PROTEINS Structure, Function, and Genetics 8:305-308 (1990)
Short Communication
Preliminary Crystallographic Analysis of Class 3 Rat Liver Aldehyde Dehydrogenase John P. Rose,' John Hempel? Ingrid KUO? Ronald Lindahl? and Bi-ChengWang'*3 Departments of 'Crystallography, 2Biochemistry, and 3Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 4Department of Biochemistry and Molecular Biology, University of South Dakota, Vermillion, South Dakota 57069
ABSTRACT NAD-linked aldehyde dehydrogenases (AlDH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AlDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group p2,, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A,and p = 110.5'. Assuming one dimer per asymmetric unit, the value V, is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the K = 180 section at JI = 90' and = 71" and 341", indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.
+
Key words: aldehyde dehydrogenase, crystals, X-ray analysis INTRODUCTION NAD-linked aldehyde dehydrogenases (AlDH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. The human liver cytosolic (class 1) and mitochondria1 (class 2) AlDHs' are responsible for the clearance of ethanol-derived acetaldehyde. These two classes of enzyme, which have 68% sequence exist as homotetramers (500 residues per subunit). Comparable AlDHs, with 9095% homology to the human enzymes can be isolated from horse, beef, sheep, and rat liver cytosol and m i t ~ c h o n d r i a . ~In- ~addition, a "high K,," aromatic aldehyde-preferring NAD or NADP utilizing AlDH has been isolated from rat hepatoma and from rats treated with 2,3,7,8-tetrachlorodibenzo-pdioxin.*s9 This class (class 3) of AlDHs, which exists as a dimer having 452 residues per subunit" (MW 49,8361, may also be isolated from the bladder," 0 1990 WILEY-LISS, INC.
lungs, and stomach'' of normal rats. Despite the low (< 30%) homology of the class 3 enzyme to the class 1and 2 enzymes, there is reason to believe, based on secondary structural predictions, that the tertiary structures of the three classes of AlDH subunits are a t least grossly similar,13 as might be expected.14 Although the identification of functional residues and regions in AlDHs has been derived from chemical modification and sequence corn par is on^,'^,^^,^^ no three-dimensional structure of an AlDH has been reported. We report here the crystallization conditions and preliminary crystallographic results for a class 3 AlDH which was isolated from an E . coli expression system.17
METHODS Catalytically active rat liver class 3 AlDH was isolated from an E. coli (BH101) expression system transformed with pTAlDH as described elsewhere,17modified to include a CM-Sepharose step in the chromatography. Transformed E. coli were grown in a 10 liter fermentor and harvested by centrifugation. The harvested cells were stored a t -7O"C, thawed, resuspended in 10 mM sodium acetate, 2 mM EDTA, 0.1% 2-mercaptoethanol, pH 5.5, and sonicated ( 4 x 15 sec bursts). Cellular debris was removed by centrifugation at 48,OOOg for 30 min in a Ti-50.2 rotor. The supernatant was dialyzed against the above buffer and loaded onto a CMSepharose 6B column equilibrated with this buffer. The column was eluted with a linear gradient of NaCl (0.0-1.0 M) and active fractions (benzaldehyde/NADP assay17) were pooled and loaded onto a 5'-AMP Sepharose column equilibrated with 25 mM potassium phosphate, 1mM EDTA, 0.1% 2-mercaptoethanol. The column was washed with the above buffer and active AlDH fractions were eluted with
Received January 11, 1990; revision accepted May 29, 1990. Address reprint requests to Professor Bi-Cheng Wang, Department of Crystallography, University of Pittsburgh, 3943 OHara Street, Pittsburgh, PA 15260.
306
J.P. ROSE ET AL.
from 50 p,l droplets of protein solution [0.7mg/ml in 1.5% (w/v) PEG-80001 containing 0.05% (w/v) mercaptoethanol, 0.5 mM EDTA, 0.25 mg/ml NAD, and 12.5 mM PIPES/NaOH (pH 6.2) placed in wells of a 9-well spot plate. The plate was then placed in a sandwich box containing a reservoir of the above buffer solution containing 2.5% PEG.
RESULTS AND DISCUSSION
Fig. 1.
Crystals of Class 3 A1 DH under polarized light.
0.5 mg/ml NAD. From a 10 liter fermentation, 20.3 mg AlDH was obtained with a specific activity of 85 Vlmg. Crystals of AlDH were grown by vapor diffusion''
From crystallization set-ups, small rectangular crystals formed in a few days and grew to a size up to 0.8 x 0.3 x 0.2 mm in 2 weeks (Fig. 1).For X-ray analysis, a crystal was mounted in a thin-walled glass capillary containing a small amount of mother liquor to prevent dehydration and sealed with diffusion pump oil. Oscillation diffraction images (Fig. 2) show diffraction to 2.8 A resolution, with some reflections observed to a resolution of 2.5 A. A data set to 3 A resolution was collected on a Siemens XlOO area detector system using double mirror focused 5 kW CuKa X-rays generated from a
Fig. 2. X-Ray diffraction pattern (1" oscillation) of a class 3 AlDH crystal, recorded on a Siemens area detector ( D = 12 cm, 0 = 20"). The arrow points to a reflection at 2.5 A resolution. For X-ray diffraction studies, crystals were mounted in thin-walled capillaries with some mother liquid to prevent dehydration.
CRYSTALS OF ALDEHYDE DEHYDROGENASE
A
307
tice vectors were observed in an exhaustive search using the indexing option in Program REFINE of the XENGEN2O system. Therefore, the crystals belong to a monoclinic space group and one peak represents the direction of a dimeric 2-fold axis and the other is generated by the interaction between the pseudo- and crystallographic 2-folds. A heavy atom search is underway in order to carry out a full crystallographic study on AlDH.
ACKNOWLEDGMENTS This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (AA-06985) to J.H., and from the NIH Division of Research Resources (RR-02877) and the National Institute of General Medical Sciences (GM-17528)to B.C.W. REFERENCES Fig. 3. A stereographic projection (k = 180 section) of the self-rotation function for class 3 A1 DH crystals, showing the pseudo-2-fold axes. Calculations were performed using ROTRANZ4and data between 30 to 4 A with a Patterson radius of 30 8 . The 41 axis is pointing to the viewer and C$ = 0" is along the c-axis.
Rigaku RU200 rotating anode. The data collection was performed using the Harvard COLLECT routines,lg with each data frame (0.25") exposed for 3 minutes. Crystal orientation, integration, and scaling were performed using the XENGEN2O program suite. Analysis of the three-dimensional data set indicates a monoclinic space group with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and p = 110.5". Systematic absences in OK0 for k f 2n suggest that the space group is P2,. Assuming one dimer per asymmetric unit, the value Vm21is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%,which is in the normal range for protein crystals. A self-rotation function22study was carried out in order to determine the relationship of the dimer axis with respect to the crystal axes. Data from 30 to 4 A were used in the calculation, producing significant rotation peaks (Fig. 3) on the K = 180 section, where K is the rotation angle?3 The rotation peaks appearing a t $ = go", = 71" and 341" have peak height of about 58% of that observed for the peak at $ = O", the crystallographic 2-fold. The fact that these peaks are nearly orthogonal could indicate an orthogonal space group. However, such an assignment is not possible because their peak heights are nonequivalent, which would indicate that the peaks at = 90" are noncrystallographic 2-folds. Furthermore, one of the rotation peaks is not parallel with any reasonable interlattice vectors. Finally, no orthogonal lat-
+
+
1. Weiner H., Flynn, T.G. (eds.) Nomenclature of mammalian aldehyde dehydrogenase. "Enzymology and Molecular Biology of Carbonyl Metabolism 2." New York Alan R. Liss, 1989:xix-xxi. 2. Hempel, J., von Bahr-Lindstrom, H., Jornvall, H. Aldehyde dehydrogenase from human liver: Primary structure of the cytoplasmic isoenzyme. Eur. J . Biochem. 141:21-35, 1984. 3. Hempel, J., Kaiser, R., Jornvall, H. Mitochondrial aldehyde dehydrogenase from human liver: Primary structure, differences in relation to the cytosolic enzyme and functional correlations. Eur. J. Biochem. 153:13-28,1985. 4. Johansson, J., von Bahr-Lindstrom, H., Jeck, R., Woenckhaus, C., Jornvall, H. Mitochondrial aldehyde dehydrogenase from horse liver: Correlations of the same species variants for both the cytosolic and the mitochondrial forms of a n enzyme. Eur. J . Biochem. 172:527-533,1988. 5. Farres, J., Guan, K.-L., Weiner, H. Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences. Eur. J . Biochem. 180:67-74, 1989. 6. Crow, D.E., Kitson, T.M., MacGibbon, A.K.H., Batt, R.D. Intracellular localization and properties of aldehyde dehydrogenase from sheep liver. Biochim. Biophys. Acta 350: 121-128,1974, 7. DUM, T.J., Koleske, A.J., Lindahl, R., Pitot, H.C. Phenobarbital-inducible aldehyde dehydrogenase in the rat. J. Biol. Chem. 264:13057-13065, 1989. 8. Feinstein, R.N., Cameron, E.D. Aldehyde dehydrogenase activity in a rat hepatoma. Biochem. Biophys. Res. Commun. 48:1140-1146,1972. 9. Deitrich, R.A., Bludeau, P., Stock, T., Roper, M. Induction of different rat liver supernatant aldehyde dehydrogenases by phenobarbital and tetrachlorodibenzo-p-dixoin.J. Biol. Chem. 252:6169-6176,1977. 10. Jones, D.E., Brennan, M.D., Hempel, J., Lindahl, R. Cloning and complete nucleotide sequence of a full length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 85:1782-1786,1988. 11. Lindahl, R. Identification of hepatocarcinogenesis-associated aldehyde dehydrogenase in normal rat urinary bladder. Cancer Res. 46:2502-2506,1986. 12. Koivusalo, M., Aarnio, M., Baumann, M., Rautoma, P. NAD(P)-linked aromatic aldehyde preferring cytoplasmic aldehyde dehydrogenases in the rat. Constitutive and inducible forms in liver, lung, stomach and intestinal mucosa. Prog. Clin. Biol. Res. 290:19-33,1989. 13. Hempel, J., Harper, K., Lindahl, R. Inducible class 3 aldehyde dehydrogenase from rat heptaocellular carcinoma liver: Disand 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated tant relationship to the class 1 and 2 enzymes from mammalian liver cytosoUmitochondria. Biochemistry 28:11601167,1989.
308
J.P. ROSE ET AL.
14. Richardson, J.S. The anatomy and taxonomy of protein structure. Adv. Protein Chem. 34167-339,1981. 15. Hempel, J., Jornvall, H. Aldehyde dehydrogenasesstructure. In: “Human Metabolism of Alcohol,” Vol. 11, Batt, R.D. (eds.). Boca Raton, F L CRC Press, Crow, K.E., 1989 77-88. 16. Jornvall, H., Persson, B., Krook, M., Hempel, J . Alcohol and aldehyde dehydrogenases. In: “Molecular Pathology of Alcoholism,” London: Oxford University Press, in press. 17. Harper K.,Jones, D.E., Brennan, M.D., Lindahl, R. Characterization of a functional recombinant rat liver aldehyde dehydrogenase: Expression as a non-fusion protein in E. coli. Biochem. Bionhvs. Res. Commun. 152:940-947.1988. 18. McPherson, A. “P;e$aration and Analysis of Protein Crystals.” New York John Wiley, 1982: 96-97. 19. Blum, M., Metcalf, P., Harrison, S.C., Wiley, D.C. A system for collection and on line integration of X-ray diffrac-
20.
21. 22. 23. 24.
tion data from a multiwire area detector. J . Appl. Crystallogr. 20:235-242, 1987. Howard, A.J., Gilliland, G.L., Finzel, B.C., Poulos, T.L., Ohlendorf, D.H., Salemme, F.R. The use of an imaging proportional counter in macromolecular crystallography. J. Appl. C ~ s t a l l o g r .20:383-387, 1987. Matthews, B.M. Solvent content of protein crystals. J. Mol. Biol. 33:491-497, 1968. Crowther, R.A. The fast rotation function. In: “The Molecular Replacement Method,” Rossmann, M.G. (ed.).New York: Gordon and Breach, 1972: 174-178. Rossmann, M.G., Blow, D.M. The detection of sub-units within the crystallographic assymetric unit. Acta Crystallogr. 1524-31, 1962. Craven, B.M. Computer program ROTRAN. TR-78-2, Department of Crystallography, University of Pittsburgh, Pittsburgh, PA 15260 1978.
Short Communication
Preliminary Crystallographic Analysis of Class 3 Rat Liver Aldehyde Dehydrogenase John P. Rose,' John Hempel? Ingrid KUO? Ronald Lindahl? and Bi-ChengWang'*3 Departments of 'Crystallography, 2Biochemistry, and 3Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 4Department of Biochemistry and Molecular Biology, University of South Dakota, Vermillion, South Dakota 57069
ABSTRACT NAD-linked aldehyde dehydrogenases (AlDH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AlDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group p2,, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A,and p = 110.5'. Assuming one dimer per asymmetric unit, the value V, is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the K = 180 section at JI = 90' and = 71" and 341", indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.
+
Key words: aldehyde dehydrogenase, crystals, X-ray analysis INTRODUCTION NAD-linked aldehyde dehydrogenases (AlDH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. The human liver cytosolic (class 1) and mitochondria1 (class 2) AlDHs' are responsible for the clearance of ethanol-derived acetaldehyde. These two classes of enzyme, which have 68% sequence exist as homotetramers (500 residues per subunit). Comparable AlDHs, with 9095% homology to the human enzymes can be isolated from horse, beef, sheep, and rat liver cytosol and m i t ~ c h o n d r i a . ~In- ~addition, a "high K,," aromatic aldehyde-preferring NAD or NADP utilizing AlDH has been isolated from rat hepatoma and from rats treated with 2,3,7,8-tetrachlorodibenzo-pdioxin.*s9 This class (class 3) of AlDHs, which exists as a dimer having 452 residues per subunit" (MW 49,8361, may also be isolated from the bladder," 0 1990 WILEY-LISS, INC.
lungs, and stomach'' of normal rats. Despite the low (< 30%) homology of the class 3 enzyme to the class 1and 2 enzymes, there is reason to believe, based on secondary structural predictions, that the tertiary structures of the three classes of AlDH subunits are a t least grossly similar,13 as might be expected.14 Although the identification of functional residues and regions in AlDHs has been derived from chemical modification and sequence corn par is on^,'^,^^,^^ no three-dimensional structure of an AlDH has been reported. We report here the crystallization conditions and preliminary crystallographic results for a class 3 AlDH which was isolated from an E . coli expression system.17
METHODS Catalytically active rat liver class 3 AlDH was isolated from an E. coli (BH101) expression system transformed with pTAlDH as described elsewhere,17modified to include a CM-Sepharose step in the chromatography. Transformed E. coli were grown in a 10 liter fermentor and harvested by centrifugation. The harvested cells were stored a t -7O"C, thawed, resuspended in 10 mM sodium acetate, 2 mM EDTA, 0.1% 2-mercaptoethanol, pH 5.5, and sonicated ( 4 x 15 sec bursts). Cellular debris was removed by centrifugation at 48,OOOg for 30 min in a Ti-50.2 rotor. The supernatant was dialyzed against the above buffer and loaded onto a CMSepharose 6B column equilibrated with this buffer. The column was eluted with a linear gradient of NaCl (0.0-1.0 M) and active fractions (benzaldehyde/NADP assay17) were pooled and loaded onto a 5'-AMP Sepharose column equilibrated with 25 mM potassium phosphate, 1mM EDTA, 0.1% 2-mercaptoethanol. The column was washed with the above buffer and active AlDH fractions were eluted with
Received January 11, 1990; revision accepted May 29, 1990. Address reprint requests to Professor Bi-Cheng Wang, Department of Crystallography, University of Pittsburgh, 3943 OHara Street, Pittsburgh, PA 15260.
306
J.P. ROSE ET AL.
from 50 p,l droplets of protein solution [0.7mg/ml in 1.5% (w/v) PEG-80001 containing 0.05% (w/v) mercaptoethanol, 0.5 mM EDTA, 0.25 mg/ml NAD, and 12.5 mM PIPES/NaOH (pH 6.2) placed in wells of a 9-well spot plate. The plate was then placed in a sandwich box containing a reservoir of the above buffer solution containing 2.5% PEG.
RESULTS AND DISCUSSION
Fig. 1.
Crystals of Class 3 A1 DH under polarized light.
0.5 mg/ml NAD. From a 10 liter fermentation, 20.3 mg AlDH was obtained with a specific activity of 85 Vlmg. Crystals of AlDH were grown by vapor diffusion''
From crystallization set-ups, small rectangular crystals formed in a few days and grew to a size up to 0.8 x 0.3 x 0.2 mm in 2 weeks (Fig. 1).For X-ray analysis, a crystal was mounted in a thin-walled glass capillary containing a small amount of mother liquor to prevent dehydration and sealed with diffusion pump oil. Oscillation diffraction images (Fig. 2) show diffraction to 2.8 A resolution, with some reflections observed to a resolution of 2.5 A. A data set to 3 A resolution was collected on a Siemens XlOO area detector system using double mirror focused 5 kW CuKa X-rays generated from a
Fig. 2. X-Ray diffraction pattern (1" oscillation) of a class 3 AlDH crystal, recorded on a Siemens area detector ( D = 12 cm, 0 = 20"). The arrow points to a reflection at 2.5 A resolution. For X-ray diffraction studies, crystals were mounted in thin-walled capillaries with some mother liquid to prevent dehydration.
CRYSTALS OF ALDEHYDE DEHYDROGENASE
A
307
tice vectors were observed in an exhaustive search using the indexing option in Program REFINE of the XENGEN2O system. Therefore, the crystals belong to a monoclinic space group and one peak represents the direction of a dimeric 2-fold axis and the other is generated by the interaction between the pseudo- and crystallographic 2-folds. A heavy atom search is underway in order to carry out a full crystallographic study on AlDH.
ACKNOWLEDGMENTS This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (AA-06985) to J.H., and from the NIH Division of Research Resources (RR-02877) and the National Institute of General Medical Sciences (GM-17528)to B.C.W. REFERENCES Fig. 3. A stereographic projection (k = 180 section) of the self-rotation function for class 3 A1 DH crystals, showing the pseudo-2-fold axes. Calculations were performed using ROTRANZ4and data between 30 to 4 A with a Patterson radius of 30 8 . The 41 axis is pointing to the viewer and C$ = 0" is along the c-axis.
Rigaku RU200 rotating anode. The data collection was performed using the Harvard COLLECT routines,lg with each data frame (0.25") exposed for 3 minutes. Crystal orientation, integration, and scaling were performed using the XENGEN2O program suite. Analysis of the three-dimensional data set indicates a monoclinic space group with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and p = 110.5". Systematic absences in OK0 for k f 2n suggest that the space group is P2,. Assuming one dimer per asymmetric unit, the value Vm21is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%,which is in the normal range for protein crystals. A self-rotation function22study was carried out in order to determine the relationship of the dimer axis with respect to the crystal axes. Data from 30 to 4 A were used in the calculation, producing significant rotation peaks (Fig. 3) on the K = 180 section, where K is the rotation angle?3 The rotation peaks appearing a t $ = go", = 71" and 341" have peak height of about 58% of that observed for the peak at $ = O", the crystallographic 2-fold. The fact that these peaks are nearly orthogonal could indicate an orthogonal space group. However, such an assignment is not possible because their peak heights are nonequivalent, which would indicate that the peaks at = 90" are noncrystallographic 2-folds. Furthermore, one of the rotation peaks is not parallel with any reasonable interlattice vectors. Finally, no orthogonal lat-
+
+
1. Weiner H., Flynn, T.G. (eds.) Nomenclature of mammalian aldehyde dehydrogenase. "Enzymology and Molecular Biology of Carbonyl Metabolism 2." New York Alan R. Liss, 1989:xix-xxi. 2. Hempel, J., von Bahr-Lindstrom, H., Jornvall, H. Aldehyde dehydrogenase from human liver: Primary structure of the cytoplasmic isoenzyme. Eur. J . Biochem. 141:21-35, 1984. 3. Hempel, J., Kaiser, R., Jornvall, H. Mitochondrial aldehyde dehydrogenase from human liver: Primary structure, differences in relation to the cytosolic enzyme and functional correlations. Eur. J. Biochem. 153:13-28,1985. 4. Johansson, J., von Bahr-Lindstrom, H., Jeck, R., Woenckhaus, C., Jornvall, H. Mitochondrial aldehyde dehydrogenase from horse liver: Correlations of the same species variants for both the cytosolic and the mitochondrial forms of a n enzyme. Eur. J . Biochem. 172:527-533,1988. 5. Farres, J., Guan, K.-L., Weiner, H. Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences. Eur. J . Biochem. 180:67-74, 1989. 6. Crow, D.E., Kitson, T.M., MacGibbon, A.K.H., Batt, R.D. Intracellular localization and properties of aldehyde dehydrogenase from sheep liver. Biochim. Biophys. Acta 350: 121-128,1974, 7. DUM, T.J., Koleske, A.J., Lindahl, R., Pitot, H.C. Phenobarbital-inducible aldehyde dehydrogenase in the rat. J. Biol. Chem. 264:13057-13065, 1989. 8. Feinstein, R.N., Cameron, E.D. Aldehyde dehydrogenase activity in a rat hepatoma. Biochem. Biophys. Res. Commun. 48:1140-1146,1972. 9. Deitrich, R.A., Bludeau, P., Stock, T., Roper, M. Induction of different rat liver supernatant aldehyde dehydrogenases by phenobarbital and tetrachlorodibenzo-p-dixoin.J. Biol. Chem. 252:6169-6176,1977. 10. Jones, D.E., Brennan, M.D., Hempel, J., Lindahl, R. Cloning and complete nucleotide sequence of a full length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 85:1782-1786,1988. 11. Lindahl, R. Identification of hepatocarcinogenesis-associated aldehyde dehydrogenase in normal rat urinary bladder. Cancer Res. 46:2502-2506,1986. 12. Koivusalo, M., Aarnio, M., Baumann, M., Rautoma, P. NAD(P)-linked aromatic aldehyde preferring cytoplasmic aldehyde dehydrogenases in the rat. Constitutive and inducible forms in liver, lung, stomach and intestinal mucosa. Prog. Clin. Biol. Res. 290:19-33,1989. 13. Hempel, J., Harper, K., Lindahl, R. Inducible class 3 aldehyde dehydrogenase from rat heptaocellular carcinoma liver: Disand 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated tant relationship to the class 1 and 2 enzymes from mammalian liver cytosoUmitochondria. Biochemistry 28:11601167,1989.
308
J.P. ROSE ET AL.
14. Richardson, J.S. The anatomy and taxonomy of protein structure. Adv. Protein Chem. 34167-339,1981. 15. Hempel, J., Jornvall, H. Aldehyde dehydrogenasesstructure. In: “Human Metabolism of Alcohol,” Vol. 11, Batt, R.D. (eds.). Boca Raton, F L CRC Press, Crow, K.E., 1989 77-88. 16. Jornvall, H., Persson, B., Krook, M., Hempel, J . Alcohol and aldehyde dehydrogenases. In: “Molecular Pathology of Alcoholism,” London: Oxford University Press, in press. 17. Harper K.,Jones, D.E., Brennan, M.D., Lindahl, R. Characterization of a functional recombinant rat liver aldehyde dehydrogenase: Expression as a non-fusion protein in E. coli. Biochem. Bionhvs. Res. Commun. 152:940-947.1988. 18. McPherson, A. “P;e$aration and Analysis of Protein Crystals.” New York John Wiley, 1982: 96-97. 19. Blum, M., Metcalf, P., Harrison, S.C., Wiley, D.C. A system for collection and on line integration of X-ray diffrac-
20.
21. 22. 23. 24.
tion data from a multiwire area detector. J . Appl. Crystallogr. 20:235-242, 1987. Howard, A.J., Gilliland, G.L., Finzel, B.C., Poulos, T.L., Ohlendorf, D.H., Salemme, F.R. The use of an imaging proportional counter in macromolecular crystallography. J. Appl. C ~ s t a l l o g r .20:383-387, 1987. Matthews, B.M. Solvent content of protein crystals. J. Mol. Biol. 33:491-497, 1968. Crowther, R.A. The fast rotation function. In: “The Molecular Replacement Method,” Rossmann, M.G. (ed.).New York: Gordon and Breach, 1972: 174-178. Rossmann, M.G., Blow, D.M. The detection of sub-units within the crystallographic assymetric unit. Acta Crystallogr. 1524-31, 1962. Craven, B.M. Computer program ROTRAN. TR-78-2, Department of Crystallography, University of Pittsburgh, Pittsburgh, PA 15260 1978.
