Claudia A. Telakowski-Hopkins

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAs by 3-methylcholanthrene.

Abstract Liver poly(A + )-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.

Sequence analysis and regulation of rat liver glutathione S-transferase mRNAs

We have utilized polysomal immunoadsorption techniques to purify the rat liver glutathione S-transferase mRNAs. Using the purified mRNAs as template, cDNA clones complementary to the Ya, Yb1, and Yc mRNAs have been constructed. The cDNA clones have been utilized in RNA blot hybridization and nuclear run-off assays to demonstrate that the Ya and Yb mRNAs are elevated 8 and 5-fold, respectively by phenobarbital; whereas the Yc mRNA is elevated only 2.0-fold. The elevation in glutathione S-transferase mRNAs is due in part to transcriptional activation of the corresponding genes. Nucleotide sequence analysis of the three glutathione S-transferase clones suggest that the Ya and Yc genes represent one rat liver glutathione S-transferase gene family whereas the Yb genes represent a second distinct glutathione S-transferase gene family. The construction of these cDNA clones will allow identification and characterization of the glutathione S-transferase structural genes as well as aid in the identification of regulatory elements that are responsible for transcriptional activation of the genes by xenobiotics.

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.

Isolation and characterization of monoclonal antibodies against rat liver epoxide hydrolase

We have established hybridoma cell lines which secrete monoclonal antibody to rat liver microsomal epoxide hydrolase. All of the monoclonal antibodies formed against epoxide hydrolase are mouse immunoglobulin subclass IgG1. Utilizing double immunodiffusion analysis, we have found that the monoclonal antibodies bind to and precipitate epoxide hydrolase in solubilized rat liver microsomes. Despite the ability of the monoclonal antibodies to precipitate rat liver microsomal epoxide hydrolase, they do not inhibit the catalytic activity of the enzyme but rather stimulate it. The monoclonal antibodies do not precipitate epoxide hydrolase in microsomes isolated from hamster, rabbit, monkey, or human. When rabbit reticulocyte lysates are programmed with rat liver mRNA, the primary translation product of epoxide hydrolase can be immunoprecipitated from the translation system using the monoclonal antibodies. The ability of the antibodies to recognize in vitro synthesized epoxide hydrolase should make them amenable to identify recombinant bacterial clones expressing this protein. The monoclonality of these antibodies and their specificity should provide a useful way of identifying, purifying, and quantitating rat liver epoxide hydrolase as well as examine the expression of the gene(s) encoding epoxide hydrolase in normal rats and in rats exposed to carcinogenic xenobiotics.

Biosynthesis of Endoplasmic Reticulum Membrane Proteins

The intent of this review is to discuss recent literature on the biosynthesis of endoplasmic reticulum (ER) membrane proteins, with a focus on two mechnisms that appear to be operative during insertion of these proteins into the ER membrane (i.e., cotranslational and posttranslational insertion). During the past few years, the biosynthesis of membrane proteins in general has received much attention, particularly in regard to the events involved in the synthesis, processing, transfer, and insertion of proteins into or across their appropriate membrane (for reviews, see [1–5]). Although a significant literature has accumulated on the biosynthesis of secretory proteins, only a limited number of studies to date have focused on the biosynthesis of ER membrane proteins. This has been due, in part, to the inherent difficulties in purifying to homogeneity many intrinsic ER membrane proteins.

Expression and Sequence Analysis of Rat Liver Glutathione S-Transferase Genes

The rat liver glutathione S-transferases are a family of enzymes which catalyze the conjugation of the reduced sulfhydryl group of glutathione with various electrophiles. In addition, the transferases bind with high affinity various exogenous hydrophobic compounds as well as potentially toxic endogenous compounds such as bilirubin and heme (1–3). The enzymes are comprised of binary combinations of at least six major subunits, Yα, Ya, Ybl, Yb2, Yc and Yn, which can be separated by onedimensional SDS-polyacrylamide gel electrophoresis (4–6).