Isolation of a hepatic iodothyronine 5'-monodeiodinase by nondenaturing agarose gel electrophoresis.
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 5'-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 5'-MD and devised conditions for nondenaturing agarose gel electrophoresis for isolation of a 5'-MD protein. SRLMP (up to 120 micrograms 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 5'-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 5'-MD activity was detected in fraction 6 (Rf, 0.375). This fraction accounted for about 30% of total 5'-MD activity and only 6% of protein in the gel. The gel slice containing the peak 5'-MD activity was next subjected to a second electrophoresis at pH 7.6 for 4 h in a 90 degrees different direction. The most 5'-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 5'-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 5'-MD activity. Immunized rabbit immunoglobulin G bound up to 70% of 5'-MD activity in SRLMP, and the binding effect varied in a dose-dependent manner. Western blot analysis revealed that the anti-5'-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.(ABSTRACT TRUNCATED AT 400 WORDS)
Sakane
S
S
Department of Medicine, University of California School of Medicine, Los Angeles 90024.
Chopra
I J
IJ
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D002793:Cholic Acids; D003902:Detergents; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007074:Immunoglobulin G; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats
D000178:Acrylamides; D000936:Antigen-Antibody Complex; D002793:Cholic Acids; D003902:Detergents; D007074:Immunoglobulin G; D020106:Acrylamide; D007453:Iodide Peroxidase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate