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Isolation of a Hepatic Iodothyronine 5’-Monodeiodinase by Nondenaturing Agarose Gel Electrophoresis^*

Department of Medicine, University of California School of Medicine Los Angeles, California 90024

Address reprint request to: Inder J. Chopra, M.D., Department of Medicine, University of California Los Angeles Center for Health Sciences, Los Angeles, California 90024.

Abstract

We have examined the application of nondenaturing agarose gel electrophoresis for isolation of the catalytically active iodothyronine 5’-monodeiodinase (5’-MD) in rat liver microsomes. Preliminary studies showed that most ingredients of conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, including acrylamide, SDS, and bromophenol blue, markedly inhibited ’-MD activity in solubilized rat liver microsomal proteins (SRLMP). We replaced these inhibitory components with those that had little or no inhibitory effect on the ’-MD and devised conditions for nondenaturing agarose gel electrophoresis for isolation of a ’-MD protein. SRLMP (up to 120 fig protein/well), prepared by preincubation with 5 mM 3-[(3-cholamidopropyl)dimethylammonio 1-propanesulfonate] (CHAPS) and 1% 2-mercaptoethanol, were subjected to electrophoresis in 0.35% agarose gel in 20 mM Tris-HCl, 10 mM EDTA, pH 6.6 buffer containing 2 mM CHAPS and 1 mM thioglycolic acid. Electrophoresis was carried out for 14 h at 4 C using 2.5 V/cm. After phoresis, the gel was sliced into 16 fractions, and homogenates of each fraction were tested for ’-MD activity by incubation with 0.6 nM [¹²⁵I]rT₃ for 30 min at 37 C in the presence of 10 mM dithiothreitol. The peak ’-MD activity was detected in fraction 6 (Rf, 0.375). This fraction accounted for about 30% of total ’-MD activity and only 6% of protein in the gel. The gel slice containing the peak ’-MD activity was next subjected to a second electrophoresis at pH 7.6 for 4 h in a 90· different direction. The most ’-MD activity was demonstrated in the third of 8 gel fractions. SDS-polyacrylamide gel electrophoresis of this fraction demonstrated only one 55 K protein in most experiments and occasionally an additional 35 K protein. The specific activity of ’-MD in this fraction of greater than 11 pmol I-/mg protein-h was at least 2.3-fold that in SRLMP. Rabbits were immunized with agarose gel containing the peak ’-MD activity. Immunized rabbit immunoglobulin G bound up to 70% of ’-MD activity in SRLMP, and the binding effect varied in a dose-dependent manner. Western blot analysis revealed that the anti-’-MD antibody bound only one 55 K protein among innumerable proteins in SRLMP. A specific antiprotein disulfide isomerase antibody bound a slightly larger, 57 K protein in SRLMP. Isoelectric focusing of SRLMP and its Western blot analysis with anti-’-MD antibody demonstrated a prominent band at isoelectric point (pi) 5.7. We conclude that hepatic ’-MD is a 55 K protein with a pi of 5.7 (Endocrinology 127: 2709–2715, 1990)

Footnotes

^* Supported by USPHS grant DK-16155 from the National Institutes of Health, Bethesda, MD.

Received May 17, 1990.

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