Date of Award

Fall 1980

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

The regulatory (B1) subunit of ribonucleoside diphosphate reductase from calf thymus has been purified to homogeneity, as demonstrated by the observation of a single band on SDS-polyacrylamide electrophoresis gels. The purification procedure involved the use of conventional techniques under conditions known to induce specific changes in the structural and binding properties of the enzyme. The first half of the purification included homogenization, precipitation with streptomycin sulfate, followed by precipitation with ammonium sulfate and ion-exchange chromatography on DEAE-cellulose. The partially purified enzyme was next dissociated by chromatography on DEAE-cellulose in the presence of ATP and magnesium ion. The elution of the more negatively charged B1-ATP complex was effected with a buffer of higher ionic strength than that required for elution of the complementary (B2) subunit. In contrast to the very poor yields reported following dissociation of other mammalian ribonucleotide reductases, dissociation of the calf thymus protein gave a 43% yield. The relatively high value may reflect the greater stability of the subunits from the calf thymus enzyme.

The regulatory (B1) subunit was purified by sequential affinity chromatography on ATP-agarose in the presence and absence of magnesium ion. The binding of magnesium ion to the B1 subunit markedly reduced its affinity for the ligand. Consequently, most contaminating ATP-binding proteins were retained by the column and removed at this stage. The B1 preparation was further purified by rechromatography of the sample on a second ATP-agarose column (no magnesium ion present) and ion-exchange chromatography of DEAE-cellulose. Concentration by chromatography on a column of hydroxylapatite gave an essentially quantitative yield of purified B1 subunit.

While no further purification was conducted on the B2 subunit, its molecular weight and Stokes' radius were determined by gel filtration on columns of Biogel A 1.5 M (Mr = 86,900; r(,0) = 35.9 A) and Sephacryl S-300 (Mr = 99,800; r(,0) = 35.8 A). The unique role of protein B2 as the iron-containing subunit in ribonucleoside diphosphate reductase was established by treatment of the partially purified enzyme and its constituent subunits with EDTA. Exposure of protein B1 to EDTA had no effect on enzyme activity while treatment of the intact enzyme or protein B2 with the chelator eliminated the activity entirely. Activity was completely restored to the EDTA-treated enzyme and B2 subunit upon the addition of Fe(II) or Fe(III). Which of the oxidation states of iron is the more efficient activator could not be ascertained with the incompletely purified enzyme used in these studies.

Two bands were observed following disc gel electrophoresis on 5% polyacrylamide gels of the hydroxylapatite fractions. In order to determine if the two bands represented different aggregated states of the B1 subunit, the hydroxylapatite fractions were subjected to electrophoresis on 10% polyacrylamide gels containing SDS. The observation of a single band with an identical R(,f) in each fraction is consistent with a model in which the two bands observed in the non-denaturing gels represent the monomer and dimer forms of protein B1. The molecular weight of the monomer was estimated to be 79,400 by electrophoresis on a 7.5% polyacrylamide gel containing SDS.

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