- Research article
- Open Access
Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity
© Bootorabi et al; licensee BioMed Central Ltd. 2008
- Received: 03 September 2008
- Accepted: 27 November 2008
- Published: 27 November 2008
Acetaldehyde, the first metabolite of ethanol, can generate covalent modifications of proteins and cellular constituents. However, functional consequences of such modification remain poorly defined. In the present study, we examined acetaldehyde reaction with human carbonic anhydrase (CA) isozyme II, which has several features that make it a suitable target protein: It is widely expressed, its enzymatic activity can be monitored, its structural and catalytic properties are known, and it contains 24 lysine residues, which are accessible sites for aldehyde reaction.
Acetaldehyde treatment in the absence and presence of a reducing agent (NaBH3(CN)) caused shifts in the pI values of CA II. SDS-PAGE indicated a shift toward a slightly higher molecular mass. High-resolution mass spectra of CA II, measured with and without NaBH3(CN), indicated the presence of an unmodified protein, as expected. Mass spectra of CA II treated with acetaldehyde revealed a modified protein form (+26 Da), consistent with a "Schiff base" formation between acetaldehyde and one of the primary NH2 groups (e.g., in lysine side chain) in the protein structure. This reaction was highly specific, given the relative abundance of over 90% of the modified protein. In reducing conditions, each CA II molecule had reacted with 9–19 (14 on average) acetaldehyde molecules (+28 Da), consistent with further reduction of the "Schiff bases" to substituted amines (N-ethyllysine residues). The acetaldehyde-modified protein showed decreased CA enzymatic activity.
The acetaldehyde-derived modifications in CA II molecule may have physiological consequences in alcoholic patients.
- Schiff Base
- Carbonic Anhydrase
- Carbonic Anhydrase Activity
- Acetaldehyde Concentration
- Acetaldehyde Reaction
Acetaldehyde, a product of ethanol metabolism, has been suggested to play a pivotal role in the toxicity of ethanol to human tissues [1–3]. It can form covalent, stable or unstable adducts with amino acids and nucleophilic biomolecules [4–10]. The adduct formation may result in changes in the physicochemical properties of proteins, nucleic acids, and lipids, disturb normal cellular functions, and create adverse immunological responses [8, 11–15].
In the present study, we used carbonic anhydrase (CA) isozyme II as a model to investigate the effects of acetaldehyde reaction with the protein. CA II is a well characterized enzyme expressed in several organs, including the brain, stomach, gut, kidney, and reproductive organs, and it is highly abundant in erythrocytes . It is one of the most efficient enzymes known in the animal kingdom, catalyzing the reversible hydration of carbon dioxide at a rate of 1.4 × 106 molecules per second . CA II was our choice of model protein, because its structural and catalytic properties are well known and it contains 24 lysine residues (out of 260 amino acids), each a potential site for acetaldehyde reaction. Currently, there are also valid methods to monitor CA II catalytic activity to assess the functional consequences of acetaldehyde reaction. Importantly, CA II catalytic activity is essential for several physiological processes such as gastric acid formation, alkalization of pancreatic juice and bile, renal proton secretion, bone resorption, and cerebrospinal fluid secretion . Although proteomic in vivo evidence of this modification is still lacking, one would expect a wide variety of adverse effects in these physiological processes, if CA II activity was disturbed due to acetaldehyde reaction in alcoholic patients.
Production of recombinant human CA II
Labelling of CA II with acetaldehyde
Human blood samples and recombinant human CA II enzyme were treated with various concentrations of acetaldehyde either in the presence or absence of a reducing agent, NaBH3(CN). All reagents were maintained and pipeting was performed at +4°C to minimize acetaldehyde evaporation. The sample tubes containing 1/10 diluted (in H2O) blood or CA II enzyme with or without acetaldehyde in H2O were tightly sealed and incubated at 37°C for 2 hr. Then 10 mM NaBH3(CN) or equal volume of H2O was added to each sample tube, and the incubation at +37°C was continued for 22 hr. After the incubation the samples were quickly cooled down to +4°C.
Isoelectric focusing and SDS-PAGE
IEF was carried out using Novex Pre-Cast vertical IEF gels (pH 3–10) (Invitrogen, Carlsbad, CA) containing 2% ampholytes. One μg samples of recombinant CA II protein untreated or treated with 10 mM NaBH3(CN) and various acetaldehyde concentrations were applied to each lane. The electrophoreses were performed in an Xcell SureLock™ Mini-Cell unit (Invitrogen) at a constant power of 2 W per gel for 2 hr with a voltage limit of 500 V. The polypeptides were visualized using Colloidal Blue staining kit (Invitrogen).
All the reagents for SDS-PAGE were from Invitrogen except for the protein markers that were obtained from Bio-Rad Laboratories (Richmond, CA). The electrophoreses were performed in an Xcell SureLock™ Mini-Cell unit (Invitrogen) under reducing conditions, using NuPAGE™ 10% Bis-Tris gels. The polypeptide bands were stained using Colloidal Blue staining kit (Invitrogen).
CA activity measurements
CA catalytic activity was first determined using a slightly modified end-point titration method of Maren et al. [44, 45]. Briefly the steps included: 150 ng of human CA II or 1 μl of 1/10 diluted blood sample was added to 500 μl of ice-cold assay buffer (20 mM imidazole, 5 mM Tris (BASE), 0,2 mM p-nitrophenol). The cuvette containing the sample and assay buffer was placed in a Lambda 35 UV/VIS (Perkin Elmer Instruments, Waltham, MA) spectrophotometer and 500 μl of ice-cold CO2-saturated H2O was added into the cuvette. The exact time for the yellow color disappearance was counted. In the control experiments without the CA II enzyme, the color disappeared in 50 sec. Statistical significance of the enzyme activity results was assessed using Student's t test and was denoted as p values.
The enzymatic activities of native recombinant human CA II and the enzyme treated with 100 mM acetaldehyde under reducing conditions were also assayed using an Applied Photophysics (Leatherhead, UK) stopped-flow instrument. Reaction was measured using 0.2 mM phenol red as an indicator, in 10 mM Hepes, 0.1 M Na2SO4, pH 7.5, for a period of 10–100 sec. To determine the kinetic parameters, CO2 concentration ranged from 1.7 to 17 mM. Kinetic parameters were obtained from Lineweaver-Burk plots, as reported earlier, and represent the mean from at least three different determinations.
Sample preparation for mass spectrometry
Due to a low tolerance of ESI for high salt concentrations, protein samples were first desalted using a PD-10 column (Amersham-Biosciences, Billingham, UK) equilibrated in advance with 10 mM ammonium acetate (pH 6.8) buffer and ten 1-mL fractions were collected. Fractions 3–5 were combined and concentrated to ~500 μL using an Ultrafree-0.5 (10-kDa cut-off) centrifugal filter device (Millipore, Billerica, MA, USA). Protein concentrations were determined by absorbance at 280 nm using a calculated extinction coefficient of ε280 = 50070 M-1 cm-1. For mass spectrometry, samples were further diluted with acetonitrile/water/acetic acid (49.5:49.5:1.0, v/v) solution to an approximate concentration of 5 μM.
All experiments were performed on a 4.7-T Bruker BioAPEX-Qe Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Billerica, MA, USA), interfaced to an external Apollo-II™ electrospray ionization (ESI) source. Protein samples were directly infused at a flow rate of 1.5 μL min-1. ESI-generated ions were externally accumulated in an RF-hexapole ion trap for 400 ms and transmitted through a high-voltage optics region to an Infinity ICR cell for "sidekick" trapping, conventional "RF-chirp" excitation and broadband detection. For each spectrum, a total of 256 co-added (512-kWord) time-domain transients were zero-filled once prior to fast Fourier transform and magnitude calculation. Frequency-to-m/z calibration was performed externally with respect to the ions of an ES Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) calibration mixture. All data were processed using Bruker XMASS 7.0.8 software.
Isoelectric focusing and SDS PAGE
Kinetic and inhibitory properties of CA II measured by the stopped-flow method
KI (acetazolamide) (nM)
Native CA II
CA II + AA + NaBH 3 (CN)
Figure 6B presents an expanded view on the charge state 24+ of the mass spectra of native CA II (Figure 6A) as well as CA II incubated in the presence of NaBH3(CN), acetaldehyde (AA), and both. In the presence of NaBH3(CN), the mass spectrum was identical to that observed for native CA II. In contrast, when reacted with 100 mM acetaldehyde in the absence of NaBH3(CN), the mass of CA II increased by ~26 Da (mm.a. = 29124.09 ± 0.09 Da), consistent with a "Schiff base" formation between acetaldehyde and a primary amino group, e.g., ε-NH2 of lysine (Δmtheor = +26.02 Da). This reaction was highly specific given that the modified protein form was present for as much as 90% as compared to the unmodified form. In the presence of NaBH3(CN), a more drastic change was observed. Evenly distributed new signals appeared in the mass spectrum, with a mass increase of ~n × 28 Da, n ranging from 9 to 19 (for the three most abundant forms, mm.a. = 29462.51 ± 0.06 Da (n = 13), 29490.54 ± 0.05 (n = 14), and 29518.59 ± 0.05 Da (n = 15)). This is consistent with the formation of multiple stable covalent acetaldehyde adducts with CA II, most probably N-ethyllysine residues (Δmtheor = +28.03 Da).
Acetaldehyde has been demonstrated to be able to bind to several different proteins, both in vitro and in vivo. The structural modifications due to acetaldehyde binding may affect the conformation, acid-base properties, and/or hydrogen-bonding patterns of amino acids on the surface or within the active site of an enzyme, thereby disrupting the normal protein function. Although several lines of previous investigations during the last two decades have focused on the generation of acetaldehyde adducts, it has turned out to be a great challenge to explore the functional consequences of these acetaldehyde-induced modifications without proper functional assays. Acetaldehyde is a very reactive compound, and thus, it has been particularly difficult to determine the presence of unstable adducts formed under non-reductive conditions.
The present results showed that human CA II is a good target protein for acetaldehyde modification. It has a high number of lysine residues (24 in total) in its primary sequence. The highly sensitive mass spectrometric method indicated that, under reducing conditions, each CA II enzyme molecule had reacted with up to 19 (14 on average) acetaldehyde molecules to form stable covalent adducts (N-ethyllysine residues). Although such a high number of acetaldehyde was bound to the enzyme, the modified protein still retained much of the enzymatic activity. Using the modified Maren's method combined with a spectrophotometric analysis, we found that the enzyme activity decreased 23.7% under supraphysiological (100 mM) acetaldehyde concentrations in reducing conditions. The stopped-flow CA activity assay showed a significantly greater decrease in the enzyme activity when compared to the spectrophotometric assay. Even though these assay methods produced different rates for the acetaldehyde-induced inhibition, both techniques clearly indicated that acetaldehyde binding reduces the catalytic activity.
Interestingly, only a single acetaldehyde molecule had reacted with CA II under non-reductive conditions, forming the unstable covalent adduct (''Schiff base''). Due to high mass resolution inherent for the FT-ICR technique, unequivocal differentiation between the ''Schiff base'' (+26.02 Da) and the substituted amine (+28.03 Da), i.e. ~2-Da mass difference at 29 kDa, was possible (Figure 1). According to the mass spectrometry, over 90% of the enzyme molecules were modified with acetaldehyde. However, repeated measurements with the acetaldehyde-treated sample, after being stored two weeks at 4°C, indicated the presence of an unmodified CA II only, consistent with unstable character of the formed ''Schiff base''. To further localize the modification site, both native and acetaldehyde-treated CA II samples were subjected to on-line pepsin digestion prior to mass spectrometry. Although the resulting peptide maps had 100% sequence coverage, the observed peptides were markedly larger (up to 10 kDa) in the presence of acetaldehyde, possibly due to decreased protease activity, making spectral assignments occasionally ambiguous. However, two of the observed 6.3-kDa peptic peptides had a mass difference of 26.01 Da, suggesting the modification site within the first 56 residues. Owing to the experimental difficulties in obtaining good tandem mass spectra for these large peptic peptides, more experiments with different proteases are warranted for the further identification of the modification site in CA II.
Both SDS-PAGE and isoelectric focusing suggested that 100 mM acetaldehyde might induce degradation of CA II protein under non-reductive conditions. However, mass spectrometry did not show any evidence for the accelerated degradation of CA II (i.e., no change in the absolute intensity of the intact protein or evidence of the peptide signals), suggesting that the smear observed in isoelectric focusing may have resulted from chemical interactions between ampholytes and acetaldehyde under non-reductive conditions.
More detailed structural studies are needed to characterize the acetaldehyde-induced modifications of CA II. This enzyme, although only weakly expressed in liver [46, 47], represents an excellent model for both structural and functional studies of acetaldehyde modification. Its crystal structure and kinetic properties have been reported by several groups [48–53]. These studies can be expanded to other CA isozymes that are more highly expressed in the liver, including CA III, VA, and CA XIV [54–56]. These enzymes may also be susceptible to modification because acetaldehyde can reach over 100 μM concentration in the liver of alcoholic patients , even though concentrations are lower, though still in a micromolar range, in the blood [58, 59].
Accurate acetaldehyde determination from biological samples has been extremely challenging. It is widely accepted that single doses of ethanol do not significantly increase blood free acetaldehyde concentrations [60, 61]. Nonetheless, such doses may elevate acetaldehyde levels within intracellular compartments or cell membranes. Baraona et al.  tested the blood of 5 healthy individuals and 6 alcoholic patients and showed that most of the blood acetaldehyde was present in the erythrocytes after alcohol consumption. In vivo, acetaldehyde concentration within the erythrocytes is about 3–10 times higher than in the plasma [62, 63]. Although acetaldehyde concentrations are probably highest in the liver, the site of ethanol metabolism, acetaldehyde modified protein epitopes have been also located to other organs and cell types. Positive immunohistochemical staining for acetaldehyde adducts has been demonstrated in the brain, heart, skeletal muscle, and erythrocytes [14, 64–66]. All of these tissues and cells contain several CA isozymes that may become functionally impaired because of adduct formation. Therefore, these modifications deserve further study to determine the exact submolecular defects caused by acetaldehyde in each isozyme.
In the present study, we showed that acetaldehyde, the first metabolite of ethanol, can modify the ubiquitous enzyme, carbonic anhydrase. Mass spectrometric analysis indicated that one of the primary NH2 groups (e.g., in lysine side chain) in the CA isozyme II had reacted with acetaldehyde under non-reducing condition, consistent with a "Schiff base" formation. In reducing conditions, each CA II molecule had reacted with 9–19 (14 on average) acetaldehyde molecules, consistent with further reduction of the "Schiff bases" to substituted amines (N-ethyllysine residues). The latter structural change led to decreased enzyme activity, which may have important physiological consequences in alcohol abusers.
This work was supported by grants from the EU projects Euroxy (CTS) and DeZnIT (CTS, SP), the Finnish Foundation for Alcohol Studies (ON), National Institutes of Health, Grant DK-40163 (WSS), and the Academy of Finland, Grant 108533 (JJ). The authors thank Jukka Lehtonen, Aulikki Lehmus and Ritva Romppanen for skilful technical assistance.
- Lieber CS: Medical disorders of alcoholism. N Engl J Med. 1995, 333 (16): 1058-1065.View ArticlePubMedGoogle Scholar
- Niemela O, Parkkila S, Yla-Herttuala S, Halsted C, Witztum JL, Lanca A, Israel Y: Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation. Lab Invest. 1994, 70 (4): 537-546.PubMedGoogle Scholar
- Freeman TL, Tuma DJ, Thiele GM, Klassen LW, Worrall S, Niemela O, Parkkila S, Emery PW, Preedy VR: Recent advances in alcohol-induced adduct formation. Alcohol Clin Exp Res. 2005, 29 (7): 1310-1316.View ArticlePubMedGoogle Scholar
- Tuma DJ, Klassen LW: Immune responses to acetaldehyde-protein adducts: role in alcoholic liver disease. Gastroenterology. 1992, 103 (6): 1969-1973.PubMedGoogle Scholar
- Stevens VJ, Fantl WJ, Newman CB, Sims RV, Cerami A, Peterson CM: Acetaldehyde adducts with hemoglobin. J Clin Invest. 1981, 67 (2): 361-369.PubMed CentralView ArticlePubMedGoogle Scholar
- Niemela O: Aldehyde-protein adducts in the liver as a result of ethanol-induced oxidative stress. Front Biosci. 1999, 4: D506-513.View ArticlePubMedGoogle Scholar
- Israel Y, Hurwitz E, Niemela O, Arnon R: Monoclonal and polyclonal antibodies against acetaldehyde-containing epitopes in acetaldehyde-protein adducts. Proc Natl Acad Sci USA. 1986, 83 (20): 7923-7927.PubMed CentralView ArticlePubMedGoogle Scholar
- Sorrell MF, Tuma DJ: The functional implications of acetaldehyde binding to cell constituents. Ann N Y Acad Sci. 1987, 492: 50-62.View ArticlePubMedGoogle Scholar
- Worrall S, de Jersey J, Nicholls R, Wilce P: Acetaldehyde/protein interactions: are they involved in the pathogenesis of alcoholic liver disease?. Dig Dis. 1993, 11 (4–5): 265-277.View ArticlePubMedGoogle Scholar
- Nicholls R, de Jersey J, Worrall S, Wilce P: Modification of proteins and other biological molecules by acetaldehyde: adduct structure and functional significance. Int J Biochem. 1992, 24 (12): 1899-1906.View ArticlePubMedGoogle Scholar
- Mauch TJ, Donohue TM, Zetterman RK, Sorrell MF, Tuma DJ: Covalent binding of acetaldehyde selectively inhibits the catalytic activity of lysine-dependent enzymes. Hepatology. 1986, 6 (2): 263-269.View ArticlePubMedGoogle Scholar
- Mauch TJ, Tuma DJ, Sorrell MF: The binding of acetaldehyde to the active site of ribonuclease: alterations in catalytic activity and effects of phosphate. Alcohol Alcohol. 1987, 22 (2): 103-112.PubMedGoogle Scholar
- Viitala K, Makkonen K, Israel Y, Lehtimaki T, Jaakkola O, Koivula T, Blake JE, Niemela O: Autoimmune responses against oxidant stress and acetaldehyde-derived epitopes in human alcohol consumers. Alcohol Clin Exp Res. 2000, 24 (7): 1103-1109.View ArticlePubMedGoogle Scholar
- Niemela O, Parkkila S: Alcoholic macrocytosis – is there a role for acetaldehyde and adducts?. Addict Biol. 2004, 9 (1): 3-10.View ArticlePubMedGoogle Scholar
- Viitala K, Israel Y, Blake JE, Niemela O: Serum IgA, IgG, and IgM antibodies directed against acetaldehyde-derived epitopes: relationship to liver disease severity and alcohol consumption. Hepatology. 1997, 25 (6): 1418-1424.View ArticlePubMedGoogle Scholar
- Fowles LF, Beck E, Worrall S, Shanley BC, de Jersey J: The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides. A kinetic study with implications for protein modification in alcohol abuse. Biochem Pharmacol. 1996, 51 (10): 1259-1267.View ArticlePubMedGoogle Scholar
- Niemela O: Distribution of ethanol-induced protein adducts in vivo: relationship to tissue injury. Free Radic Biol Med. 2001, 31 (12): 1533-1538.View ArticlePubMedGoogle Scholar
- Tuma DJ, Hoffman T, Sorrell MF: The chemistry of acetaldehyde-protein adducts. Alcohol Alcohol Suppl. 1991, 1: 271-276.PubMedGoogle Scholar
- Donohue TM, Tuma DJ, Sorrell MF: Acetaldehyde adducts with proteins: binding of [14C]acetaldehyde to serum albumin. Arch Biochem Biophys. 1983, 220 (1): 239-246.View ArticlePubMedGoogle Scholar
- San George RC, Hoberman HD: Reaction of acetaldehyde with hemoglobin. J Biol Chem. 1986, 261 (15): 6811-6821.PubMedGoogle Scholar
- Tuma DJ, Jennett RB, Sorrell MF: The interaction of acetaldehyde with tubulin. Ann N Y Acad Sci. 1987, 492: 277-286.View ArticlePubMedGoogle Scholar
- Jennett RB, Sorrell MF, Johnson EL, Tuma DJ: Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain. Arch Biochem Biophys. 1987, 256 (1): 10-18.View ArticlePubMedGoogle Scholar
- Jennett RB, Saffari-Fard A, Sorrell MF, Smith SL, Tuma DJ: Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium. Life Sci. 1989, 45 (16): 1461-1466.View ArticlePubMedGoogle Scholar
- Xu DS, Jennett RB, Smith SL, Sorrell MF, Tuma DJ: Covalent interactions of acetaldehyde with the actin/microfilament system. Alcohol Alcohol. 1989, 24 (4): 281-289.PubMedGoogle Scholar
- Tuma DJ, Newman MR, Donohue TM, Sorrell MF: Covalent binding of acetaldehyde to proteins: participation of lysine residues. Alcohol Clin Exp Res. 1987, 11 (6): 579-584.View ArticlePubMedGoogle Scholar
- Jennett RB, Sorrell MF, Saffari-Fard A, Ockner JL, Tuma DJ: Preferential covalent binding of acetaldehyde to the alpha-chain of purified rat liver tubulin. Hepatology. 1989, 9 (1): 57-62.View ArticlePubMedGoogle Scholar
- Klassen LW, Tuma DJ, Sorrell MF, McDonald TL, DeVasure JM, Thiele GM: Detection of reduced acetaldehyde protein adducts using a unique monoclonal antibody. Alcohol Clin Exp Res. 1994, 18 (1): 164-171.View ArticlePubMedGoogle Scholar
- Braun KP, Cody RB, Jones DR, Peterson CM: A structural assignment for a stable acetaldehyde-lysine adduct. J Biol Chem. 1995, 270 (19): 11263-11266.View ArticlePubMedGoogle Scholar
- Braun KP, Pavlovich JG, Jones DR, Peterson CM: Stable acetaldehyde adducts: structural characterization of acetaldehyde adducts of human hemoglobin N-terminal beta-globin chain peptides. Alcohol Clin Exp Res. 1997, 21 (1): 40-43.PubMedGoogle Scholar
- Lin RC, Shahidi S, Lumeng L: Production of antibodies that recognize the heterogeneity of immunoreactive sites in human hemoglobin chemically modified by acetaldehyde. Alcohol Clin Exp Res. 1993, 17 (4): 882-886.View ArticlePubMedGoogle Scholar
- Gaines KC, Salhany JM, Tuma DJ, Sorrell MF: Reaction of acetaldehyde with human erythrocyte membrane proteins. FEBS Lett. 1977, 75 (1): 115-119.View ArticlePubMedGoogle Scholar
- Peterson CM, Nguyen LB: Clinical implications of acetaldehyde adducts with hemoglobin. Prog Clin Biol Res. 1985, 183: 19-30.PubMedGoogle Scholar
- Niemela O, Israel Y: Hemoglobin-acetaldehyde adducts in human alcohol abusers. Lab Invest. 1992, 67 (2): 246-252.PubMedGoogle Scholar
- Niemela O, Israel Y, Mizoi Y, Fukunaga T, Eriksson CJ: Hemoglobin-acetaldehyde adducts in human volunteers following acute ethanol ingestion. Alcohol Clin Exp Res. 1990, 14 (6): 838-841.View ArticlePubMedGoogle Scholar
- Savolainen MJ, Baraona E, Lieber CS: Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins. Life Sci. 1987, 40 (9): 841-846.View ArticlePubMedGoogle Scholar
- Wickramasinghe SN, Gardner B, Barden G: Cytotoxic protein molecules generated as a consequence of ethanol metabolism in vitro and in vivo. Lancet. 1986, 2 (8511): 823-826.View ArticlePubMedGoogle Scholar
- Behrens UJ, Hoerner M, Lasker JM, Lieber CS: Formation of acetaldehyde adducts with ethanol-inducible P450IIE1 in vivo. Biochem Biophys Res Commun. 1988, 154 (2): 584-590.View ArticlePubMedGoogle Scholar
- Jukkola A, Niemela O: Covalent binding of acetaldehyde to type III collagen. Biochem Biophys Res Commun. 1989, 159 (1): 163-169.View ArticlePubMedGoogle Scholar
- Zhu Y, Fillenwarth MJ, Crabb D, Lumeng L, Lin RC: Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4–3-ketosteroid 5 beta-reductase. Hepatology. 1996, 23 (1): 115-122.PubMedGoogle Scholar
- Parkkila S: An overview of the distribution and function of carbonic anhydrase in mammals. Exs. 2000, 79-93. 90Google Scholar
- Lehtonen J, Shen B, Vihinen M, Casini A, Scozzafava A, Supuran CT, Parkkila AK, Saarnio J, Kivela AJ, Waheed A, et al.: Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family. J Biol Chem. 2004, 279 (4): 2719-2727.View ArticlePubMedGoogle Scholar
- Hu PY, Waheed A, Sly WS: Partial rescue of human carbonic anhydrase II frameshift mutation by ribosomal frameshift. Proc Natl Acad Sci USA. 1995, 92 (6): 2136-2140.PubMed CentralView ArticlePubMedGoogle Scholar
- Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H: Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands. J Histochem Cytochem. 1990, 38 (7): 941-947.View ArticlePubMedGoogle Scholar
- Maren TH: A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther. 1960, 130: 26-29.PubMedGoogle Scholar
- Sundaram V, Rumbolo P, Grubb J, Strisciuglio P, Sly WS: Carbonic anhydrase II deficiency: diagnosis and carrier detection using differential enzyme inhibition and inactivation. Am J Hum Genet. 1986, 38 (2): 125-136.PubMed CentralPubMedGoogle Scholar
- Parkkila S, Parkkila AK, Juvonen T, Rajaniemi H: Distribution of the carbonic anhydrase isoenzymes I, II, and VI in the human alimentary tract. Gut. 1994, 35 (5): 646-650.PubMed CentralView ArticlePubMedGoogle Scholar
- Pan PW, Rodriguez A, Parkkila S: A systematic quantification of carbonic anhydrase transcripts in the mouse digestive system. BMC Mol Biol. 2007, 8: 22-PubMed CentralView ArticlePubMedGoogle Scholar
- Supuran CT: Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov. 2008, 7 (2): 168-181.View ArticlePubMedGoogle Scholar
- Liljas A, Kannan KK, Bergsten PC, Waara I, Fridborg K, Strandberg B, Carlbom U, Jarup L, Lovgren S, Petef M: Crystal structure of human carbonic anhydrase C. Nat New Biol. 1972, 235 (57): 131-137.View ArticlePubMedGoogle Scholar
- Hakansson K, Carlsson M, Svensson LA, Liljas A: Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes. J Mol Biol. 1992, 227 (4): 1192-1204.View ArticlePubMedGoogle Scholar
- Duda D, Govindasamy L, Agbandje-McKenna M, Tu C, Silverman DN, McKenna R: The refined atomic structure of carbonic anhydrase II at 1.05 A resolution: implications of chemical rescue of proton transfer. Acta Crystallogr D Biol Crystallogr. 2003, 59 (Pt 1): 93-104.View ArticlePubMedGoogle Scholar
- Di Fiore A, Scozzafava A, Winum JY, Montero JL, Pedone C, Supuran CT, De Simone G: Carbonic anhydrase inhibitors: binding of an antiglaucoma glycosyl-sulfanilamide derivative to human isoform II and its consequences for the drug design of enzyme inhibitors incorporating sugar moieties. Bioorg Med Chem Lett. 2007, 17 (6): 1726-1731.View ArticlePubMedGoogle Scholar
- Fisher SZ, Maupin CM, Budayova-Spano M, Govindasamy L, Tu C, Agbandje-McKenna M, Silverman DN, Voth GA, McKenna R: Atomic crystal and molecular dynamics simulation structures of human carbonic anhydrase II: insights into the proton transfer mechanism. Biochemistry. 2007, 46 (11): 2930-2937.View ArticlePubMedGoogle Scholar
- Parkkila S, Halsted CH, Villanueva JA, Vaananen HK, Niemela O: Expression of testosterone-dependent enzyme, carbonic anhydrase III, and oxidative stress in experimental alcoholic liver disease. Dig Dis Sci. 1999, 44 (11): 2205-2213.View ArticlePubMedGoogle Scholar
- Nagao Y, Platero JS, Waheed A, Sly WS: Human mitochondrial carbonic anhydrase: cDNA cloning, expression, subcellular localization, and mapping to chromosome 16. Proc Natl Acad Sci USA. 1993, 90 (16): 7623-7627.PubMed CentralView ArticlePubMedGoogle Scholar
- Parkkila S, Kivela AJ, Kaunisto K, Parkkila AK, Hakkola J, Rajaniemi H, Waheed A, Sly WS: The plasma membrane carbonic anhydrase in murine hepatocytes identified as isozyme XIV. BMC Gastroenterol. 2002, 2: 13-PubMed CentralView ArticlePubMedGoogle Scholar
- Eriksson CJ: Determination of hepatic acetaldehyde and its biphasic relationship to the ethanol concentration in rats. Adv Exp Med Biol. 1980, 132: 459-467.PubMedGoogle Scholar
- Matthewson K, Al Mardini H, Bartlett K, Record CO: Impaired acetaldehyde metabolism in patients with non-alcoholic liver disorders. Gut. 1986, 27 (7): 756-764.PubMed CentralView ArticlePubMedGoogle Scholar
- Lindros KO, Stowell A, Pikkarainen P, Salaspuro M: Elevated blood acetaldehyde in alcoholics with accelerated ethanol elimination. Pharmacol Biochem Behav. 1980, 13 (Suppl 1): 119-124.View ArticlePubMedGoogle Scholar
- Eriksson CJ, Fukunaga T: Human blood acetaldehyde (update 1992). Alcohol Alcohol Suppl. 1993, 2: 9-25.PubMedGoogle Scholar
- Eriksson CJ: Measurement of acetaldehyde: what levels occur naturally and in response to alcohol?. Novartis Found Symp. 2007, 285: 247-255. discussion 256–260.View ArticlePubMedGoogle Scholar
- Baraona E, Di Padova C, Tabasco J, Lieber CS: Red blood cells: a new major modality for acetaldehyde transport from liver to other tissues. Life Sci. 1987, 40 (3): 253-258.View ArticlePubMedGoogle Scholar
- Hernandez-Munoz R, Ma XL, Baraona E, Lieber CS: Method of acetaldehyde measurement with minimal artifactual formation in red blood cells and plasma of actively drinking subjects with alcoholism. J Lab Clin Med. 1992, 120 (1): 35-41.PubMedGoogle Scholar
- Niemela O, Parkkila S, Worrall S, Emery PW, Preedy VR: Generation of aldehyde-derived protein modifications in ethanol-exposed heart. Alcohol Clin Exp Res. 2003, 27 (12): 1987-1992.View ArticlePubMedGoogle Scholar
- Niemela O, Parkkila S, Koll M, Preedy VR: Generation of protein adducts with malondialdehyde and acetaldehyde in muscles with predominantly type I or type II fibers in rats exposed to ethanol and the acetaldehyde dehydrogenase inhibitor cyanamide. Am J Clin Nutr. 2002, 76 (3): 668-674.PubMedGoogle Scholar
- Rintala J, Jaatinen P, Parkkila S, Sarviharju M, Kiianmaa K, Hervonen A, Niemela O: Evidence of acetaldehyde-protein adduct formation in rat brain after lifelong consumption of ethanol. Alcohol Alcohol. 2000, 35 (5): 458-463.View ArticlePubMedGoogle Scholar
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