For many years this laboratory has been developing and using radiation target analysis, a method for determining the mass of biologically active molecules. The laboratory has developed a variety of experimental techniques to properly utilize the inactivation of biochemically active material by ionizing radiation, specifically that from high-energy electrons. Radiation target theory has been successfully extended to complex biological systems. These techniques and theories have been utilized in studies of enzymes, receptors, transporters, and other structures of wide interest in the biomedical field. Often these radiation studies resulted in discovery of unexpected features or properties of these biologically active materials. The objectives of present studies are to determine the precise nature of damage in macromolecules caused directly by ionizing radiation and to utilize this knowledge in the application and extension of radiation target analysis. Several projects have been completed during the past year. One of these is a radiation study of ribonucleotide reductase, a crucial enzyme in all cells and in viral infection. It is composed of two large subunits and two small subunits, the latter of which contains a stable free radical which is crucial for function. A radiation study of this enzyme from herpes simplex virus (HSV)revealed the active structure was less than the entire tetrameric molecule. Enzymatic activity only requires one large and one small subunit. Additional radiation studies of the isolated individual subunits revealed that the subunit interactions observed in the intact enzyme were missing in the recombinant subunits, indicating that the structure of the native enzyme is significantly different from the recombinant forms. Radiation effects on the recombinant small subunit resulted in the disappearance of the free radical (and consequent loss of enzymatic activity) due to radiation products which were created elsewhere in the sample. This effect was only observed in the isolated small subunit and did not occur in the intact holoenzyme or in any other protein. It was suggested that the free radical is accessible to the solvent in the isolated small subunit, but not in the holoenzyme. A separate project concerns the enzyme reverse transcriptase which is essential for retroviral replication. In HIV, there are two immunoreactive forms of the enzyme. One is a 66 kDa structure and the other is a 51 kDa form which arises from proteolysis of the large polypeptide. Radiation inactivation of these individual polypeptides and the enzymatic activities (RNA-dependent and DNA-dependent DNA polymerases) have been studied in monomeric, homdimeric and heterodimeric forms of the enzyme. All dimeric forms of the enzyme show a tightly-coupled structure in which radiation-deposited energy is transferred to both subunits. The monomeric form shows that only one subunit is required for enzymatic activity. The method of radiation target analysis continues to reveal unique molecular properties of biologically active structures. Often these properties were unanticipated from studies using other techniques. This new knowledge has given a greater understanding of the mechanisms of these biological functions. In some cases these properties offer the possibility of external control or modification of these active structures.
