Nitrogen Regulation of Catabolic Enzymes of Neurospora Crassa
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I ! 77-2398 FACKLAM, Thomas John, 1950- NITROGEN REGULATION OF CATABOLIC ENZYMES OF NEUROSPORA CRASSA. The Ohio State University, Ph.D., 1976 Chemistry, biological Xerox University MicrofilmsAnn , Arbor, Michigan 48106 NITROGEN REGULATION OF CATABOLIC ENZYMES OF Neurospora crassa DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Thomas John Facklam, B.S. The Ohio State University 1976 Reading Committee Approved by G. A. Marzluf, Ph.D. L. F. Johnson, Ph.D. T. J. Byers, Ph.D. Advisor / / \ (J Developmental Biology Progra ACKNOWLEDGEMENTS I wish to thank Dr. George Marzluf for his patient assistance and guidance during my graduate training. I extend special appreciation to my wife, Nancy, for her support and encouragement during the last four years. I gratefully acknowledge the financial support of the Developmental Biology Program. ii VITA June 28, 1950 Born - Buffalo, New York 1972 B.S., Cornell University, Ithaca, New York. 1973-1974 Graduate Teaching Associate, Department of Zoology, The Ohio State University, Columbus, Ohio. 1974-1976 N.I.H. Developmental Biology Traineeship The Ohio State University, Columbus, Ohio. r PUBLICATIONS "Nitrogen Regulation of Amino Acid Metabolism in Neurospora crassa." Genetics 80:s29 (1975). iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii VITA, iii LIST OF TABLES v LIST OF FIGURES vi INTRODUCTION, 1 METHODS AND MATERIALS 26 Growth of organism Enzyme assays Allantoinase isolation Affinity column Polyacrylamide gel electrophoresis Molecular weight determination RESULTS................................................................ 38 Growth of Neurospora on various nitrogen sources Amino acid uptake Growth of amino acid transport mutants Smino acid transport in pm-g and pm-n Regulation of arginine, ornithine and proline catabolic enzymes Allantoinase stabilization Isolation of allantoinase Molecular weight Multi-enzyme complex Michaelis constant Effects of cations on activity Presence of metals in allantoinase Effect of sulfhydryl reagents Feedback inhibition Thermal stability Sensitivity to proteases Characteristics of type II activity In vitro turnover DISCUSSION............................................................. 110 BIBLIOGRAPHY 127 iv LIST OF TABLES 1. Growth of wild-type and amr on various nitrogen sources. 2. Growth of amino acid transport mutants on various amino acids. 3. Arginase specific activity. 4. Ornithine transaminase specific activity. 5. Pyrroline-5-carboxylate dehydrogenase specific activity. 6. Proline oxidase specific activity. 7. Fractionation of cell-free extracts by (NH^^SO^. precipitation. 8. Lack of association of allantoicase and uricase with allantoinase. 9. Effect of cations on allantoinase activity. 10. Effect of chelators on allantoinase activity. 11. Effect of reducing substances on allantoinase. 12. Effect of potential feedback. 13. Allantoinase sensitivity to proteases. 14. Properties of type II activity. 15. SDS treatment of allantoinase. 16. Protease activity of allantoinase preparation. 17. Treatment of allantoinase by aln extract. 18. Summary of nitrogen control. v LIST OF FIGURES 1. Degradative pathway of purines. 2. Degradative pathway of arginine, ornithine and proline. 3. Flow diagram of allantoinase isolation. 4. Amino acid uptake of wild-type and amr. 5. Uptake of arginine, phenylalanine and aspartate by pm-n. 6. Uptake of arginine, phenylalanine and aspartate by pm-g. 7. Allantoinase stability in vivo. 8. Allantoinase stability in vivo in the presence of cyclohemimide. 9. Allantoinase stability in vitro. 10. Allantoinase stability in vitro in the presence of protease inhibitors. 11. Allantoinase stability in vitro in the presence of sulfhydryl reagents. 12. Elution of Sephadex G-150 column. 13. Elution of Sephadex G-100 column. 14. Polyacrylamide gels of G-150 and G-100 allantoinase preparations, 15. Molecular weight determination of allantoinase. 16. Molecular weight determination by SDS gel electrophoresis. 17. Lineweaver-Burk plot of allantoinase. 18. Thermal stability of allantoinase I and II. 19. Thermal stability with and without presence of substrate. 20. Stability of G-150 allantoinase. 21. Stability of G-100 allantoinase. 22. Molecular weight determination of type II. vi 23. Gel-filtration of SDS dialyzed allantoinase. 24. Degradation of allantoinase. 25. Model of nitrogen regulation. vii INTRODUCTION Neurospora crassa, a member of the fungal class Ascomycetes, is a typical eukaryote and can provide a model for studying regulatory mechanisms in higher organisms. Neurospora is a typical eukaryote in that it con tains mitochondria, a nucleus, ribosomes, seven chromosomes, poly A-con- taining mRNA (1), histones, and other structures and metabolic functions attributable to higher eukaryotes. As a heterotroph, Neurospora can utilize such simple compounds as acetate, glycerol, and glucose as carbon sources. Nitrate, ammonium, and various amino acids are able to provide the cells with nitrogen (2). Neurospora crassa is the best genetically characterized eukaryote, second only to Drosophila melanogaster. The combination of a well defined genetics, plus the ease with which it grows on completely defined media make Neurospora crassa an ideal organism with which to study regulation. Neurospora crassa is able to utilize most amides, amines, purines, and many amino acids as nitrogen sources. One aspect of my research was to examine nitrogen control of amino acid catabolism, with particular attention to arginine and proline metabolic degradation. I have also studied the closely related complex regulation of the purine catabolic enzyme, allantoinase, whose synthesis requires simultaneous induction and catabolic derepression. I will first review salient features of the regulatory mechanisms possessed by Neurospora crassa and related eukaryote organisms, and related aspects of nitrogen metabolism in these forms. 1 2 Then, my specific research objectives will be described and major conclusions will be summarized. Davis and his coworkers have studied extensively the control of arginine and ornithine metabolism in Neurospora. Their work has lead to a description of a regulatory mechanism where metabolites and enzymes are channeled and compartmentalized. The effect of this arrangement is to maintain high local concentrations of metabolites as well as to protect them from their catabolic enzymes. A second effect is to allow the regulation of independent metabolic pathways containing identical inter mediates. In Neurospora crassa the arginine and pyrimidine biosynthetic pathways utilize such regulatory mechanisms. The common intermediate shared by both the arginine and pyrimidine pathways is carbamyl phosphate (CAP). Carbamyl phosphate is synthesized by two carbamyl phosphate synthetases (CPSase), one for pyrimidines (CPSase P) and one for the arginine pathway (CPSase A). CPSase A is located in the mitochondria and is fully repressible by arginine (4,5). CPSase P has been localized in the nucleus complexed to arginine trans- carbamylase (ATCase)(6). The bifunctional CPSase P within the nucleus is feedback inhibited by UTP and is derepressed under the condition of low uridine (7,8). The enzyme, by being compartmentalized within the nucleus, is situated where the nucleoside triphosphates are pooled and quickly used, thus providing for a most rapid and sensitive regulation. Both CPSases provide for a separate pool of CAP, but an accumulation of CAP in one pathway can result in an overflow into the other pathway (4). This overflow effect is particularly evident with mutations lacking a particular CPSase; this deprives one pathway of CAP, but the defficiency is relieved if the second pool overflows (9, 10). 3 The channeling of the pyrimidine CAP pool could be accomplished by binding CAP to the CPSase-ATCase aggregate (4,5). This form of molecu lar compartmentation (11) would commit the CAP to the pyrimidine path way via ATCase tp synthesize ureidosuccinate. In the arginine pathway, CAP is believed to be confined in the mito chondria along with CPSase A (12,13). The intramitochondrial concentra tion of CAP is sufficiently high for efficient use by the next enzyme, ornithine carbamyltransferase, also located within the mitochondria (4,7). For arginine biosynthesis CAP is compartmentalized to increase its local concentration as well as keep it segregated from being metabo lized by ATCase. CPSase A, which is very sensitive to repression by arginine, is active in the presence of large concentrations of intracellular arginine. CPSase A is insensitive to feedback inhibition by arginine (5). Along with CPSase A and ornithine carbamyltransferase, ornithine acetyltrans- ferase and two other enzymes which synthesize ornithine, are located within the mitochondria (12). The product of ornithine carbamyltrans ferase, citrulline, is exported from the mitochondria into the cytoplasm where the remainder of the arginine biosynthetic enzymes reside. The catabolic enzymes are also found in the cytoplasm (12). Arginine, both exogenous and biosynthetic, goes directly towards protein synthesis, bypassing a large sequestered cellular pool. The arginine which fails to be incorporated into protein enters this pool (14). Labeling studies indicate that once exogenous arginine enters the intracellular pool it remains there and is exchanged only very slowly. This maintains a low arginine concentration within the cytoplasm insufficient to maintain catabolism. Weiss (15) has