1) Background: Exposure of biologically active materials to ionizing radiation at very low temperature leads to damage to macromolecules. Biochemical functions are lost at a rate directly dependent on the molecular mass of the active structures. This is the basis for radiation target analysis which is used to determine the size of the functional unit of enzymes, receptors, transporters and others that carry out important biological activities. These studies revealed new and unexpected aspects of biomedically important systems including basic differences between proteins and ribonucleic acid in their responses to ionizing radiation. Other biophysical techniques are used to learn other aspects of macromolecular function in living cells: ultracentrifugation of intact Euglena cells results in stratification of cellular components based on their respective densities. These cells are living and can recover, grow and divide. 2) Objective of present studies: a: Fundamental studies of the actions of ionizing radiation on macromolecules continue in order to define the exact nature of the damage in different species of molecules. Analyses of these effects by radiation target theory establishes a radiation-sensitive mass associated with measured biological activities. This reveals a fresh perspective in the structure-function relationship in these macromolecules. b: Application of the radiation technique to enzymes, binding sites, and transporters to determine the size of their active structures, which often is less than the mass of the entire complex. c: Analysis of the recovery processes in stratified Euglena cells to determine the mechanisms involved and the time course of restoration of normal cellular function. 3) Results during the past year: a: To test whether radiation damage can appear across disulfide bridges, immunglobulin G (IgG) was irradiated. IgG is composed of two 52 kDa polypetides and two of 24 kDa. In the rabbit, there are three disulfide bridges between these subunits; these disulfide bridges can be chemically cleaved before radiation exposure. Radiation damage to each of the subunits was examined in both native and reduced irradiated samples. In both cases, radiation damage was confined to a single subunit; there was no evidence for the transfer of radiation energy across a disulfide bridge. The 52 kDa subunits were destroyed individually whether or not they were linked by disulfide bridges to other subunits. The smaller subunits revealed a complex radiation response, indicating that small doses of radiation were less effective than high doses. It was shown that radiation damage to the larger subunits released fragments which moved with the smaller subunits on electrophoresis. The mathematics of this model were found to agree quantitatively with the observations. These results raised many new questions which will be pursued. b: Target analysis was applied to the radiation inactivation of the enzyme reverse transcriptase. It revealed that the DNA template/primer substrate acted as a scaffold for the protein to form dimers. c: The recovery of stratified Euglena was found to depend on the g-force to which they had been exposed and the length of exposure. Centrifugal forces up to 150 000 x g and exposure times up to 18 hours were tested. The recovery process was due to a normal change in cell shape: contractile structures in the cell envelope caused the elongated cells to become more spherical; relaxation permitted the cells to become more extended. The internal contents of the cell became redistributed in this process. After one-two hours recovery time, the previously stratified cells were indistinguishable from untreated Euglena and metabolic activity was restored. 4) Conclusions and Significance: a) Radiation studies of IgG showed that molecular damage was restricted to the polypeptide which suffered a radiation interaction, even if the polypeptide was linked to another via a disulfide bridge. This result differs from disulfide-linked ricin in which damage was observed in both chains only when the disulfide bridge was intact. While radiation damage appears throughout a polyptide no matter where the radiation event occurred, the mechanism(s) by which damage appears in adjacent polypeptides differs among proteins. b: Radiation studies established the previously-unknown size of reverse transcriptase structures involved in the function of this enzyme. It also revealed a novel finding: the role of DNA in the dimerization of polypeptide subunits. c: The recovery of Euglena from centrifugation-induced stratification depends primarily on a natural process of changes in cell shape. The mechanism of this contractile operation is not yet defined, nor is the role of this process in normal cell metabolism. However, the delay in the recovery process indicates a need either for reorientation of individual protein molecules within the organelle complexes or reestablishment of contacts between different organelle complexes and other subcellular structures.
