Date of Award

Winter 1982

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

The catalytic (M2) and regulatory (M1) subunits of calf thymus ribnucleotide reductase have been purified to homogeneity. Each runs as a single band in SDS-polyacrylamide gel electrophoresis with polypeptide molecular weights of 84,000 for M1 and 58,000 for M2. Additional evidence for homogeneity was obtained using high performance liquid gel chromatography. The native M1 subunit is predominately monomeric (R(,s) = 37 A) under experimental conditions while the M2 subunit is oligomeric (R(,s) = 53 A). The quaternary structure of the native M2 protein was evaluated by chemical cross-linking with dimethyl suberimidate. The results demonstrate that native M2 protein exists in a dimer-tetramer equilibrium. The amino acid composition of the complementary subunits of the ribonucleotide reductase has been determined. Significant compositional homology exists between the M1 polypeptide and its E. coli (B1) counterpart. However, comparative analytical peptide mapping reveals that the M1 and B1 polypeptides have different primary structures. Atomic absorption analysis indicates that protein M2 contains 3 g-atoms Fe/mole monomer, in contrast to the 1 g-atom Fe/mole monomer reported for protein B1. The role of protein M2 associated iron is not yet clear, since addition of exogenous iron to ribonucleotide reductase preparation results in a nearly two-fold enzyme activation. The M2 subunit has a UV-visible absorption spectrum with a major peak at 404 nm but this spectrum is different from that of the B2 subunit of E. coli ribonucleotide reductase. This chromophore is also observed in intact calf thymus enzyme preparations. Addition of ATP, a positive allosteric effector, to intact enzyme results in an increase in the extinction coefficient of the visible chromophore. This spectral enhancement may reflect a shift in equilibrium toward an active protein M1-M2 complex and/or a structural change at the chromophore. EPR spectroscopic experiments at 77 K did not detect a free radical species in the calf thymus enzyme analogous to that which occurs in the E. coli ribonucleotide reductase. Thus, significant differences between the ribonucleotide reductases from these eukaryotic and prokaryotic sources are indicated.

Download

Share

COinS