; R o o t E n t r y F R 5 C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l 5 5 4 @ F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT METABOLIC BIOCHEMISRY Shelley A. Graves Shelley A. Graves @ 7 e = e 7 7 j j j j j j j ? 1 w y y y ' T Z K ? j ? j j j j w ~ " j j j j w Metabolic Biochemistry Panel Summary Brouwer, M. MCB 9506050 The panel recognized the benefits of working with the blue crab as a model system for studying intracellular copper trafficking in eukaryotes. The proposal is well focused and clearly articulated. The collaborations, especially with the research group led by Dr. Ian M. Armitage, are seen as significantly strengthening the project. It is felt that the present research program will yield new, interesting, and important structural information on copper-metallothionein (CuMT) isoforms. Form 7: This is an excellent research proposal which is now being recommended for funding after being resubmitted a third time to the program. The PI has made careful use of revi ewers comments and prepared a well-organized, clearly written, compelling proposal. The panel was convinced of the advantages of working with the blue crab to obtain important information about Cu-metabolism applicable to higher animal systems. The proposal received three excellent and one very good rating. I am pleased to recommend this invertebrate project for funding. Brouwer, M. MCB-9506050 Copper is an essential but toxic element. It is proposed to elucidate how this metal is converted intracellularly into a biologically useful form, while avoiding copper s toxic side effects. Blue crabs will be used as a model system. The blue crab is dependent on the copper-protein hemocyanin (Hc) for oxygen transport. Four copper complexes function as a first line of defense against copper toxicity in the crab: copper-glutathione (Cu(I)-GSH), and three isoforms of the copper-metallothionein (CuMT) family. CuMT-I and II are virtually identical. CuMT-III is distinctive. Cu(I)-GSH can deliver copper for the activation of apohemocyanin and apoMT. It is unknown whether CuMTs can participate in intracellular copper exchange. The first objective then is to evaluate the hypothesis that CuMT-I/II and/or CuMT-III may transfer copper to apoHc with GSH acting as a metal-exchange mediator and GSSG as a copper-releasing agent. Our second objective is to determine the metal-binding properties and three-dimensional structure of the two distinct CuMT isoforms by 2 dimensional-NMR spectroscopy, in collaboration with Dr. Ian Armitage at Yale University. Antioxidant enzymes, including the ubiquitous copper-zinc superoxide dismutase (CuSnSOD), in higher animal systems form a second line of defense against copper-induced oxidative cell damage. However, it has not been possible to detect CuZnSOD in crabs and decapod crustacea in general. The third objective of the project is to determine whether the cytosolic SOD in crabs is a novel protein, or a new, hitherto unknown member of the CuZnSOD family. %%% Copp er is an essential but toxic trace metal that has been implicated in oxidative damage to DNA, proteins, and cell membranes. The long-term objective of this study is to gain a better understanding of the control of intracellular trafficking of copper, and of the molecular mechanisms involved in defense against copper-mediated oxidative damage. Studies will be performed with the blue crab, Callinectes sapidus, which has a high requirement for copper in their respiratory pigment, hemocyanin. There appear to be three metallothionein (low-molecular weight metal-detoxifying proteins) (CuMT) isoforms in the blue crab, which are structurally and functionally different from each other. It is unknown, however, whether the CuMTs participate in intracellular copper-exchange processes. To find out if they do, copper-free hemocyanin will be treated with different CuMTs in an attempt to restore normal hemocyanin function. Secondly, two-dimensional nuclear magnetic resonance (NMR) techniques will be used to determine the structure of blue crab CuMTs. Studies will be performed in collaboration with Dr. Ian Arnitage at Yale University. In addition to oxygen transport, it is believed that Cu operates in special enzymes together with zinc (Zn) to protect cells against certain toxic effects of oxygen. This may occur in a special enzyme called Cu/Zn superoxide dismutase (SOD). Consequently, the third objective of this project is to determine if such an activity exists in the blue crab, as in higher animal systems, and, if so, isolate and purify the CuZnSOD in an attempt to determine its structure and mechanism of operation. *** ;
