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I ‘A 329;“ ‘ {Ht ‘ 'v -\ ,J This is to certify that the dissertation entitled THE IDENTIFICATION AND CHARACTERIZATION OF INDOLE-B-ACETYL-MYO-INOSITOL HYDROLASE FROM VEGETATIVETISSUE OF _Z_§_A MAYS L. presented by Prudence J. Hall has been accepted towards fulfillment of the requirements for A Ph.D. degree in Botany & Plant Pathology \Za TS Bahia; Major professor Date (I‘HJ\83 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 IV‘ESI_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .—,—. your record. FINES will be charged if book is returned after the date stamped below. EM: Wrist!" (yawn new Ii” 1'. ,;‘ -.: a ~ ._ ;,, r“ 'tJ lwWfl \Enmwunlen». meg-u USE out? THE IDENTIFICATION AND CHARACTERIZATION OF INDOLE-3-ACETYL-fl19-INOSITOL HYDROLASE FROM VEGETATIVE TISSUE OF gEA MAYS L. By Prudence J. Hall A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1983 ABSTRACT ’D —.} _/ THE IDENTIFICATION AND CHARACTERIZATION OF 5-? INDOLE-3-ACETYLffl19-INOSITOL HYDROLASE _ FROM VEGETATIVE TISSUE OF ZEA MAYS L. «j’g/ //' By Prudence J. Hall The presence of conjugates of the plant growth hormone indole-3- acetic acid (IAA) in vegetative tissue of plants in excess of the free acid is postulated to play a role in maintenance of endogenous hormone levels. In shoots of Zea mays L. seedlings, esters of IAA predominate and it is believed that these esters are enzymatically hydrolyzed to provide free IAA to the growing shoot. One ester has been isolated from corn shoots and was identified as indole-3-acetylgmyQ-inositol (IAInos). This dissertation describes the partial purification and characterization of an enzyme from corn shoots which hydrolyzes myo—inositol esters of IAA. The enzyme was partially purified by column chromatography using abaminohexyl agarose, hydroxylapatite, and Sephadex 6-100. In addition to the hydrolysis of IAInos, the enzyme preparation hydrolyzed the methyl esters of IAA and naphthyleneacetic acid, and the synthetic ester, a-naphthyl acetate. The rate of enzymatic hydrolysis was unaffected by added cofactors, sulfhydryl reagents, and metal cations, except for Mn2+which was somewhat inhibitory. Glycerol appeared to inhibit the appearance of free IAA, but other experiments demonstrated the possibility of a transesterification reaction. The hydrolysis of IAInos can be demonstrated in extracts from seeds and roots in addition to shoots. Additional data is presented Prudence J. Hall demonstrating the localization of enzymatic activity in the coleoptilar portion of the shoot. A calculation based on published data showed that the relative amount of esterified IAA in coleoptiles as opposed to mesocotyls parallels the distribution of enzymatic activty. IAA esterified to myg-inositol is present in substrate preparations as chemically and biologically resolvable isomers. Examination of substrate in reaction mixtures before and after enzymatic hydrolysis demonstrated that the chemically resolvable isomers disappeared at different rates. This leads to the suggestion that the endogenous rate of hydrolysis of IAInos is controlled in part by the abundance of some isomers relative to others. While the presence of hormone conjugates in plant tissues is recog- nized, and many studies have been published on their characterization and synthesis, the data presented in this dissertation constitutes the first report of an enzyme hydrolyzing a conjugate of IAA. To George T. Jones, Professor of Botany, Oberlin College and to Rich, Ethan, Eric and Kitty 11 ACKNOWLEDGEMENTS I wish to acknowledge the assistance of the members of my guidance committee, Norman Good, Derek Lamport and William Wells. I have appreciated their comments and advice. The guidance, encouragement and support of my major professor, Robert S. Bandurski, is gratefully acknowledged. It has been a priviledge to work in his laboratory. The interest and support of all those people in Dr. Bandurski's laboratory in the years I have been there - especially Aga Schulze, Jerry Cohen, Volker Magnus, Bill Pengelly and John Chisnell, must be acknowledged. Their friendship and their willingness to share their ideas have been most appreciated. I also acknowledge the support and encouragement of my husband and sons. They have encouraged me throughout my graduate studies and have helped out in every way. TABLE OF CONTENTS Page LIST OF TABLES...................................................... vi LIST OF FIGURES................................................ ..... vii LIST OF ABBREVIATIONS............................................... x GENERAL INTRODUCTION AND LITERATURE REVIEW.......................... 1 Conjugates of IAA: roles and metabolism......................... 3 Esterases: activities and identification in plants.............. 11 References 0.0.0.0...OOOOOOOOOOOOCOOOOOOOOOOOOOIOOOOOOOOOOOOO0.0. 17 EXPERIMENTAL I: PARTIAL PURIFICATION OF IAA-Mlg-INOSITOL HYDROLASE FROM ZEA MAYS L. VEGETATIVE TISSUECOOOOOOOOOOOOOOOOOOO0.0. 22 Abstract.O...OIOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOIOOOI. ....... O... 23 IntrOductionOC ..... .0.0.00.00.00.00.0IOOCOOOOOOOOOOOOOOOOOOOOOOOO 24 Mater1a15 and HethOdSOO0...0.0....0.00.00.0000...OOOOOOOOOOOOOOOO 26 Chemicals.0.0...O...0.0.0.0.0...0.0.0.0000...OOOOOOOOOOOOOOOOO 26 Preparation Of Plant Material0.00...0.0.0....OOOOOOOOOOOOOOOOO 26 BUfferSOOOCOOOOOO0.000.0.0000...0.00.00.00.00...OOOOOOOOOOOOOO 27 Protein Concentration......................................... 27 Polyacrylamide Gel Electrophoresis............................ 27 Esterase Activity Staining.................................... 27 Assay of IAInos Hydrolase Activity............................ 28 Assay of a-Naphthyl Acetate Esterase Activity................. 29 Results.......................................................... 30 Polyacrylamide Gel Electrophoresis............................ 33 Molecular Weight Estimates.................................... 39 HydrOIytic ActiVitieSOO0.0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 39 Denaturation.................................................. 42 Other Sources of IAInos Hydrolase Activity.................... 42 DiSCUSSIOnOOOoooooooooooooooooooocoo-0000000000000000000000000... 45 ReferenCESOOOOOOOOOIOOOOOOOO0.0....0...00...0.0000000000000000.0. 49 EXPERIMENTAL II: CHARACTERIZATION OF IAA-MXQ-INOSITOL HYDROLASE AS ISOLATED FROM ZEA MAYS L. VEGETATIVE TISSUE ........... 52 AbStraCto00000000000oocoone...00000000000000.0000000000.00...coo. 53 IntrOductionOOOOOOOOOO0.0...0......0.0.0.0000...OOOOOOOOOOOOOOOOI 54 Materials and MethOdSOO00.0.0.0....0.0.0.00.0...OOOOOOOOOOOOOOOOO 56 ChemicaISIOOOOOOOOO00....OOOOOOOIOOOOOOOOOOOOO00.000.000.00... 56 BUfferSOOOOOOOOOO...00......OOOOOOOIOOOCOOOOOOIOOOOOOOOOOOOOCO 56 P1ant ExtraCtSooooooo000.000.0000.000000000000000000000000000. 56 iv Protein Concentrations........................................ IAInos Hydrolase Activity..................................... Hydrolysis of a-Naphthyl Acetate.............................. Preparation of Methyl Esters.................................. Hydrolysis of Methyl IAA (Me-IAA)............................. Hydrolysis of Methyl Esters of NAA, PAA and BA................ HPLC Resolution of IAInos Isomers and IAA..................... Results.......................................................... Characterization of IAInos Hydrolase.......................... pH optimum................................................. Tim course....0...OCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0...... Enzyme concentration....................................... Effect of additions to the reaction mixture................ Effect of added substrate.................................. Preferential hydrolysis of isomers......................... Esterolytic Activityes of IAInos Hydrolase and Esterase....... Hydrolysis of a-naphthyl acetate........................... Hydrolysis of Me-IAA....................................... Hydrolysis of other esters................................. Effects of glycerol........................................ Hydrolytic activities in coleoptiles as compared to mesocotyls.................................. Discussion.................................................... ReferenceSOC0.0.0.0000...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO CONCLUDING DISCUSSIONCOOOOOCOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOO0.... ReferenCESOO0..OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...0.00.00.00.00... APPENDIX A: pH Optima for HydrOIySESOOOO...OOOOOIOOOOOOOO...0...... pH optimum for 3H-IAInOS HYdr01y51SOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO pH Optimum for a-Naphthyl Acetate Hydrolysis..................... APPENDIX B: Affinity Chromatography...........u..................