Natalie Cohen, BS - US grants

This is a Research Opportunities for Women, Research Planning Grant, to support preliminary studies and other activities related to the development of a research project.

Some soluble cellular enzymes in their natural environment have kinetic properties that are not evident under the conditions used in most enzymological studies. There is evidence that some soluble pathway-related enzymes are organized in cells, some associated with membranes, such that the pathway intermediates are channeled. These are important features of cells. The long term objective of this work is to obtain comprehensive information on the properties and organization of soluble enzymes in situ; this would extend our knowledge and understanding of normal cell structure and function and of regulatory mechanisms, as well as of the deviations from these which occur in disease. Several pathway-specific intermediates of urea synthesis are channeled, indicating that the enzymes of the pathway are organized in situ. The cytoplasmic urea cycle enzymes in permeabilized hepatocytes will be characterized with regard to their requirements for aspartate, ornithine, and ATP. Experiments involving inhibition of specific transporters and competition between endogenous and exogenous substrates will be done to obtain information on the channelling of intermediates. Immunohistochemical methods will be used to locate the cytoplasmic urea cycle enzymes in intact and permeabilized hepatocytes, and possibly in Bri j-treated and "nude" hepatocytes. Preparations of inner and outer mitochondrial membranes and of contact sites will be used to determine whether ornithine transcarbamylase, the ornithine transporter, and the arginase of the outer membrane form a complex; evidence will be obtained from sedimentation analysis, immunoblotting, and patterns of cross-linking.

The Gordon Research conference on Macromolecular Organization and CellFunction has met since 1987; this will be the eighth conference in this series. The most recent one met at Queens College, Oxford University, U.K. in September, 1998. This proposal requests partial funding for the next conference, to be held again in Oxford, August 6-11, 2000. This conference focuses on the intracellular organization of cellular
components, the principles and mechanisms underlying that organization, and the essential role of such organization in cell function and its regulation. The first conferences concentrated on the role of enzyme organization in metabolic regulation, but the subject matter has been greatly broadened over the years, so that it now includes virtually all aspects of cell function. Macromolecular organization is critical not
only for the maintenance of various static cell structures, but also for the dynamic events that are coupled to key processes such as chromatin remodeling, transcription, RNA editing and transport, nuclear import/export, translation, protein degradation, cytoskeletal function, organelle transport, signal transduction, metabolic pathways, and apoptosis. Conference topics include the mechanisms of macromolecular interactions, and the responses of such mechanisms to changes in metabolic or hormonal status. The functional implications of interactions, such as channeling of metabolic intermediates or of signaling molecules, or the translocation of cellular components are of central interest, as are the characteristics of macromolecular complexes, both stable and transient. In previous conferences it has become very clear that many interactions may be altered or even lost when cells or tissues are disrupted for study, and a major interest is the development and application of methods to analyze interactions in the
living cell. This is an interdisciplinary conference. It attracts not only experimental scientists with interests and expertise ranging from biochemistry, cell biology, and molecular biology to biophysics, physics, and instrumentation, but also theoreticians and modelers. The interdisciplinary atmosphere produces excellent discussions and scientific interactions that are stimulating and highly valued by the participants. Eighteen excellent speakers and eight prominent discussion leaders have already
accepted invitations to participate in the formal sessions of the next conference. A number of slots have been kept open for the most recent developments in this rapidly moving field.