Student Theses and Dissertations

Date of Award

1968

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

The experiments described in this thesis were undertaken in order to define and purify proteins that might have a specific function in nerve activity. The studies were limited to rat brain proteins soluble in dilute aqueous buffers at neutral pH. Fractionation procedures for these proteins and two means of identifying individual proteins of interest in the fractions were developed.

The soluble proteins were divided into two classes according to their solubility in mildly acidic conditions: the pH 5 soluble and pH 5 insoluble fractions. Two different large anatomical areas of the brain referred to as "cortex" and "brainstem," contained the same amount of pH 5 soluble proteins but differed considerably in the pH 5 insoluble protein content. Using analytical disk electrophoresis on polyacrylamide gels as a tool for examining individual components of the pH 5 soluble fraction, the following comparisons were made: Brain extracts from a single animal species gave a characteristic pattern, which differed qualitatively from extracts of other tissues and from serum. Extracts of brains from different animal species all differed from each other, but different strains of rats were identical. Finally, the patterns obtained with different regions of the central nervous system of a given animal were identical, but peripheral nerve differed.

One means of characterizing the soluble brain proteins was by studying their in vivo biosynthesis during variations in electrical activity. For this purpose, an experimental system was employed which utilized the phenomena of unilateral spreading cortical depression in conscious animals. It was found that after administration of tritiated leucine the specific activity of "cortical" proteins was about 16% lower in the depressed hemisphere than in the control hemisphere of the same animal, while the specific activity of "brainstem" proteins remained the same on both sides. The pH 5 soluble and insoluble fractions were affected to the same extent. Further subfractionation of the pH 5 soluble proteins failed to show individual proteins whose biosynthesis was altered to a greater extent than the average over all soluble proteins. The significance of these results is discussed with respect to the use of biosynthetic criteria for identifying proteins of importance in nerve tissue.

The second means of characterizing individual brain proteins was the investigation of their tissue specificity, using immunological methods. For this purpose antisera to rat brain extracts were prepared. About twelve antigenic components could be demonstrated by immunoelectrophoresis; most were present in extracts of other tissues as well. The various antigens, most of which were in the pH 5 soluble fraction, could be separated according to their electrophoretic mobility by zone electrophoresis on starch. Preparation of antisera to the six subfractions of the soluble proteins obtained in this manner made possible the identification and purification of a protein, designated antigen ⍺, which could not be demonstrated in other tissues.

Antigen ⍺ was found to be an acidic protein, consisting of a single type of subunit having a molecular weight of about 39,000. Evidence was obtained that indicated that the molecule exists as a dimer stabilized through disulfide bonds. Higher-order aggregates were also observed. The protein is found in both the central and peripheral nervous systems of the rat, and immunological evidence for the existence of related proteins in other animals was also obtained. The properties of antigen a were compared to those of certain other brain proteins, especially the "S-l00" protein, which is the only other highly purified soluble protein found only in the central and peripheral nervous system.

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