Carbonyl Reductases and Pluripotent Hydroxysteroid Dehydrogenases of the Short-Chain Dehydrogenase/Reductase Superfamily

Dissertation / Doktorarbeit von Frank Hoffmann

Introduction:

Metabolic reduction is the counterpart to oxidative pathways and plays an important role in the phase-I metabolism of carbonyl group bearing substances. Carbonyl reduction means the formation of a hydroxy group from a reactive aldehyde or ketone moiety and is generally regarded as an inactivation or detoxification step since the resulting alcohol is easier to conjugate and to eliminate. Not only are these carbonyl-containing compounds widespread in the environment and enter the body as xenobiotics and environmental pollutants, but they can also be generated endogenously through normal catabolic oxidation and deamination reactions. Many endogenous compounds such as biogenic amines, steroids, prostaglandins and other hormones are metabolized through carbonyl intermediates. In addition, lipid peroxidation within the cell results in the production of reactive carbonyls such as acrolein, 4-hydroxynonenal, 4-oxononenal and malon-dialdehyde, while oxidative damage to DNA generates base propenals. Dietary sources of carbonyl-containing compounds are diverse and include aldehydes found in fruits as well as the breakdown product of ethanol, acetaldehyde. Pharmacologic drugs represent further sources of exposure to carbonyl-containing compounds.

From the pharmacologist’s point of view, carbonyl reduction has been shown to be of significance in various inactivation processes of drugs bearing a carbonyl group. On the other hand, the carbinols formed may retain therapeutic potency, thus prolonging the pharmacodynamic effect of the parent drug, or, in some instances, a compound gains activity through carbonyl reduction.

From the toxicologist’s point of view, carbonyl reduction plays an important role in the toxification of drugs such as daunorubicin and doxorubicin (cf. chapter 4), whereas numerous reports corroborate the concept of carbonyl-reducing enzymes being involved in detoxification processes of endogenous and xenobiotic reactive carbonyl compounds.

Compared with the oxidative cytochrome P450 (CYP) system, carbonyl-reducing enzymes had, for a long time, received considerably less attention. However, the advancement of carbonyl reductase molecular biology has allowed the identification and characterization of several carbonyl-reducing enzymes, including pluripotent hydroxysteroid dehydrogenases that are involved in xenobiotic carbonyl compound metabolism, in addition to catalyzing the oxidoreduction of their physiologic steroid substrates.

Table of Contents:

Text Sample:

Chapter III.8, Detection of Overexpressed Proteins:

In order to control the purity of the enzyme preparations or to perform Western blot analysis, polyacrylamide gel electrophoresis under denaturing conditions was performed to separate proteins according to their molecular weights. According to their different amino acid (aa) composition proteins carry different electric charges. SDS incorporates in constant rates into the proteins masking their individual charge from their aa-composition. After SDS-treatment the proteins differ only in their molecular mass and move in the constant electric field towards the anode.

Preparation of the gel:

The electrophoresis was employed using the BioRad electrophoresis apparatus Mini Protean II cell (BioRad, München, Germany). The gels (80 mm × 65 mm × 0,75 mm) composition is indicated in ‘materials’. Cleaning of the glass plates was performed using 70% ethanol. Assembling of the electrophoreses apparatus was carried out according to the manufacturers instructions. For the vertical gel electrophoresis first the resolving gel was prepared. Polymerization of the solution is started by APS and TEMED which therefore should be added immediate before the solution is filled between the glass plates. After polymerization the stacking gel was prepared and filled in. In a final step the comb is placed in the glass plates.

To obtain a resolving of proteins between 20 and 70 kDa a 10% acrylamide/bisacrylamide gel was prepared.

Electrophoresis:

Before electrophoresis the protein samples were diluted 1:2 with protein loading buffer and denatured for 5 min at 95oC. Subsequently, 3-20 µl of the samples were loaded to the gel. The electrophoresis chamber was completely filled with Tris/glycine-electrophoresis buffer. The electrophoresis was started with a voltage of 80 V until the bromphenol blue marker reached the top of the separating gel then performed at 150 V until the tracking dye reached the bottom of the separating gel. As molecular weights standards in SDS-PAGE different Molecular Weight Astandards have been used.

After electrophoresis, the proteins were visualized by staining the gel at room temperature for several hours in a solution of Coomassie Brillant blue R 250. Destaining was carried out at room temperature in a mixture of water/isopropanol/acetic acid (81:12:7).

Determination of Protein Concentrations:

Protein concentrations were determined with the BioRad Protein Microassay, a dye-binding assay based on the differential colour change of a dye in response to various concentrations of protein (Bradford, 1976). The principle of this method is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brillant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Each time the assay was performed, a standard curve was prepared using bovine serum albumin as protein standard (0-10 mg ml-1); A595 was corrected for the blank. 0.8 ml of appropriately diluted samples or bovine serum albumin were mixed with 0.2 ml dye protein reagent and the absorbance of the solution at 595 nm was measured at room temperature with a spectrophotometer (Kontron) after 10 minutes.

Protein Immunodetection by Western blot Analysis:

After electrophoresis, the protein samples were transferred from an unstained SDS-polyacrylamide gel to polyvinylidene fluoride membrane (ProBlott PVDF-Membran, Applied Biosystems, Weiterstadt, Germany) using a Semy-dry transfer system (Trans-Blot SD, BioRad Laboratories, München, Germany). The transfer was performed using 3 tiers of whatman-paper saturated with cathode-buffer, the protein gel, a Hybond-C membrane saturated with anode-buffer and additional 6 tiers of whatman paper saturated with anode-buffer. Protein transfer could be monitored using a prestaned protein standard. The Tris/glycine buffer system was used as transfer buffer, as indicated in ‘Materials’.

The electroblotting was performed applying a constant current of 0.8 mA/cm2 for 2 h. Following the electrotransfer of proteins, the membrane was blocked for at least 30 min with 5% (w/v) dried milk in PBST with gentle shaking at room temperature. After washing the membrane for 3 times with PBST the membrane was then incubated at 40C over night with 1:10.000 dilutions of a rabbit anti-3α-HSD/CR antibody in 2.5 % (w/v) dried milk in PBST. After washing 3 × 10 min with PBST, in order to remove the unbound primary antibody, the membrane was incubated for 2 h in 2.5 % (w/v) dried milk in PBST with 1:20000 dilutions of alkaline phosphatase-conjugated anti- (rabbit IgG) antibody. Excess of conjugates was removed by washing the membrane 3 × 10 min with PBST. Immunodetection was performed with the protocol for the ECF detection kit provided by Amersham Biosciences using the Kodak X-OMAT filmsto detect the light emitted during the reaction of the substrate ECF with the alkaline phosphatase-linked to the secondary antibody.

To detect the His-tag conjugated to the 3α-HSD/CR-protein anti-His-antibody, mouse made, (Amersham Biosciences) was used in 1:5000 dilution. The second antibody, anti mouse IgG, peroxidase conjugate, sheep-made was used in 1:50.000 dilution.

Detection of chemiluminescence with x-ray radiographs:

After incubation with ECL-reagent the membrane was put into a transparency film and inserted together with the x-ray-film into the autoradiography cassette. Different exposure times were tested to yield the best contrast and less background.

Enzyme Linked ImmunoSorbent Assay (ELISA):

The Enzyme-Linked Immunosorbent Assay (ELISA) is a useful and powerful method to detect proteins as antigens in the range of ng/ml to pg/ml in solutions by an antibody. Based on the principle of antibody-antibody interaction, this test allows for easy visualization of results and can be completed without the additional concern of radioactive materials use. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.

As the main principle a first antibody is specific for the protein to be detected and the second antibody which is specific for the first antibody bears a linked enzyme. The linked enzyme causes a chromogenic or fluorogenic substrate to produce a signal.

As a standard 200, 100, 50, 25, 12.5, 6.25 ng of purified 3α-HSD/CR protein was applied in coating buffer into the wells. 200µl of 12 different samples were tested according to the scheme in Fig. III-1. Each sample was diluted 1:100 in wash-buffer. The coating buffer fixes the antigen to the surface of the wells. After 30 min incubation at 370C all wells are washed with 250 µl of wash buffer. 200µl of anti-3α-HSD/CR-antibody, diluted 1:1000 in wash buffer, are added to the samples. After washing for 3 times with wash buffer excess of unbound antibodies are removed and only the antibody-antigen complexes remains attached to the well. A second antibody (DAKO, anti rabbit, pig made) was added in 1:1000 dilution (in wash buffer) which will bind to any antigen-antibody complexes. The second antibody is coupled to the substrate-modifying enzyme HRP. After 30 min incubation at 370C and washing for 4 times with 250µl of wash buffer the substrate (100µl ABTS, Böhringer) was added to the wells which are converted by the enzyme to elicit a chromogenic signal. After final incubation for 30 min at 370C the chromogen was measured at 404 nm. The reference was measured at 490 nm. The enzyme (HRP) linked to the second antibody acts as an amplifier: even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.