Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

The β-bungarotoxin-binding protein from chick brain: binding sites for different neuronal K channel ligands co-fractionate upon partial purification

β-Bungarotoxin (β-Butx) is a presynaptically active neurotoxin which blocks neuronal A-type K⁺ channels. Here, the efficient solubilisation and about 300-fold purification of the β-Butx-binding protein from chick brain were achieved by detergent extraction at high ionic strength followed by chromatography on DEAE Affigel Blue, β-Butx Affigel 102 and wheat germ agglutinin Sepharose. Binding of ¹²⁵I-labelled β-Butx to the purified protein was inhibited by two other K⁺ channel ligands, dendrotoxin I and mast cell-degranulating peptide. It is concluded that the β-Butx-binding protein is a member of a family of voltage-gated K⁺ channels which exhibit varying affinities for different polypeptide ligands.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

Characterization of the alpha-Amylases Synthesized by Aleurone Layers of Himalaya Barley in Response to Gibberellic Acid

The gibberellic acid (GA₃)-induced α-amylases from the aleurone layers of Himalaya barley (Hordeum vulgare L. cv Himalaya) have been purified by cycloheptaamylose-Sepharose affinity chromatography and fractionated by DEAE-cellulose chromatography. Four fractions (α-amylases 1-4) were obtained which fell into two groups (A and B) on the basis of a number of characteristics. Major differences in serological characteristics and in proteolytic fingerprints were found between group A (α-amylases 1 and 2) and group B (α-amylases 3 and 4). Also, the lag time for appearance of group B enzyme activity was longer than for group A, and the appearance of group B required higher GA₃ levels than group A. The components of each group behaved similarly, although differences in proteolytic fingerprints were detected.

These results together with those from other studies indicate that GA₃ differentially controls the expression of two α-amylase genes or groups of genes giving rise to two groups of α-amylases with many different properties.

     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.     

A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase

An extracellular β-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS–PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of β-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 °C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0–9.0 and at 55 °C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K_m of 4.3 mM for p-nitrophenol-β-d-xylopyranoside and was competitively inhibited by xylose with a K_i value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of β-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added β-xylosidase were about 1.5–1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a β-xylosidase from Paecilomyces thermophila.     

Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Alzheimer's disease-linked mutations in presenilin-1 result in a drastic loss of activity in purified γ-secretase complexes

Background

Mutations linked to early onset, familial forms of Alzheimer''s disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1–42/Aβ1–40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1–42 production, decreasing Aβ1–40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.

Methodology/Principal Findings

We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1–40, Aβ1–42 and APP intracellular domain productions in vitro.

Conclusion/Significance

Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1–42/Aβ1–40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer''s disease.     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Purification of DNA-origami nanostructures by rate-zonal centrifugation

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.     

Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields

Background

p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca²⁺-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood.

Methodology/Principal Findings

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca²⁺ wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine.

Conclusion

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.     

Extraction and purification of phycocyanin from Calothrix sp.

The extraction and purification of phycocyanin from Calothrix sp., cyanobacteria isolated from rice fields in Cuernavaca, Morelos, Mexico is described. Phycocyanin was extracted with 2 mg of lysozyme/g wet biomass, and purified by anion chromatography using Q-Sepharose fast-flow (Pharmacia^®, 1.5 cm×10 cm) column and hydrophobic interaction chromatography with methyl macro-prep (Bio-Rad^®, 1.5 cm×20 cm) column. The purified protein showed a pI of 5.2 and has two subunits with apparent molecular mass of 21–17 kDa each. The estimated molecular mass of native purified phycocyanin was 114 kDa, suggesting a stereochemistry of (β)₃.     

Organization of 3β-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358–1364] of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and cytochrome P450_scc (cyt. P450_scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3β-HSD and cyt. P450_scc is observed during the purification of 3β-HSD from mitochondria. The behaviour of 3β-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450_scc. Immunoprecipitations from mitochondria with either anti-cyt. P450_scc or anti 3β-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 μM between 3β-HSD and P450_scc was obtained. These observations suggest that P450_scc and 3β-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.     

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells: purification, characterization and subcellular localization

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells was purified to homogeneity as judged by SDS-PAGE and silver staining of a single 32 kD band. A second, more hydrophobic product of the purification protocol contained additional bands at 45 and 80 kD. The 17β-estradiol dehydrogenase activities of both products exceeded those for 17-one reduction by more than 260-fold. Activities of 3-, 3β- and 20-dehydrogenases were absent in either fraction. Polyclonal and monoclonal antibodies raised against the 32 kD protein and the more hydrophobic product precipitated the enzymatic activity and reacted with the 32 and 80 kD bands, but not with the 45 kD band in Western blots. The subcellular localization of the enzyme was studied in sections of intact cells and of isolated organelles using gold sol coated with F(ab′)₂ fragments of monoclonal antibody F1. Gold particles were found exclusively over cytoplasmic vesicles of 120–150 nm diameter with electron-dense contents.     

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH₄)₂SO₄ and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem. 250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res. 9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.     

Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase

α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp⁶⁶⁶), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys⁴⁹⁹ and Cys⁵⁸⁷ is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.     

Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor

The filamentous fungus Aspergillus versicolor produced large amounts of mycelial β-xylosidase activity when grown on xylan or xylose as the only carbon source. The presence of glucose drastically decreased the level of β-xylosidase activity, while cycloheximide prevented the induction of the enzymes by xylan or xylose. The β-xylosidases induced by xylose or xylan were purified by a simple protocol involving DEAE-cellulose chromatography and ammonium sulphate precipitation. The purified enzymes were acidic proteins, with carbohydrate contents of 21% for that induced by xylose, and 47% for that induced by xylan. Their apparent molecular masses, estimated by gel filtration, and optimal temperatures for β-xylosidase activities, were about 60 and 100 kDa, and 40 and 45 °C, respectively, for the enzymes induced by xylose and xylan. Xylose-induced β-xylosidase exhibited an optimum pH of 6.0, while that of the xylan-induced enzyme was 5.5. Both purified β-xylosidases exhibited also β-galactosidase, β-glucosidase and -arabinosidase activities. In addition to synthetic substrates, the enzymes hydrolysed xylobiose and xylotriose, suggesting a physiological role. K_M values for p-nitrophenyl β- -xylopyranoside were 0.32 mM, for the xylose-induced β-xylosidase, and 0.19 mM for the xylan-induced one. Xylose competitively inhibited both β-xylosidases, with K_I values of 5.3 and 2.0 mM, for the enzymes induced by xylose or xylan, respectively.     

Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms

Background

Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.

Methodology/Principal Findings

In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.

Conclusion/Significance

Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Expression of human 17β-hydroxysteroid dehydrogenase in mammalian cells

The insert of 1278 bp containing the entire coding region of cDNA encoding human 17β-hydroxysteroid dehydrogenase (17β-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17β-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3β,17β-diol are interconverted at a lower rate. The present data suggest the existence of two 17β-HSDs.