Michael Dunn, Ph.D. - US grants

Funding is requested to make possible the participation of thirteen American scientists in an international workshop on "Comparative Analysis of Catalytic Mechanisms of Zinc Enzymes", to be held June 17-22, 1985 at San Miniato (Pisa), Italy. The objective of the workshop is to bring together chemists, biophysicists, biochemists and molecular biologists whose research is directed toward the elucidation of structure-function relationships in zinc metalloenzyme catalysis. The workshop will emphasize the discussion of catalytic mechanism for those zinc enzymes for which there is available detailed information about structure and mechanism (e.g., carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase). Attention will also be given to less well characterized zinc enzymes such as RNA polymerase and EcoR1 endonuclease. Assessments of the impact of different experimental methods in solving various aspects of mechanism will be made, and attention will be given to the formulation of new questions for future investigation. Proceedings will be published as brief papers either in book or journal form. The rationale for holding such a workshop at this time stems from recent technological advances both in physical biochemistry and in molecular biology. This workshop will provide a forum for exchanging iformation and ideas among the diverse group of scientists committed to the elucidation of the catalytic mechanisms of zinc metalloenzymes.

We propose three major areas of investigation: 1) Glomerular and mesangial cell contractility will be studied using a computer-based image analysis microscopic system. We will assess the contractile effects of thromboxane A2, leukotriene C4 and D4, and platelet activating factor on isolated rat glomeruli and rat glomerular mesangial cells in culture. The glomerular contractile actions of these compounds will be blocked with specific antagonists to thromboxane receptors and leukotriene receptors. The relaxant effect of PGE2 or PGI2 will be studied and the role of intracellular cyclic AMP as the mediator of mesangial relaxation will be evaluated. The importance of calcium entry or translocation of intracellular calcium as a mediator of mesangial contraction in response to the aforementioned contractile agents will also be studied; 2) Studies of glomerular immune injury will use several models of nephrotoxic serum nephritis induced by antiglomerular basement membrane antibodies. Glomerular synthesis of leukotriene B4, C4 and D4 will be measured in normal and immune-injured glomeruli. The actions of LTC4 and LTD4 on glomerular function in vivo will be evaluated, after intrarenal infusion in the rat, with measurements of glomerular filtration rate, renal blood flow and urinary protein excretion. The therapeutic efficacy of lipoxygenase inhibitors in nephrotoxic serum nephritis will be evaluated as will the value of a leukotriene D4 receptor antagonist; 3) Renal medullary prostaglandin synthesis will be measured in salt-sensitive, hypertensive Dahl rats. Using cell culture techniques, we will determine whether reduced PGE2 synthesis is in renal medullary interstitial cells or renal papillary collecting tubular cells. Renal medullary interstitial cell synthesis of anti-hypertensive polar renomedullary lipid (platelet activating factor) will be measured and compared between hypertensive and normotensive rats. Transepithelial salt transport will be assessed in renal papillary collecting tubular cell monolayers and we will determine whether this is the site of enhanced sodium reabsorption in hypertensive salt-sensitive rats. The effects of prostaglandins and platelet activating factor on renal papillary collecting tubular cell transport will be measured.

The goals of this research are (1) to understand the roles of allosteric interactions in the regulation of metabolite transfer between sequential enzymes in a metabolic pathway, (2) to understand the relationship between enzyme conformation change, substrate specificity, and catalysis, and (3) to explore the significance of direct metabolite transfer in systems which contain more than one type of catalytic site. Tryptophan synthase systems from E. coli and S. typhimurium (bienzyme complexes) and yeast and blue-green algae (multienzymes) will be studied utilizing rapid mixing kinetic techniques in combination with absorbance and fluorescence spectrophotometry, and high resolution nuclear magnetic resonance. %%% The efficiency of cellular metabolism requires the existence of multienzyme complexes which catalyze a sequence of chemical changes on metabolites. The efficiency derived from not having to recapture intermediates from the exterior milieu is suspected to be the primary reason for such highly organized processing. The proposed research explores an ideal model system for such linked enzyme-catalyzed processing of substrates. In the case of tryptophan synthase, substrate is passed through a "tunnel" between the enzyme active sites. The studies planned will examine how the two active sites interact with each other in order to optimize catalysis and transfer of metabolites between them. A great deal will potentially be learned through this work about the way this complex protein machinery works and why it is designed as it is.

We propose experiments to study glomerular mesangial cells, in culture, in order to evaluate membrane receptors and signal transduction pathways activated by the vasoconstrictors, endothelin (ET) and thromboxane A2 (TxA2). We will quantitate receptor number, affinity and specificity and correlate receptor characteristics with cellular responses to these ligands. We will also evaluate the role of GTP binding proteins to link receptors to plasma membrane enzymes such as adenylate cyclase and phospholipases A, C and D. The role of G proteins will be measured in both intact cells and with cell membranes using stable GTP analogues. The capacity of phospholipase D to mediate cellular responses to ET and TxA2 will be studied as will the possibility that phospholipase C will use phospholipid substrates in addition to polyphosphoinositides. In subsequent studies, we will evaluate the importance of these signal transduction pathways to mediate the cellular responses of contraction and proliferation. Specifically, we will attempt to mimic proliferative and contractile actions of ET and TxA2 through changes of cellular inositol phosphates, (Ca2+)i pHi and protein kinase C activity. We will also attempt to dissociate phospholipase C activation from the membrane receptor through EJ-ras transfection of the mesangial cells. These manipulations will allow dissection of the relative importance of these signal transduction events either singly or in combination to mediate mesangial contraction and proliferation. Finally, we will search for a genetic defect of mesangial and vascular smooth muscle cells in genetically hypertensive strains. We propose that hyperresponsiveness of the phospholipase C signalling pathways may mediate enhanced vasoconstriction and reduced glomerular filtration. This theory will be tested using cultured vascular smooth muscle and mesangial cells with an assessment not only of their contractile and proliferative responses to contractile agonists but also a comparison of phospholipase C activation with measurements of (Ca2+)i, pHi, release of inositol phosphates and stimulation of protein kinase C.

The proposed research is based on the hypothesis that autacoids such as platelet activating factor and eicosanoids are important mediators and modulators of glomerular immune injury. We believe that the autocrine and paracrine actions of these autacoids alters glomerular mesangial function both in vitro ad in vivo and, hence, plays an important role int he development of glomerular immune injury. We will evaluate the roles of platelet activating factor and eicosanoids (leukotrienes, thromboxane and prostaglandins) in three models of experimental glomerular disease. These models are: nephrotoxic serum nephritis, immune complex nephropathy induced by cationic bovine gamma globulin, and murine systemic lupus erythematosus. In these models, we will assess glomerular synthesis of platelet activating factor and eicosanoids, and we will evaluate the therapeutic benefits of the following drugs: platelet activating factor receptor blockage; leukotriene D4 receptor blockage; inhibitor of leukotriene B4 and leukotriene C4 synthesis; inhibitors of thromboxane synthesis and thromboxane receptors. these agents will be administered both acutely and chronically and the beneficial effects assessed through measurements of glomerular function, glomerular morphology and glomerular synthesis of these autacoids. Additionally, we will monitor the in vivo expression of genes controlling the synthesis of fibronectin and collagen. Glomerular mesangial cell cultures from rats and humans will be used in vitro to study the effects of immune stimulation on mesangial synthesis of platelet activating factor, eicosanoids, and matrix proteins including fibronectin and collagens. Immune stimulation will be induced with immune complexes, membrane attack complex, and leukocytes. These experiments will enhance our understanding of mesangial function and biochemistry under conditions which mimic immunologic injury in vivo. We wish to pursue in depth, the effects of platelet activating factor on cultured mesangial cells. These experiments will quantitate and characterize platelet activating factor receptors, G protein linkage of receptors and phospholipases, and intracellular events of signal transduction including phospholipase A2 and phospholipase C-mediated changes. Furthermore, we will test the hypothesis that both positive and negative feedback actions exist mediated through protein kinase C and cyclic AMP-protein kinase A.

Site-specifically mutated insulins have enormous potential value both ad hormones with improved physical, chemical and biological properties for the treatment of diabetes mellitus and as subjects for the study of insulin structure-function relationships. The Novo Research Institute (Denmark) had made available to this project large amounts (100 mg to >1 gm) of insulin mutants which vary widely in physical-chemical properties and in biological potency. Objectives: The long-term objective of this project is to further our knowledge of structure-function interrelationships for human insulin and proinsulin. The specific objectives are to answer the following questions: (a) How different are the solution structures of biologically active monomeric insulin and aggregated forms of metal-free native human insulin? (b) Is there a structure-function correlation between monomeric insulin mutants of widely varying biological activity? (c) Is there a significant difference in conformation between biologically active (monomeric) insulin and biologically inactive proinsulin? (d) How does protein concentration, ionic strength, pH and mutation at specific loci alter the aggregation and fibrillation behavior of metal-free insulin? (e) What hexamer conformation states are induced in solution by the lyotropic anions (SCN- ,l-,Br-, Cl-) and by phenol? (f) How are the energetics of the phenol and lyotropic anion-induced conformational transitions influenced by site- specific mutation? Relevance: The project is important to three areas of insulin therapy: (1) Understanding the relationship between insulin structure and the recognition of insulin by receptors; information critical to the rational design of improved insulins via genetic and chemical synthesis. (2) Understanding the effects of pH, ionic strength and site-specific mutation on the aggregation and fibrillation of insulin; information that is important for the improved design of insulin formulations for the treatment of insulin-dependent diabetics. (Insulin fibrillation is a serious obstacle to the development of a safe, implantable infusion pump for the delivery of insulin). (3) Understanding the dynamics of the lyotropic anion and phenol-induced conformation changes in the insulin hexamer; information that will assist efforts to design improved slow release formulations. Methods: NMR investigations of signatures of three-dimensional structure for various monomeric insulin species will use standard one- and two- dimensional methods (selective irradiation, magnetization transfer, multiple quantum filters, homonuclear 1H and heteronuclear 1H-15N multiple quantum correlation spectroscopy). 1H FT NMR, stopped-flow rapid-mixing kinetics and Doppler shift (dynamic photon correlation) light scattering spectroscopy will be used to study insulin aggregation and fibrillation and conformational transitions in the insulin hexamer.

This award from the Chemistry Research Instrumentation and Facilities (CRIF) Program and the Major Research Instrumentation (MRI) Program and the Office of Multidisciplinary Activities (OMA) will assist the Department of Chemistry at University of California @ Riverside to upgrade a 300 MHz and a 500 MHz NMR Spectrometer. This equipment will enhance research in a number of areas including the following: (1) total synthesis of natural products, (2) NMR characterization of pollutant transport through porous media, (3) structure-function played by H-bonded protons with large H-chemical shifts in the insulin hexamer, (4) conformation analysis and the relationship to biological activity, (5) stereochemistry of side chain crosslinking in peptides and proteins, and (6) synthesis, structural and biological studies of biomedically significant natural products. Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution. Access to state-of-the-art NMR spectrometry is essential to chemists who are carrying out frontier research. The results from these NMR studies are useful in the areas such as polymers, catalysis, and in biology.

DESCRIPTION: Long Term Objectives. Elucidate fundamental principles of catalytic efficiency, allosteric regulation and substrate channeling in enzyme catalysis. Specific Aims. The work to be undertaken involves studies of the pyridoxal phosphate-requiring bacterial enzyme tryptophan synthase to accomplish the following: (a) Determine the mechanism of action of monovalent cations (MVC) in catalysis and regulation. (b) Establish a detailed mechanism for the roles of structural elements in the transmission of allosteric signals between subunits of the bienzyme complex. (c) Determine the catalytic function of protons thought to be involved in low-barrier H-bonds (LBHBs). Background. The tryptophan synthase bienzyme complex (alpha2-beta2) catalyzes the last two steps in the synthesis of L-Trp. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha and beta sites. Reactions at the beta sites are hypothesized to switch alpha2-beta2 between low and high activity forms and "open" and "closed" conformations. This prevents the escape of the common metabolite, indole, and ensures that the alpha and beta reaction occur in phase. The binding of MVCs to a specific site (separate from the catalytic sites) activates specific steps in the catalytic pathway of tryptophan synthase. The MVC effect is hypothesized to result from selective lowering of the activation energies of certain steps through stabilizing interactions between the MVC and protein conformations complementary to the corresponding activated complexes. MVC binding also is essential to allosteric communication. Isotope effects suggest the involvement of an LBHB in tryptopha synthase catalysis. LBHBs are hypothesized to be strong bonds (12 to 24 kcal/mol) which lower activation energies, or stabilize conformation states. Methods. These hypotheses will be tested via the use of rapid kinetics, isotop effects, 1H NMR, mutants enzymes, and fluorescent probes. Significance to Huma Health and Disease. This work will contrbute new knowledge of structure-function relationships in enzyme catalysis and regulation concerning (a) The mechanism of action of MVCs. (b) The roles of conformation change in catalysis, allosteric regulation, and substrate channeling. (c) The roles, if any, of LBHBs in protein function.

DESCRIPTION (provided by applicant): Lonq-Term Objectives: Elucidate fundamental relationships of chemical catalysis, enzyme conformational dynamics, allosteric regulation, and enzyme structure to the mechanism of metabolite transfer between enzyme pairs (substrate channeling) in a metabolic cycle. Specific Aims: To achieve a detailed mechanistic description of the allosteric regulation of substrate channeling in the bacterial tryptophan synthase bienzyme complex. The roles played by the binding of allosteric effectors, the formation of intermediates, and the binding of a monovalent cation cofactor in the regulation of channeling will be determined. Backqround: The tryptophan synthase bienzyme complex catalyzes the last two steps in the biosynthesis of L-tryptophan. Catalysis is intimately related to allosteric signaling and metabolite transfer between the alpha- and beta-sites. Hypotheses To Be Tested: (a) The alpha- and beta-subunits of the bienzyme complex switch between "open", "partially open" and "closed" conformation states during the catalytic cycle in response to binding and covalent reaction of substrates. (b) Certain covalent states of the beta-site give rise to a slow switching from the closed to the open conformation state(s) that is functionally important for substrate channeling. (c) Monovalent cation (MVC) binding plays a cofactor role that modulates the catalytic and conformational energetics of the complex. (d) The BetaAsp305-BeataArg141 salt bridge is critically important for the allosteric regulation and reaction specificity of the complex. Methods: The hypotheses will be tested via the use of rapid kinetics, equilibrium binding, 19F NMR, mutant enzymes, substrate analogues, optical spectroscopy and x-ray crystallography. The project Significance to Human Health and Disease derives from the new knowledge about structure-function relationships for the class of protein nanostructures consisting of enzyme-enzyme complexes that are organized into metabolic pathways. These nanostructures are subject to non-traditional forms of allosteric regulation that are poorly understood. Four project areas are of fundamental interest: (a) The roles of protein structural elements and protein conformational dynamics in substrate channeling between enzyme pairs in metabolic pathways. (b) The roles played by protein conformational dynamics in biological function, (c) the roles of monovalent cations in the modulation of protein function, (d) the role played by substrates as allosteric effectors in substrate channeling.

An award has been made to Cameron University under the direction of Dr. Michael T. Dunn to integrate a collection of plant specimens from the Wichita Mountains Wildlife Refuge Herbarium into the Cameron University Herbarium. New cabinets will be purchased for safe storage of plant and paleobotanical specimens. The flora of the wildlife refuge represents a unique snapshot of floral diversity in the area over the past few decades. Students will be hired to assist with entering data into a database for plants from the refuge. The integration of the two collections will safeguard the specimens being incorporated and enhance the value of the college's herbarium for teaching and research.