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 brought to fruition during the past year. The enzyme hyaluronan synthase is of great medical importance in human infections by streptococcus. Radiation studies revealed that each enzyme molecule requires fifteen molecules of a specific lipid in order to function. The enzyme ribonucleotide reductase is crucial in AIDS infection. It has a unique structure involving a stable free radical. In isolated subunits, this radical confers extraordinary radiation sensitivity which is not seen in the intact holoenzyme. Another enzyme, phenylalanine hydroxylase, is especially important in humans: a defect in this enzyme is the cause of phenylketonuria in newborns. Radiation studies of this enzyme have been continuing for several years. The enzyme has complex regulation mechanisms; in the purified state, radiation studies revealed the different structures involved in several of these mechanisms. Now the technique has been applied to the enzyme in the crude state, revealing unanticipated differences from those seen previously. It appears that purification has altered the structure of the enzyme complex. 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.
