I. Purification and Partial Characterization of Chromatin Subunits. II. Structural Studies of a Membrane-Bound Acetylcholine Receptor

Author: Ross, Michael Jay

Year: 1977

Degree: Dissertation (Ph.D.)

Advisors: Stroud, Robert M.; Samson, Sten Otto; Raftery, Michael A.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/kh8k-yr31

Abstract

Investigations into the structural organization of integral membrane proteins and of protein-DNA complexes are reported. Purification and biochemical studies of chromatin subunits were carried out as preliminaries to structural studies. X-ray diffraction, electron microscopy and image processing techniques are applied to the study of membrane-bound acetylcholine receptor (AcChR) isolated from Torpedo californica.

Electrophoretic fractionation of monomer and oligomer chromatin subunits from rat liver and trout testes is reported. The nucleoprotein complexes do not dissociate or denature during electrophoresis. The bands seen in the gels contain both DNA and protein. The effects of DNA in homogeneity and histone degradation on the electrophoretic behavior of monomer subunit particles is discussed. Bands from the electrophoresis pattern of trout testes subunits were purified by preparative slab gel electrophoresis. These fractions remigrate as single peaks with unchanged mobility.

In the course of these investigations, DNAase II was studied as a possible probe of DNA-protein complexes. High concentrations of divalent cations inhibit enzymatic activity, and it is shown that the rate of catalyzed DNA hydrolysis decreases as a linear function of divalent cation concentration. Calcium, magnesium and manganese have identical effects. No activity is measurable if the cation concentration is raised above 0.08 M.

The first structural studies of a eukaryotic cell surface protein are reported. Electron microscopy of negatively stained particles of AcChR-rich membranes reveal, for the first time, the presence of large, highly ordered lattices. These lattices are characterized and the surface structure of the molecules in them studied by image reconstruction techniques to a nominal resolution of 20 Å. Large and small angle x-ray diffraction patterns from pellets of AcChR-rich membranes are reported and analyzed. From these data, the electron density of this membrane-bound protein, perpendicular to the membrane sheet, is determined. Indications of three-dimensional ordering within the pellets are also found.

The errors inherent in the use of digitized film data for image reconstructions and for analysis of x-ray diffraction are studied. Instrumental errors in the Syntex AD-1 flatbed autodensitometer are also analyzed.

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