Cytochrome b(,5) is an integral membrane protein of the endoplasmic reticulum of the hepatocyte. Previous work by the groups of Ozols and Strittmatter has determined the primary sequence and the orientation of the molecule. It is embedded on the cytoplasmic face of microsomal membranes via a hydrophobic 40 amino acid long "tail" at the carboxy terminus. The synthesis of this protein is assigned to the free polyribosome population of rat hepatocytes, and the experimental documentation of this assignment is reported. / Free and membrane-bound polyribosomes were separated by a modification of the method of Ramsey and Steele (1976) and characterized by electron microscopy. Poly A('+) RNA isolated from the total free (poly A('+) RNA(,f)) or total membrane-bound polyribosomes (poly A('+) RNA(,mb)) was translated using the heterologous wheat germ system. Fluorographic SDS-PAGE analysis demonstrated that the translation products of poly A('+) RNA(,f) had electrophoretic mobilities similar to those of liver cytosol proteins. In contrast, poly A('+) RNA(,mb) was translated into polypeptides with mobilities similar to serum proteins (the major secretory product of the hepatocyte). Antiserum to the complete cytochrome b(,5) molecule immunoprecipitated from the translation products of poly A('+) RNA(,f) a single polypeptide with electrophoretic mobility identical to that of native cytochrome b(,5) (17,500 daltons). Quantitative analysis indicated 85% synthesized from poly A('+) RNA(,f) compared to only 15% from poly A('+) RNA(,mb). Control experiments with antiserum to serum albumin demonstrated that following immunoprecipitation, a single polypeptide of M(,r) 68,000 was synthesized by poly A('+) RNA(,mb). This M(,r) was shown to be larger by 3000 daltons than that of native serum albumin; the difference being accounted by the known "signal sequence". Quantitation indicated 90% synthesized from poly A('+) RNA(,mb) and 10% from poly A('+) RNA(,f). / In conclusion, an exclusive location of the site of synthesis of microsomal cytochrome b(,5) is assigned to the free polyribosome population. This conclusion provides a ready explanation (i) for the ubiquitous distribution of the protein to most intracellular organelles; (ii) for the topographic orientation on the cytoplasmic face of membranes and (iii) for the hyrophobic tail (membrane insertion sequence) of amino acids at the carboxy terminal end of the molecule.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.68580 |
| Date | January 1980 |
| Creators | Rachubinski, Richard A. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Anatomy) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000092297, proquestno: AAINK52095, Theses scanned by UMI/ProQuest. |
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