Alkaline phosphatase from Escherichia coli has been immobilized on Sepharose CL-4B using low levels of cyanogen bromide activation in order to favour the attachment of the dimer to the support through a single covalent linkage. The level of phosphatase activity in the matrix-bound monomer, obtained after dissociation of the immobilized dimer, was dependent on the extent of cyanogen bromide activation of the support, indicating that this activity is due to contaminating dimers. The quaternary structure of the subunit, reversibly immobilized through a disulfide linkage, was determined by crosslinking the matrix-bound protein, then releasing the protein from the support and analysing it by sodium dodecyl sulfate gel electrophoresis. This method demonstrated the presence of dimeric structures whose concentration was linearly correlated to the amount of phosphatase activity in the matrix-bound subunits. / Titration of matrix-bound ('125)I-labelled subunits with soluble nascent ('131)I-labelled subunits resulted in the recovery of over 60% of the original dimer phosphatase activity and a final isotope ratio of 1.06. Titration of metal-free immobilized dimer and monomer with ('65)Zn('2+) showed that the monomer bound 0.9 equivalents of Zn('2+), while the dimer bound 4.1 equivalents of Zn('2+). These results indicate that although the monomer lacks catalytic activity, it exists as a highly folded structure containing sites for Zn('2+) binding and subunit interactions. Chemical modification with ethoxyformic anhydride has demonstrated that three histidines per subunit are modified in the soluble enzyme with a concomitant loss of catalytic activity. Zn('2+) ions protect the enzyme from modification as well as from inactivation, thus implicating all three histidines in Zn('2+) binding. Zn('2+) also protects the monomer against this modification providing independent evidence for Zn('2+) binding in renatured subunits. These techniques, developed in order to characterize subunits of alkaline phosphatase, are generally applicable to other oligomeric enzymes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.68616 |
| Date | January 1981 |
| Creators | McCracken, Susan. |
| 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 Biochemistry) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000137729, proquestno: AAINK54851, Theses scanned by UMI/ProQuest. |
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