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Immune checkpoint inhibitors are effective in treating a variety of malignancies, including metastatic bladder cancer. A generally accepted hypothesis suggests that immune checkpoint inhibitors induce tumor regressions by reactivating a population of endogenous tumor-infiltrating lymphocytes (TILs) that recognize cancer neoantigens. Although previous studies have identified neoantigen-reactive TILs from several types of cancer, no study to date has shown whether neoantigen-reactive TILs can be found in bladder tumors. To address this, we generated TIL cultures from patients with primary bladder cancer and tested their ability to recognize tumor-specific mutations. We found that CD4 + TILs from one patient recognized mutated C-terminal binding protein 1 in an MHC class II-restricted manner. This finding suggests that neoantigen-reactive TILs reside in bladder cancer, which may help explain the effectiveness of immune checkpoint blockade in this disease and also provides a rationale for the future use of adoptive T cell therapy targeting neoantigens in bladder cancer.

A new N-methyl D aspartate neurotransmitter receptor interacting protein has been identified by yeast two-hybrid screening of a mouse brain cDNA library. C-terminal binding protein 1 (CtBP1) was shown to associate with the intracellular C-terminal regions of the N-methyl D aspartate receptor subunits GluN2A and GluN2D but not with GluN1-1a cytoplasmic C-terminal region. In yeast mating assays using a series of GluN2A C-terminal truncations, it was demonstrated that the CtBP1 binding domain was localized to GluN2A 1157-1382. The GluN2A binding domain was identified to lie within the CtBP1 161-224 region. CtBP1 co-immunoprecipitated with assembled GluN1/GluN2A receptors expressed in mammalian cells and also, in detergent extracts of adult mouse brain. Co-expression of CtBP1 with GluN1/GluN2A resulted in a significant decrease in receptor cell surface expression. The family of C-terminal binding proteins function primarily as transcriptional co-repressors. However, they are also known to modulate intracellular membrane trafficking mechanisms. Thus the results reported herein describe a putative role for CtBP1 in the regulation of cell surface N-methyl D aspartate receptor expression.

Invariant natural killer T (iNKT) cells exhibit potent antitumor effects upon activation by recognizing a specific glycolipid antigen. We previously performed phase I-II clinical studies to utilize iNKT cells using α-galactosylceramide-pulsed dendritic cells and identified leukotriene B4 12-hydroxydehydrogenase (LTB4DH) as a biomarker highly expressed in T cells derived from non-small cell lung cancer (NSCLC) patients who showed prolonged survival in respond to the iNKT cell immunotherapy. Because LTB4DH expression correlated with prolonged survival of NSCLC patients, we considered LTB4DH to play a role in iNKT cell immunotherapy. We herein demonstrate that the overexpression of LTB4DH in CD4 + or CD8 + T cells increases interferon-γ production and tumoricidal activity in the presence of prostaglandin E 2 . Moreover, the expression of granzyme a, granzyme b, and perforin mRNA was increased in LTB4DH-overexpressing cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD + ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD + ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD + ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.Several metabolic factors affect cellular glucose metabolism as well as the innate inflammatory response. Here, the authors show that glucose metabolism regulates pro-inflammatory responses through effects on the cytosolic NADH:NAD+ ratio and the NAD(H)-sensitive transcription co-repressor CtBP.

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Background: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown., Methods: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA)., Results: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH., Conclusions: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.

The cannabinoid receptor 1 (CBR1) is being widely investigated because of its specific structure and functions compared with other cannabinoid receptors. In this study, we immunized BALB/c mice with synthesized human CBR1 polypeptide and obtained a novel monoclonal antibody (MAb) against human CBR1. Analysis through enzyme-linked immunosorbent assay (ELISA), spot-ELISA, Western blot, and immunohistochemistry revealed that the MAb was specifically against recombinant human CBR1 protein, and its subtype and affinity constant (Kaff) were IgG2b/k and 7.85 × 10(8) M/L, respectively. Using this MAb we found that CBR1 is expressed on HL-7702 cells and lipid tissue, raising the possibility that the CBR1 may take a role in glucose and lipid metabolism. Thus, this antibody might facilitate studies for pathophysiology of diseases associated with glucose and lipid metabolism abnormality.

Background: Anti-p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200-kDa protein (p200) of the dermal-epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C-terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti-p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L(-1) salt-split skin by indirect immunofluorescence microscopy and labelling of a 200-kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories., Objectives: To develop a simple, sensitive and specific diagnostic tool for anti-p200 pemphigoid., Methods: Sera from patients with anti-p200 pemphigoid (n = 35), bullous pemphigoid (BP, n = 101), epidermolysis bullosa acquisita (EBA, n = 10), antilaminin 332 mucous membrane pemphigoid (MMP, n = 14), pemphigus vulgaris (PV, n = 51) and healthy volunteers (HV, n = 131) were tested by a novel enzyme-linked immunosorbent assay (ELISA) that employed a recombinant monomeric C-terminal fragment of human laminin γ1 (hLAMC1-cterm) expressed in Escherichia coli., Results: Serum reactivity with hLAMC1-cterm was detected in sera from 24 of 35 (69%) patients with anti-p200 pemphigoid, two of 101 (2%) with BP, 0 of 10 with EBA, two of 14 (14%) with anti-laminin 332 MMP, 0 of 51 with PV, and 0 of 131 HV., Conclusions: This novel ELISA will facilitate the diagnosis of anti-p200 pemphigoid., (© 2010 The Authors. BJD © 2010 British Association of Dermatologists 2010.)

Genetically modified crops have resistance to abiotic stress by introduction of choline oxidase protein. In the present study, the safety of choline oxidase protein derived from Arthrobacter globiformis was assessed for toxicity and allergenicity. The protein was stable at 90 degrees C for 1 h. Toxicity studies of choline oxidase in mice showed no significant difference (p > 0.05) from control in terms of growth, body weight, food consumption, and blood biochemical indices. Histology of gut tissue of mice fed protein showed normal gastric mucosal lining and villi in jejunum and ileum sections. Specific IgE in serum and IL-4 release in splenic culture supernatant were low in choline oxidase treated mice, comparable to control. Intravenous challenge with choline oxidase did not induce any adverse reaction, unlike ovalbumin group mice. Histology of lung tissues from choline oxidase sensitized mice showed normal airways, whereas ovalbumin-sensitized mice showed inflamed airways with eosinophilic infiltration and bronchoconstriction. ELISA carried out with food allergic patients' sera revealed no significant IgE affinity with choline oxidase. Also, choline oxidase did not show any symptoms of toxicity and allergenicity in mice.

Aim: To generate monoclonal antibodies (mAb) against glyoxylate reductase/hydroxypyruvate reductase (GRHPR)., Methods: Normal human liver tissues were homogenized, and human liver cytosolic proteins were isolated by centrifugation. The total human liver cytosolic proteins were used to immunize BALB/c mice to prepare mAb by hybridoma technique. The mAbs were detected by ELISA, Western blot, and immunohistochemistry. The antibody specificities were identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening., Results: One hybridoma cell line, ADB291, secreting specific mAb against GRHPR was established. The Ig subclass of the mAb was IgG1(kappa). Data from immunohistochemistry showed that ADB291 can recognize hepatocyte cytoplasm. ADB291 mAb was used to isolate its protein antigen by IP. Proteins captured by the mAb were loaded to SDS-PAGE and subjected to Western blot and MALDI-TOF MS analysis. lambda expression Uni-ZAP XR pre-made liver cDNA library was screened with ADB291 hybridoma supernatants. All of our data demonstrated that ADB291 mAb specially reacted with GRHPR., Conclusion: A hybridoma cell line stably secreting specific mAb against GRHPR is established. The specific mAb against GRHPR would be very useful for the studies of GRHPR functions and distribution.

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

Background: A number of antigens have been characterized and proposed as potential candidates for immunocontraception. P26h, a 26-kDa hamster sperm protein located on the acrosomal cap, is known to be involved in sperm-zona pellucida interactions. Furthermore, in vivo fertilization can be blocked by active immunization of male hamsters against P26h or maltose-binding protein recombinant P26h (MBP-P26h)., Objective: The aim of this study was to investigate the immune response and reproductive function of female hamsters immunized against MBP-P26h., Results: Active immunization against MBP-P26h resulted in anti-P26h circulating antibodies, with enzyme-linked immunosorbent assay (ELISA) titers showing interindividual variability. The antibodies produced by the animals immunized against MBP-P26h reacted with the native P26h protein in ELISA, in Western blot analysis and in immunostaining performed on cauda epididymal spermatozoa. Mating of immunized female hamsters resulted in a significant decrease in the number of viable fetuses only in females with high titers of anti-P26h circulating antibodies., Discussion: This result is in agreement with the sperm-zona pellucida binding assay's results. Indeed, sera collected from immunized animals, and not from control animals, significantly blocked sperm-zona pellucida binding in vitro. Histological studies showed that active immunization did not cause any pathology in the reproductive tissues., Conclusion: These findings suggest that P26h is a potential candidate for the development of a contraceptive vaccine in both males and females.

The genes for the large subunit of [NiFe] hydrogenase 2, (hybB) and for L-1,2 propanediol oxidoreductase (fucO), were identified in an Actinobacillus (A.) pleuropneumoniae serotype 7 strain. Based on the hypothesis that adaptation to anaerobic conditions in damaged lung tissue may play a role in A. pleuropneumoniae persistence in host tissues, deletion mutants with a deletion in the hybB or the fucO gene were constructed and examined in an aerosol infection model. Deletion of the hybB or fucO genes appeared to have no significant effect on A. pleuropneumoniae virulence.

Despite the various contraceptive methods available, an effective and inexpensive method remains to be established. Immunocontraception may help to achieve this goal. P26h has been proposed as a candidate for the development of a male contraceptive vaccine. P26h, a hamster sperm protein, interacts with the zona pellucida. Furthermore, in vivo fertilization can be blocked completely by active immunization of male hamsters against P26h. Maltose binding protein (MBP)-P26 shares antigenic determinants with the native P26h present on cauda epididymal spermatozoa. The aim of the present study was to reproduce the immunocontraceptive properties of native P26h by immunizing male hamsters against a recombinant P26h fused with a maltose binding protein (MBP). Active immunization of male hamsters with the MBP-P26h showed that specific anti-P26h circulating IgGs could be generated. Mating of immunized male hamsters with superovulated females resulted in a significant decrease, 20-25%, in the fertilization rate. This result is in agreement with results from in vitro sperm-zona pellucida binding assays. Indeed, the anti-recombinant P26h IgGs showed lower inhibitory properties when compared with anti-native P26h IgG. Despite the high anti-P26h IgG titres generated in hamsters, histological studies showed that active immunization has no pathological sequelae to the reproductive tissues. The potential of P26h as a candidate for a contraceptive vaccine is discussed.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter., (Copyright 2001 Academic Press.)

The coding sequences of three single-chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybrida, and the DFR-encoding sequence were cloned in two-hybrid vectors. The vectors were transformed in the yeast strain HF7c (his3-200, trp1-901, leu2-3) and the scFv-DFR interaction was analyzed by measuring yeast growth on medium without histidine. ScFv-G4 and, to a lesser extent, scFv-A4 could interact with DFR in the yeast nucleus. On the contrary, scFv-H3 showed no interaction with its antigen in yeast. The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two-hybrid test. This suggests that it is possible to evaluate the antigen-scFv interaction in a reducing subcellular environment with the two-hybrid test. Therefore, the yeast two-hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serine-glyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

An Aspergillus parasiticus cDNA library was screened with monoclonal antibody raised against a purified A. parasiticus 43-kDa protein demonstrating norsolorinic acid reductase (NOR) activity. One immunopositive clone contained a cDNA insert of 1,418 bp. DNA sequence analysis of this cDNA identified an open reading frame of 1,167 bp that represented the norA gene. The deduced amino acid sequence of the norA coding region consisted of 388 residues capable of encoding a polypeptide of 43.7 kDa. Southern blot analysis of genomic DNA from A. parasiticus indicated that there may be an additional copy of norA. Western blot (immunoblot) analysis of crude protein extracts of A. parasiticus mycelia demonstrated a band of reactivity at 43 kDa only when the fungus was grown in a medium conducive to aflatoxin biosynthesis. Northern (RNA) blot analysis of total RNA from the fungus demonstrated a band of hybridization at about 1.5 kb. As observed with the fungal NORA protein, the norA transcript was present only when the fungus was grown in medium conducive to aflatoxin biosynthesis. Hybridization of the norA cDNA with cosmid DNAs known to encompass a major portion of the A. parasiticus and Aspergillus flavus aflatoxin biosynthetic pathway gene cluster placed the norA gene coding region just upstream of the ver-1 gene. The deduced amino acid sequence of norA had 49% amino acid identity with that of an aryl-alcohol dehydrogenase (aad) gene from Phanerochaete chrysosporium.

The immunocytochemical localization of tetrameric carbonyl reductase in the mouse lung was determined by an electron-microscopical immunogold procedure using monospecific antibodies against the enzyme. The labelling of carbonyl reductase was observed within the mitochondria of the ciliated and non-ciliated cells of the bronchioles and the type II alveolar pneumocytes, and the density of labelling in the non-ciliated cells was higher than those in the other cells. No significant labelling was detected over other compartments of the epithelial cells. The labelling was undetectable in the type I alveolar cells, alveolar macrophages and connective tissue cells of the lung. These results clearly indicate the localization of carbonyl reductase to the mitochondrial matrix of these epithelial cells, of which the non-ciliated bronchiolar cells contained particularly high amounts of the enzyme.

1. The ontogenic pattern of xenobiotic carbonyl reducing activity and glucocorticoid 11 beta-oxidoreducing activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in mouse liver and kidney was examined. In addition, the expression of this enzyme was investigated by means of immunoblot analysis in the same tissues. 2. In liver, the foetus shows low or no enzyme activities. After birth both activities increase dramatically with age and remain then on a high plateau until the time of sexual maturity (4 weeks). After maturity, the enzyme activities decline to intermediate values. The developmental pattern of immunological expression of the liver enzyme corresponds well with that of the enzyme activity. 3. Considerable activities of xenobiotic carbonyl reduction and glucocorticoid 11 beta-oxidoreduction are also present after birth in all developmental stages of the kidney. However, no immunological crossreaction was found in any stages with the antibody against the liver 11 beta-HSD suggesting the presence of a structurally different isozyme form in the kidney. 4. The dramatic increase of both activities during the peri- and postnatal developmental periods suggest a potentially biological significance of the liver 11 beta-HSD isozyme in early animal life. 5. Besides being involved in 11 beta-glucocorticoid metabolism in particular the liver enzyme seems to play an additional role as xenobiotic carbonyl reductase.

3-Deoxyglucosone (3DG) is a reactive intermediate in the glucose-mediated cross-linking of proteins. An enzyme catalyzing the reduction of 3DG is thought to prevent the damage to protein by the formation of 3DG. The NADPH-dependent enzyme activity was detected in the extracts of various monkey tissues, among which kidney exhibited the highest specific activity. One dimeric enzyme with subunit M(r) of 39,000 and two monomeric enzymes with M(r) of 38,000 and 34,000 were purified from monkey kidney. The dimeric enzyme exhibited high dihydrodiol dehydrogenase activity and was immunochemically identical to dimeric dihydrodiol dehydrogenase of monkey kidney. The two monomeric enzymes exhibited aldehyde reductase activity, but were clearly distinct from each other in substrate specificity, inhibitor sensitivity, and effect of sulfate ions. One enzyme was immunologically cross-reacted with human liver aldehyde reductase, whereas sequence data of digested peptides from the other enzyme revealed > 97% identity with human placental aldose reductase. Comparison of kinetic constants among the monkey kidney enzymes and aldoketo reductases from several mammalian tissues indicated that dimeric dihydrodiol dehydrogenase and aldose reductase exhibited higher catalytic efficiency for 3DG than did aldehyde reductase, carbonyl reductase, and monomeric dihydrodiol dehydrogenase.

We report the initial identification of a 37.5-kDa putative aldoketo reductase in human colon carcinoma cells. An aminoterminal trypsin fragment was sequenced and found to be identical to bovine prostaglandin F synthase in 19 of 21 amino acids. Levels of this cytosolic human aldo-keto reductase, assessed by immunoblots using polyclonal antibodies raised against this protein, increased 30-fold in cells resistant to the Michael reaction acceptor ethacrynic acid and increased with time and ethacrynic acid concentration after treatment of wild-type cells. Induction of the reductase appeared to be cell type and drug specific. It was induced by the Michael reaction acceptors dimethyl maleate, t-butylhydroquinone, and hydroquinone but not by the nitrogen mustard chlorambucil. Ethacrynic acid and dimethyl maleate induced the reductase in a second human colon cell line but not in human prostate cells. NADPH-dependent metabolism of aldoketo reductase substrates by cytosol from colon but not prostate cells was enhanced 2-3-fold when cells were grown in the presence of either ethacrynic acid or dimethyl maleate. The discrepancy between induced reductase activity and protein levels may be due to the multiplicity of constitutively expressed NADPH-dependent reductases that compete for substrate. Ethacrynic acid-resistant cells exhibited low levels of cross-resistance to Adriamycin, mitomycin C, and the bovine prostaglandin F synthase substrates phenylglyoxal and prostaglandin D2. Thus, significant overexpression of a human aldo-keto reductase structurally related to bovine prostaglandin F synthase may result from exposure of cells to Michael reaction acceptors and may give rise to an enhanced capacity to metabolize exogenous and endogenous substrates, thereby contributing to the drug-resistant phenotype.

Five yeast strains were isolated by enrichment culture on the basis of their ability to grow on mandelate and two of these strains were identified as Rhodotorula glutinis. In addition, a range of yeasts from culture collections was screened for growth on mandelate. The results suggest that mandelate utilization is a widespread but not universal characteristic within the genus Rhodotorula. Several of the yeasts contained an inducible NAD-dependent D(-)-mandelate dehydrogenase and an inducible dye-linked (presumably flavoprotein) L(+)-mandelate dehydrogenase. All the D(-)-mandelate dehydrogenases from the yeasts showed immunological cross-reactivity with each other (as judged by both immunoinhibition and immunoblotting), as did all the yeast L(+)-mandelate dehydrogenases that were tested. Determination of N-terminal amino acid sequences of several bacterial and yeast lactate and mandelate dehydrogenases, together with the evidence from the immunological studies, confirmed and extended previous proposals that there are several major groups of such dehydrogenases: FMN-dependent, membrane-bound L(+)-lactate and L(+)-mandelate dehydrogenases (M(r) = approx. 44,000) in bacteria, mitochondrial flavocytochrome b2 L(+)-lactate and L(+)-mandelate dehydrogenases (M(r) = approx. 59,000) in yeasts, FAD-dependent, membrane-bound D(-)-lactate and D(-)-mandelate dehydrogenases in bacteria, and soluble NAD-dependent D(-)-mandelate dehydrogenases in both bacteria and yeasts.

We studied the properties of retinol dehydrogenase (11-cis-specific) from bovine retinal pigment epithelium. Detergents caused a loss of retinol dehydrogenase activity; therefore, we added 3 mM NADH as a stabilizer to solubilize this enzyme and partially purified this enzyme using Sepharose CL-6B and hydroxyapatite column chromatography. The partially-purified sample, which contained two major proteins (66 kDa, 33 kDa), had substrate preference to 11-cis and 13-cis-retinal but not to all-trans and 9-cis isomers. Monoclonal anti-33 kDa protein of retinal pigment epithelial crude extract by Western blotting. In addition, we found that monoclonal anti-retinol dehydrogenase antibody bound specifically to retinal pigment epithelium and not to Müller cells or to rod outer segments by immunohistochemical methods.

A new immunization method has been developed for the production of monoclonal antibodies. This technique uses small amounts of partially purified and weak immunogenic antigen, bound to membranes after blotting from SDS-PAGE. For this purpose two different membranes have been tested. Immobilon-P polyvinylidene difluoride (PVDF) membranes were less mitogenic than nitrocellulose membranes, and were therefore selected for the in vitro immunization using 6-pyruvoyl tetrahydropterin synthase as antigen. The in vitro immunization method was then used for the production of monoclonal antibodies against 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes on the biosynthetic pathway of tetrahydrobiopterin, the natural cofactor of the mammalian aromatic amino acid hydroxylases. The antibodies obtained were mainly of the IgM type.

A specific monoclonal antibody prepared for the 29-kDa a subunit of silkworm fat body sepiapterin reductase (SPR) was able to recognize the subunit in crude extract of fat body after SDS treatment. Although SPR from the silkworm fat body has biochemical properties similar to those reported for SPR from mammalian sources, especially rat erythrocytes, the antibody failed to recognize the 28-kDa subunit of rat erythrocyte SPR. This result indicates that SPR from silkworm fat body has a different amino-acid sequence from that of the rat erythrocyte enzyme. Sepiapterin reductase activity has not been found in crude extract of fat body from the silkworm mutant lemon. Although the antibody recognized only 29-kDa protein in the crude extract of silkworm fat body from normal strain after SDS-treatment, the antibody recognized only an approximately 80-kDa protein in the crude extract of the lemon mutant after SDS-treatment.

3 alpha-Hydroxysteroid dehydrogenase is a cytosolic, monomeric, NADPH-dependent oxidoreductase which reduces 3-keto-5-dihydrosteroids to their tetrahydro products. We present here the first partial amino acid sequence data for the human liver enzyme and show these sequences to be identical to the deduced amino acid sequence for human hepatic chlordecone reductase. In addition, these two enzymes exhibit similar substrate and cofactor specificities and immunological reactivity. The results suggest that the natural substrates for chlordecone reductase are 3-keto-5-dihydrosteroids and that these two proteins may be identical.

Carbonyl reductase (EC 1.1.1.184) is a cytosolic, monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds and a general distribution in human tissues. A carbonyl reductase closely resembling the human enzyme is exclusively expressed in rat reproductive tissues and adrenals (Iwata, N., Inazu, N. and Satoh, T. (1989) J. Biochem. 105, 556-564). In order to investigate the relationship between the human and rat enzyme, carbonyl reductase from human testis was purified to homogeneity. The enzyme was indistinguishable from carbonyl reductase from other human tissues on the basis of physicochemical properties, substrate specificity, inhibitor sensitivity and immunological reactivity. Likewise, the human and rat testis enzymes exhibited greatly overlapping substrate specificities for prostaglandins, steroids as well as many xenobiotic carbonyl compounds, and showed the same susceptibility to inhibition by flavonoids and sulfhydryl-blocking agents. Structural homology between the two enzymes was indicated by the mutual cross-reactivity of antibodies against carbonyl reductase from one species and the enzyme protein from the other species. Unlike the rat enzyme, which is confined to Leydig cells, the human enzyme was detectable in Leydig cells as well as Sertoli and spermatogenic cells.

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

The F420-dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP(+)-dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420-dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42 degrees C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP(+)-dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60 degrees C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl. The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP(+)-dependent ADH of Mb. palustre contained 4-8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4-hydroxymercuribenzoate, whereas the F420-dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10-phenanthroline or high concentrations (e.g. 100 microM) of 4-hydroxymercuribenzoate. Polyclonal antibodies against the NADP(+)-dependent ADH of Mb. palustre precipitated only the homologous ADH. A precipitation of the NADP(+)-dependent ADH of Methanocorpusculum parvum required a 10-fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP(+)-dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP(+)-dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420-dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP(+)-dependent ADHs. No immunological relation of the NADP(+)- or F420-dependent ADHs of methanogens with ADH of yeast or horse liver was found. In accordance with the immunological data, the N-terminal amino acid sequences of the NADP(+)-dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N-terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP(+)-dependent enzymes.

1. N-Terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus N.C.I.B. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53 and yeast K(+)-activated aldehyde dehydrogenase. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50161 (5 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby. West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273. 5. The extent of sequence similarity suggests that the benzyl alcohol dehydrogenases are related to each other and also to established members of the family of long-chain Zn2(+)-dependent alcohol dehydrogenases. Benzaldehyde dehydrogenase II from Acinetobacter appears to be related to the Pseudomonas TOL-plasmid-encoded benzaldehyde dehydrogenase. The yeast K(+)-activated aldehyde dehydrogenase has similarity of sequence with the mammalian liver cytoplasmic class of aldehyde dehydrogenases but not with any of the Acinetobacter or Pseudomonas enzymes. 2. Antisera were raised in rabbits against the three Acinetobacter enzymes and both of the Pseudomonas enzymes, and the extents of the cross-reactions were determined by immunoprecipitation assays with native antigens and by immunoblotting with SDS-denatured antigens. Cross-reactions were detected between the alcohol dehydrogenases and also among the aldehyde dehydrogenases. This confirms the interpretation of the N-terminal sequence comparisons and also indicates that benzaldehyde dehydrogenase I from Acinetobacter may be related to the other two benzaldehyde dehydrogenases. 3. The amino acid compositions of the Acinetobacter and the Pseudomonas enzymes were determined and the numbers of amino acid residues per subunit were calculated to be: benzyl alcohol dehydrogenase and TOL-plasmid-encoded benzyl alcohol dehydrogenase, 381; benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II, 525; TOL-plasmid-encoded benzaldehyde dehydrogenase, 538.

The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp.

Three enzyme forms (T1, T2, T3) from rat testis and two from rat vas deferens (V1, V2) of carbonyl reductase have been highly purified to apparent homogeneity. These carbonyl reductases from rat reproductive organs have several similarities in terms of molecular mass (32-33 kDa), isoelectric point (pI 5.9-6.4), immunochemical properties, cofactor requirement (NADPH dependency) and sensitivity to sulfhydryl reagents. The isoenzymes from the vas deferens (V1, V2) have similar catalytic activities, whereas those from the testis (T1, T2, T3) showed different catalytic activities from each other. All enzymes, however, reduced quinones, aromatic aldehydes and ketones, while T3, V1 and V2 were characterized as possessing high affinity towards prostaglandins. An immunoinhibition study using a specific antibody indicated that these enzymes were solely responsible for the overall catalytic activities of 13, 14-dihydro-15-oxo-prostaglandin F2 alpha, 4-benzoylpyridine, and 4-nitroacetophenone reduction and prostaglandin F2 alpha oxidation in both testis and vas deferens cytosol. The immunohistochemical staining revealed a positive immunoreactivity to antibody only in the Leydig cells of the testis, but neither the germ cells nor Sertoli cells in the seminiferous tubule. The staining also showed that the enzymes in the vas deferens were primarily localized in mucosal epithelium cells.

The isozyme A of L-2-hydroxyacid oxidase is a peroxisomal flavoenzyme that catalyzes the oxidation of short-chain aliphatic L-2-hydroxyacids in many tissues of higher organisms. A new purification procedure allowed us to obtain a 1400-fold purified enzyme from chicken liver. The N-terminal amino acid of the polypeptide chain was found to be blocked as that of spinach glycolate oxidase, contrastingly with that of rat kidney isozyme B. Its amino acid composition was comparable to that of other known L-2-hydroxyacid oxidases. Despite different substrate specificity, some immunological identity was observed between chicken liver L-2-hydroxyacid isozyme A and rat kidney isozyme B.

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.

A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate 'leaked' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.

We have cloned a full-length cDNA coding for human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using a rabbit antibody against human ADH as a first probe, by the modified method of Young and Davis [Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198]. Mixed 14-mer synthetic oligonucleotides encoding Asp-Asp-His-Val-Val and Gln-Cys-Gly-Lys-Cys were used as a second probe. These amino acid sequences are considered to be common in all three subunits (alpha, beta, and gamma) controlled by the ADH1, ADH2, and ADH3 loci. Ten lambda gt11 recombinants of 35 positive plaques obtained by antibody screening contained inserted cDNAs of 1.5-2.4 kilobase pairs and were found to exhibit positive signals by hybridization with synthetic probes. One of them, with an inserted cDNA of 1631 base pairs, contained a sequence that encodes 374 amino acid residues of the human beta 1 subunit, a chain initiation codon, a chain termination codon, and additional 3' and 5' untranslated regions. A complete amino acid sequence of the human beta 1 subunit was deduced from the cDNA.

D-Malate dehydrogenase (D-malate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.83) was purified to homogeneity from Pseudomonas fluorescens UK-1 grown on D-malate and some properties of the purified enzyme were compared with those of beta, beta-dimethylmalate dehydrogenase (3,3-dimethyl-D-malate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.84). D-Malate dehydrogenase has the molecular weight and subunit size of 140 000 and 34 000, respectively (the same as those of beta, beta-dimethylmalate dehydrogenase). The amino acid compositions of the two enzymes are similar as well. D-Malate dehydrogenase cross-reacted with anti-beta, beta-dimethylmalate dehydrogenase and beta, beta-dimethylmalate dehydrogenase cross-reacted with anti-D-malate dehydrogenase. The two dehydrogenases have similar catalytical properties. Both of the dehydrogenases were unaffected by sulphydryl reagents but were inactivated by 1,2-butanedione. NAD provided better protection against inactivation than D-malate or beta,beta-dimethyl-DL-malate.

Urea inhibits the activity of histidinol dehydrogenase from Escherichia coli B, prevents precipitation of the enzyme by specific antibodies and dissolves immunoprecipitates which were formed in the absence of urea. However, immunoadsorption of the enzyme to Sepharose-bound antibodies can take place in the presence of high concentrations (5-8 M) of urea. The immobilized antibody-bound enzyme exhibits almost full activity after removal of the urea and is inhibited by soluble specific antibodies in the presence of urea. The possibility of using immunoadsorption in the presence of urea for the study of insoluble proteins is discussed.

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.

Prostaglandin E 9-ketoreductase, NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type I), and NADP+-dependent 15-hydroxyprostaglandin dehydrogenase (type II) have been partially purified from swine renal medulla and cortex. Eleven times more NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity was found in the cortex than in the medulla. On the other hand, about twice as much NADP+-dependent dehydrogenase activity was found in the medulla than in the cortex. The prostaglandin 9-ketoreductase activities were equally distributed in the swine kidney cortex and medulla.

Through the use of specific immunoadsorbent columns, it is shown that Escherichia coli aspartokinase I-homoserine dehydrogenase I, aspartokinase II-homoserine dehydrogenase II, aspartokinase III, and homoserine kinase, enzymes involved in the same complex biosynthetic pathway, share antigenic determinants. This raises the question of a common origin for the four cibtenoirart kinases. (Aspartate kinase or ATP:L aspartate 4-phosphotransferase, EC 2.7.2.4; homoserine dehydrogenase or Lhomoserine:NADP oxidoreductase, EC 1.1.1.3; homoserine kinase or ATP:L-homoserine O-phosphotransferase, EC 2.7.1.39.)

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.

Monospecific rabbit antibodies against purified Fischer-344 rat liver alcohol dehydrogenase were produced and used to develop an enzyme-linked immunoadsorbent assay for alcohol dehydrogenase. The assay is based upon the competitive inhibition of specific antibody binding to antigen (alcohol dehydrogenase adsorbed onto plastic microtiter plates) by soluble alcohol dehydrogenase (contained in unknown sample extracts or in known standard solutions). The amount of bound antibody is determined following incubation with peroxidase-linked second antibody (goat anti-rabbit IgG antibody-peroxidase conjugate) by colorimetric measurements of peroxidase activity at 490 nm in the presence of O-phenylenediamine. The assay is highly sensitive (it detects 10-1000 ng alcohol dehydrogenase/50 microliter) and it offers a precise (interexperimental variations in samples were less than 10%), rapid (6-8 h), and specific method for measurements of alcohol dehydrogenase in tissue homogenates or cultured hepatocytes. The assay was used to study changes in alcohol dehydrogenase levels during the growth cycle of cultured hepatocytes over a 2-week period and in rat liver homogenates after starving the animals for 72 h. In cultured hepatocytes, alcohol dehydrogenase activity and immunoassayable enzyme levels decreased coordinately during lag and early log phase, from 13.2 +/- 1.2 to 5.0 +/- 1.0 micrograms enzyme/mg protein, respectively. In mid-log phase, the enzyme levels were very low (1.3 +/- 0.4 micrograms enzyme/mg protein). During stationary phase, the levels (5.7 +/- 0.6 micrograms enzyme/mg protein) increased to 35% of the levels of freshly isolated hepatocytes (15.6 +/- 1.4 micrograms enzyme/mg protein). In starved animals, the enzyme levels decreased from 7.56 +/- 0.55 to 2.97 +/- 0.27 mg enzyme/liver. These changes also coincided with decreases in activity from 8.84 +/- 0.35 to 6.56 +/- 0.68 microM/min/liver.

The extent to which treatment of horse liver alcohol dehydrogenase (ADH) by procedures known to disturb the Zn association induced conformation changes detectable by immunological techniques has been investigated. Treatment of ADH by sodium dodecyl sulphate or by total reduction and carboxymethylation leads to complete loss of reactivity with a rabbit immune serum against the native enzyme. After selective carboxymethylation the enzymatic activity was reduced but the preparation had the same immunological activity of the native enzyme. Similar results were obtained when ADH was treated with reagents known to react with the "functional" Zn atoms, such as sodium dietyldithiocarbamate, 1,10 phenanthroline, and 2,2' bipyridine. In contrast dialysis in 0.01M phosphate buffer containing 0.1mM EDTA removing the "structural" Zn atoms leads to a parallel decrease of enzymatic and immunological activity. Thus loss of "structural" Zn atoms affects the immune reactivity of ADH differently from the loss of "functional" Zn atoms.

Chemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78,000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3 s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.