Partial purification of the microsomal rat liver iodothyronine deiodinase. I. Solubilization and ion-exchange chromatography.
Rat liver microsomal fraction was treated with several non-ionic, anionic or zwitterionic detergents in order to investigate which is most suitable for subsequent purification of the iodothyronine deiodinase. Criteria for effective solubilization were (a) no or little inhibitory effect of the detergent on deiodinase activity, (b) non-sedimentable activity by centrifugation at 105,000 X g, and (c) a low molecular weight of the soluble complex as determined by Sephacryl S-300 gel filtration in the presence of detergent. Optimal solubilization was obtained by treatment of the microsomes with cholate and subsequent precipitation of dispersed protein with 30% ammonium sulfate, resulting in the removal of adhering phospholipids. Enzyme was resolubilized best with the non-ionic detergents Brij 56 or Emulgen 911 in the presence of 0.5 M NaCl. This deiodinase preparation had an isoelectric point at pH 9.3 and was further purified by subsequent chromatography on DEAE-Sephacel and CM-Sepharose. Only the Emulgen 911-dispersed enzyme was retained by the CM-Sepharose column. Further purification was investigated by chromatofocusing. This resulted in a rapid inactivation of the Emulgen 911 preparation whereas the Brij 56-soluble enzyme was ultimately purified 400 times after DEAE-Sephacel and chromatofocusing.
Mol
J A
JA
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
van den Berg
T P
TP
Visser
T J
TJ
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D007453:Iodide Peroxidase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D003902:Detergents; D007453:Iodide Peroxidase