Date of Award

Fall 1983

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

A chromatographic method was developed to isolate arginase isozymes from bovine brain and liver. The purified proteins were characterized as to their native molecular weight, subunit structure, amino acid composition, hexose content, behavior on disc gel electrophoresis, and reaction with polyclonal antibodies raised against a liver arginase antigen. An arginase-specific messenger RNA preparation was also isolated from liver polysomes using immunological techniques.

Two liver and four brain arginases were purified, having similar native molecular weights, yet different subunit compositions, and displaying varying affinities for the Mn('2+) ion that is required for complete enzymatic activity. The amino acid compositions of all six proteins are closely related, although there are some noticeable disparities, particularly with respect to alanine.

When analyzed by disc gel electrophoresis, a bovine liver or brain arginase enzyme will appear as a diffuse band. The carbohydrate moiety of these molecules may be responsible for this pattern, but a variable loss of Mn('2+) in the electric field also contributes to the diffuseness, since the migration of each isozyme was observed to change if separated by electrophoresis in the presence of added Mn('2+) or EDTA.

Several distinct antigenic determinants were recognized by anti-liver arginase antibodies. Two of the bovine brain arginase proteins identified in this work share one of these immunological sites with the liver enzyme, while the other two arginases isolated from brain tissue hold a separate determinant in common with it. Bovine liver arginase antibodies were also observed to cross-react with the arginase enzyme in a mouse liver homogenate.

An arginase enriched mRNA preparation was translated in a cell-free system. After immunoprecipitation and SDS electrophoresis, the translated products migrated to the same positions as authentic liver arginase isozymes.

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