Date of Award
Doctor of Philosophy
The purposes of this study were to purify the intermediate (10-nm) filament proteins desmin from porcine myocardium and vimentin from porcine aorta, and to examine selected properties of both proteins. Desmin was prepared from the residue remaining after extraction of actomyosin from cardiac myofibrils. The desmin was solubilized with 8 M urea, and purified by batch chromatography in 6 M urea-containing solutions on DEAE cellulose and hydroxyapatite, followed by column chromatography on DEAE-Sepharose CL-6B. Vimentin was prepared from the tunica media of porcine aorta adjacent to the heart. The vimentin was solubilized with 8 M urea from the residue remaining after extraction of actomyosin from smooth muscle myofibrils. Final purification was achieved in 6 M urea-containing solutions by batch chromatography on DEAE cellulose, followed by column chromatography on hydroxyapatite and DEAE-Sepharose CL-6B. Circular dichroic spectra of both proteins indicated an alpha-helical content of about 41%. Both proteins existed as a major, and a slightly more acidic, minor isovariant. Amino acid analysis showed that cardiac desmin was similar in composition to other mammalian desmins. Aorta vimentin is similar in composition to vimentin isolated from other sources, but differs significantly from desmin. Desmin was localized at the Z-line and intercalated disc region of cardiac myofibrils by indirect immunofluorescence. Vimentin was localized in a filamentous pattern in vascular smooth muscle cells. At physiological pH and ionic strength, both proteins assembled into filaments similar in morphology to native 10-nm filaments, but there were significant differences in the solubility properties of the two proteins. At subphysiological ionic strength, vimentin was more soluble than desmin. Both proteins were more soluble at low ionic strength in the presence of a reducing agent. At physiological ionic strength, a reducing agent had little effect. At the same ionic strength, divalent cations were more effective than monovalent cations in promoting filament elongation. Mixtures of desmin and vimentin copolymerize to form heteropolymers with solubility properties and filament lengths intermediate between desmin and vimentin homopolymers. Decoration of the heteropolymers with desmin or vimentin antibodies showed that both proteins were distributed uniformly in the filaments.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Michael Kenneth Hartzer
Hartzer, Michael Kenneth, "Purification and properties of porcine cardiac desmin and vascular smooth muscle vimentin " (1984). Retrospective Theses and Dissertations. 8171.