Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes

Previous analysis of a chimeric enzyme mBEII-IBspHI, in which the C-terminal 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) are replaced by the corresponding 284 amino acids of isoform I (mBEI), suggested that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference. In the present study, additional hybrids of mBEI and mBEII were generated and expressed in Escherichia coli BL21 (DE3) to dissect the structure/function relationships of the C-terminal regions of maize branching enzymes. A truncated form of purified mBEII-IBspHI, which lacks the C-terminal 58 amino acids, retained similar levels of V(max) in branching activity, K(m) for reduced amylose AS 320, and substrate preference for amylose than amylopectin when compared to mBEII-IBspHI. This indicates that the C-terminal extension derived from mBEI is not required for either catalysis or substrate preference. However, deletion of an additional 87 amino acids from the carboxyl terminus resulted in complete loss of activity. Replacement of the deleted C-terminal additional 87 amino acids with the corresponding 79 amino acids from mBEII restored 25% of the mBEII-IBspHI branching activity without altering substrate preference. It thus appears that a C-terminal region encompassing Leu649-Asp735 of mBEII-IBspHI is required for maximum catalytic efficiency. Another C-terminal region, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination.     

Threonine 11 of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase may interact with NAD(+) during catalysis

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH.     

Induction of NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase expression by androgens in human prostate cancer cells

Prostate cancer cells are known to express cyclooxygenases (COXs) and synthesize prostaglandins. Catabolism of prostaglandins in these cells remains to be determined. Induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key metabolic inactivation enzyme, was investigated in androgen-sensitive LNCaP cells and in hormone-independent PC3 cells. 15-PGDH was found to be induced by dihydrotestosterone or testosterone in a time- and dose-dependent manner in LNCaP but not in PC3 cells as shown by activity assay and immunoblot analysis. However, prostaglandin synthetic enzymes, COX-1 and COX-2, were not found to be induced by androgens. Induction was also achieved by 17beta-estradiol and progesterone, although to a lesser extent. Induction of 15-PGDH was not blocked by steroid receptor antagonist, RU 486, nor by antiandrogen, flutamide. However, induction was inhibited by tyrosine kinase inhibitor, genistein, and by ERK kinase inhibitor, PD 98059, but not by protein kinase C inhibitor, GF109203X. These results suggest that androgens induce 15-PGDH gene expression through an unconventional nongenomic pathway.     

Deletion mutagenesis using an 'M13 splint': the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl adenylate.

The X-ray crystallographic structure of tyrosyl-tRNA synthetase (TyrTS) comprises only the N-terminal 320 amino acids of the molecule as the C-terminal 99 amino acids are poorly ordered in the crystal. A new technique, employing a single-stranded M13 splint, has been used to direct a deletion in the cloned gene of TyrTS so as to remove the disordered C-terminal region. We find that the truncated enzyme catalyses the formation of tyrosyl adenylate with unchanged Kcat and Km values and the crystallographic model must therefore include all the binding and catalytic residues involved in tyrosine activation. However, the truncated enzyme no longer binds tRNATyr or transfers tyrosine to tRNATyr. This indicates that the structural division of TyrTS is equally a functional one: the N-terminal structural domain catalyses tyrosine activation while the disordered C-terminal domain carries major determinants in tRNA binding.     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

The role of Leu-190 in the function and stability of adenylate kinase

To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.     

Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase

The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD+-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11-cis-13-trans-eicosatetraenoic acid (15-KETE). The Km for 15-HETE was 16 microM, which was 2.5 times lower than the value obtained for PGE1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with Km values of 138 and 178 microM, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD+. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a Km of 12 microM. These data indicate that omega 6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD+-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.     

Understanding human 15-hydroxyprostaglandin dehydrogenase binding with NAD+ and PGE2 by homology modeling, docking and molecular dynamics simulation

Homology modeling, molecular docking, and molecular dynamics simulation have been performed to determine human 15-hydroxyprostaglandin dehydrogenase (15-PGDH) binding with its NAD+ cofactor and prostaglandin E2 (PGE2) substrate. The computational studies have led to a three-dimensional (3D) model of the entire 15-PGDH-NAD+-PGE2 complex, demonstrating the detailed binding of PGE2 with 15-PGDH for the first time. This 3D model shows specific interactions of the protein with the cofactor and substrate in qualitative agreement with available experimental data. Our model demonstrates the PGE2-binding cavity of the protein for the first time. The model further leads to an interesting prediction that the catalytic activity of 15-PGDH should also significantly be affected by Gln148, in addition to the previously known three catalytic residues (Ser138, Tyr151, and Lys155). The reported 3D model of 15-PGDH-NAD+-PGE2 complex might be valuable for future rational design of novel inhibitors of 15-PGDH.     

Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

The purification and properties of NADPH-dependent carbonyl reductases from rat ovary

Two carbonyl reductases have been highly purified from rat ovary to apparent homogeneity. Though they have similarities in terms of molecular weight (33,000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2 alpha. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13,14-dihydro-15-keto-prostaglandin F2 alpha. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13,14-dihydro-15-keto-prostaglandin F2 alpha reduction and prostaglandin F2 alpha oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.     

Experimental hyperthyroidism in rats suppresses in vitro prostaglandin metabolism in lung and kidney.

Metabolism of prostaglandin (PG) F2alpha and PGE2 was depressed 40--62% in 100,000 g cytoplasmic supernatants of lungs and kidneys prepared from rats made hyperthyroid by 18 daily L(-) thyroxine injections (200microgram, s--c). These hyperthyroid rats had elevated serum thyroxine levels, cardiac hypertrophy and thyroid atrophy. There were no differences in soluble protein concentrations, NAD+ utilisation by endogenous enzymes and substrates, or in the NAD+ dependence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) between the supernatants prepared from hyperthyroid rats and saline-injected controls. Thyroxine did not inhibit PG metabolism in vitro up to 260 micrometer. These results suggest that thyroxine specifically decreases intracellular levels of PG-metabolising enzymes, especially of the rate-limiting 15-PGDH. Metabolism of PGF2alpha and PGE2 by 15-PGDH was faster in smaller rats and declined with increasing animal weight. These studies imply that some of the clinical features of hyperthyroidism in man might be caused by deficiencies in PG metabolism.     

Novel prostaglandin dehydrogenase in rat skin.

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.