentrations, however, its contribution to the total aldehyde dehydrogenase activity in human liver is very small . Cytoplasmic fraction II accounts for moat of the cytoplasmic aldehyde dehydrogenase activity . Fraction II has Km values for propionaldehyde and acetaldehyde below 1 WM, as waa also found by Kraemer & Deitrich (1) sad Blair & Bodley (2) with their partially purified enzymes . A similarity also exists in substrate specificity and pH optimum (between 9 and 10) . Disulfiram caused 50% inhibition of soluble fraction II at 2 ~M concentration when 9 mM propionaldehyde was the aubatrate .
DEAE-cellulose chromatography of the mitochondrial fraction yielded two peaks which eluted very close to each other . Analyses with propionaldehyde, acetaldehyde and glycolaldehyde gave the same relative activities of the two peaks, sad the same was true for soluble fractions II and III. The substrate specificity sad kinetic constants of mitochondrial fraction I were very similar to those of soluble fraction II . The pH optimum was between 9 sad 10, and 50% inhibition was caused by 1 .5 WM disulfiram concentration when 9 mM propionaldehyde was the substrate . In rat liver the low-Km aldehyde dehydrogenase has been suggested to be located in the mitochondrial matrix compartment (3) . The moderate leakage of glutamate dehydrogenase into the cytoplasm suggests that the enzymatically similar activities of cytoplasmic fraction II and mitochoadrial fraction I may be due to the same enzyme which has leaked from the mitochondria during subcellular fractionation. 1n human liver mitochondrial suspension 20-30% higher values for aldehyde dehydrogenase activity were measured with 9 mM propionaldehyde and 18 mM acetaldehyde concentrations, respectively, than with 60 ~M con centrations . This can not be attributed to a contaminating microsomal activity nor to aubatrate activation, as shown by kinetic measurements with the partially purified enzyme fractions . Human liver mitochondria evidently do contain a high-Km aldehyde dehydrogenase, although its activity seems to be relatively low. In the fractions eluted from DEAE-cellulose, however, such high-K m activity was not detected . The properties of the microsomal aldehyde dehydrogenase of human liver are very similar to those described in rat liver ( Refa . 3 and 5) . Formaldehyde and glycolaldehyde are not good substrates sad the Km values for propionaldehyde and acetaldehyde are in the millimolar range . Previous results with rat strains selected for their ethanol preference have suggested that there may be significant strain differences in the microsomal and mitochondrial high-K m aldehyde dehydrogenasee (13) . The connection between these high-Km activities and acetaldehyde oxidation _in vivo is still, however, obscure .
Vol . 16, No . 10
Human Liver Aldehyde Dehydrogenases
1569
Acknowledg ements I am grateful to Associate Professor Martti Koivuealo for valuable diacussioas, to Dr . Matti Lempinen for providing the liver biopsy specimen and to Mrs . Eija Haasaaen and Mra . Ritva Leponiemi for the skillful technical aesietaace . This work waa supported by grants from the Sigrid Jusélius Foundation, the National Research Council for Medical Sciences sad the Foundation for Studies on Alcohol, Finland . Referenten 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
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