WAITE INSTIT'UTE tt,8.96 LIBRARY IN THB ENZYMBS OF NITRATE ASSII',IILATION SCLEROT IN T A SCLEROT I ORU I'1 by ì4askuntjir Abdul Rachim, B'Sc" Tr' A thesis submitted in fulfilment of the'requirements for the degree of Nlaster of Agricultural Science Departmenr of Agricultural Biochemistry Waite Agricultural Research Institute The UniversitY of Adelaide' FebruarY, 1986 û¡¿a"rdr-af Aî-v"f6 t- PREFACE Part of the work described in this thesis has been pre- sented at the Australian þiochemical Society Conference (Canberra, 1985) and published in the following journals: I Some properties of glutamine synthetase and glutamate synthase from ScTerotinia scl-er otiorun. M.A. Rachirn and D.J.D. Nicholas (f985) Proc. Aust. Biochen. Soc. !, 2I 2. Glutamine synthetase and glutamate synthase from Sc-lerotinia sclerotiorun. M.A. Rachim and D.J.D. Nicholas (1985) Phytochemistry 4, 254I-2548 3 Some properties of nitrate reductase from Sclerotinia scTerotiorun. M.A. Rachim and D.J.D. Nicholas (1986) Phytochenistry ë- (i" Press) 1L ACKNO\^II-EDGEMENTS f wish to express my deepest and sincerest thanks to my supervisor, Prof. D.J"D. Nicholas, chairman of the Department of Agricultural.Bio- The University of chemistry , lrlai-te Agricultural Research Institute ' and constructive criticism Adelaide for his constant encouragement ' guidance throughout the progress of the present investigation and in the preparation of manuscriPt. I would also like to thank Mr. D. Hein for his assistance with the 15N Mr. B.A. Palk for preparing the photographic prints' "*p"riments, others who l"Irs. 1"1. Brock for the skrlful typing of the thesis and to a1I helped me from time to time. I am grateful to my wife Erna for her patience and unfailing encouragenent dur-'ng the course of this investigation. The postgraduate scholarship frorn International Development Programme of Australian Universities and Colleges is gratefully acknowledged ' a1a DECLARATION I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any Univerqity. To the best of my knowledge and belief no material described herein has been previously published or written by another person except when due reference is made i-n Ëhe text. If accepted for the award of a M.Ag.Sc. degree, this thesis will be available for loan and photocopying. }{.A. RACHIM l_v ABBRBVIATIONS The abbreviations for chemicals and symbols in general follow either the tentative rules of IUPAC-IUB Commission on Biochemical Nomenclarure (Biochen. J. (1966) 101: 1-7) or the Instruction to Authors for the Phytochernistry (Phytochemistry (1983) 22'- I-7). CHBI.,I]CALS ADP Adenosine 5t-diphosphate t AMP Adenosine 5 -monophosPhate | ATP Adenosine 5 -triphosphate BSA Bovine serum albumin BV Benzyl viologen BVH Benzyi viologen (reduced) CDP CyEidíne 5'-diphosphace | CMP Cytidine 5 -rnonophosphate CMP Cytidine 5' -triphosphate CyL b557 Cytochrome b55l DCPIP 2, 6-DichlorophenolindoPhenol DEAE-ce11u1ose Diethylaminoethyl ce11ulose DTT Dithiothrei-tol EDTA Ethylenediamine tetraacetic acid FAD Flavin adenine dinucleotide FADH2 Flavin adenine dinucleoticle (reduced) FMN Flavin mononucleotide Þ-MNH2 Flavin mononucleotide (reduced) GDP Guanosine 5t-diphosphate GMP Guanosine 5r-monoPhosPhate t GTP Guanosine 5 -triphosphate IDP Inosine 5r-diphosphate | IMP Inosine 5 -monophosPhate t ITP- Tnosine 5 -triphosphate MSX L-methionine-DL-Su1 Phoximine MV Methyl viologen 14VH Methyl viologen (reduced) NADH Nicotinamide adenj-ne dinucleotide (reduced) NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized ) NADPH Nicotinamide adenine dinucleotide phospiiate (reduced ) NEM N-ethylmaleimid e p-CMB p-chloromercuriben zoaLe SDS Sodium dodecyl sulphate -SH Sulphydryl group SVD Snake venom phosPhodiesterase TCA Trichloroacetic acid Tris Tris (hydroxymethyl) aminomethane | TIDP Uridine 5 -diphosphate | IJMP Uridine 5 -monophosphate t UTP Uridine 5 -triphosphate v Svmbols and Units A absorbance OC degree Celcius (centigrade) cm centimeter g gram o Ò unit of gravitational field hr hour(s) kDa kilo dalton(s) Ki inhibitor constant Michaelis-Menten constant K^lil L licre M molar mA milliampere mg milligram min minute(s) ml millilitre mm millimeter mlul millimolar mmo 1e millimo1e(s) l4l^l molec.¡lar weight ug microgram u1 microlitre urn micrometer u¡4 micromolar ( umo 1e micromole s ) N normal nm nanometer nmole nanomole ( s ) 7" percent inorganic phosphate Pa p S l- pound per square inch U V. ultra violet v volume vl weight va TABLE OF CONTENTS Ig. lto:_ PREFACE i ACKNO\^TLEDGEMBNTS ii DBCLARATION iii ABBREVIATIONS iv vi TABLE OF CONTENTS LIST OF TABLES ix L]ST OF þ-IGURES xi SUI'{l'{ARY xiv 1. INTRODUCTION 1 1.1 The biology of ScTerotinia scTerotiorun 1 L.2 Nitrate assimilation in microorganisms 3 ,.I rl 'iÌ T.2.I Nitrate reductase 4 ¡ L.2.2 Nitrite reductase 8 I.2.3 I'letabolism of ammonia into amino acids 11 r.2.3.r Glutamine synthetase L2 r.2.3.2 Glutarnate synthase L6 1.3 Aims of the study 18 2. MATERIALS AND METHODS 19 2.I Culturing Sclerotinia scTerotiorun 19 2.I.L Grov¿th conditions I9 2.L.2 Harvesting 19 2.2 Enzyme methods 20 2.2.I Nitrate reductase assay 20 I I 2.2.L .I Colorimet::ic 20 I 2.2.I.2 SPectroPhotometric 2I 2.2.2 Nitrite reductase and hydroxylamine 2L reductase assays 2.2.2.L Colorimetric 2l t 2.2.2.2 SPectroPhotometric 23 vl1 Paee No: 2.2"3 Glutamine sYnthetase assay 23 tl 2 "2.3 .r Transferase reaction 23 2.2.3.2 Biosypthetlc reaction 24 2.2.4 Glutamate synthase assay 25 2.2.5 Determination of Michaelis-Ilenten 25 constant (Kr) 2.2.6 Determinatiön of inhibitor constant (K1 ) 25 2.2.7 Determination of molecular weight 25 2.2.7 .I Gel f iltration 25 2.2.7.2 Electrophoresis 26 2.2.8 Separation and identification of flavin 27 from nitrate reductase 2.2.9 Adenylylation and deadenylylation of 27 glutamine synthetase 2.3 General techniques 29 15N 2.3.r Incorporation of into washed felts 29 2.3.2 Preparations of columns 29 2.3.2.1 Matrex Ge1 Blue A 30 2.3.2.2 Blue Sepharose CL-68 30 1 2.3.2.3 Sepharose 68 30 ef 2.3.2.4 DEAE-cellulose 31 'iù 2,4 Other determinations 31 2.4.r Nitrite 31 2.4.2 Ammonia 32 2.4.3 Protein 32 2.5 Biochemicals, chemicals and other materials 33 2.5.r Biochemicals and chernicals 33 2.5.2 Other materials 34 3. RESULTS 35 3.1 Nitrate reductase 35 3.1.1 Purification 35 3 "r.2 Properties 36 3 | .2.r Molecular weight 36 3 I.2.2 Effect of pH 36 I 3 r.2.3 Electron donors 36 3 r.2.4 K, values for substrate 4r reductant and cofacEor ' 3.r ,2.5 CharacterízaLion of flavin 4I isolated from the Purified enzyme t 3.1..3 Inhibitor studies l+7 vl_al Paee No: 3.2 Nitrite reductase 55 3.2.r Purification 55 3.2.2 Properties 56 3.2.2,I Effect of ptl and time of incubation 56 3.2.2.2 Electron donors 60 3.2.2.3 Requirement for flavin 60 3.2.2,4 K, values for substrate ' reductant 60 and cofactor 3.2.2.5 Stoichiometries for NADPH, nitrite 66 and anmonia 3.2.3 Inhibitor studies 66 3.2.4 Inactivation of nitrite and hydroxylamine 70 reductases by NADPH in the Presence of FAD 3.3 Incorporation of [15tq]-fuUe11ed (NH4)2504 into cell- 70 nitrogen 3.4 Glutamine sYnthetase 75 3.4.1 Purification 75 3,4.2 Properties 77 3.4.2.r Molecular wei-ght 77 3.4.2.2 Effect of pH and incubation time 77 3.4,2.3 Divalent cation requirenent 77 3.4 .2.4 Nucleotide specificitY 82 3.4,2.5 K, values for substrate 82 3.4.3 Inhibitor studies B4 3.4.4 Adenylylation/deadenylylation 89 3.5 Glutamate sYnthase 95 9s 3.5. 1 Purification 3.5.2 Properties 99 3.5.2.r Molecular weight 99 3.5.2.2 Effect of pH 99 3.5.2.3 Substrate requirement 99 3.5.2.4 K, values for substrates and NADPII 99 3.5.3 Inhibitor studies 103 t12 4. DISCUSSION 4.r Nitrate reductase tr2 4,2 Nitrite reductase TT7 4.3 Pathway of ammonia assimilation L22 I 4.3.1 Glutamine synthetase t23 4.3.2 Glutamate synthase r27 5. BIBLIOGRAPHY 131 I l-x LIST OF TABLES Table Pase No: 1 Purification of nitrate reductase 2 Electron donors for the purified nitrate reductase activitY 3 Effects of metal inhibítors on the MVlI-dependent nitrate reductase activitY 4 Effects of inhibitors on NADPH-dependent nitrate reductase activitY 5 Effects of inhibitors of sulphydryl-groups and flavin respectively on MVH-nitrate reductase activity 6 Purification of nitrite reductase 7 ¿ffects of various electron donors on nitriEe reductase activitY B Effects of flavins on NAD(P)H-nitrite reductase and its associated diaphorase activities 9 Stoichiometries for NADPH, NOt and NH3 for nitrite reductase 10 Effects of metal binding agents on NADPH- dependent nitrite reductase 11 Effects of inhibitors of sulphydryl-groups and flavin on NADPH-nitrite reductase I2 Effects of preincubation of nitrite reductase with reductant, cofactor andfor substrate on its activity (MSX) 13 Effects of L-methionine-Dl-sulphoximin e - and azaserine on the incorporation of r5N- label1ed (NH4)2504 into washed cel1s t4 Purification of glutarnine synthetase 15 Transferase and biosynthetic activities with various divalent cations 16 Effects of various nucleotides on transferase and biosynthetic activities t7 Effects of various concentration of L-methionine- Dl-sulphoximine on transferase activity 18 Effects of various amino acids on glutamine synthetase activi-LY x : i ì I Pase No: Table I 91 19 Bffects of organic acids on glutamine synthetase activitY I L on 20 Effects of snake venon phosphodiesterase 96 glutamine sYnthetase activitY 2t Purification of glutamate synthase 98 22 Substrates and NADPH requirements for ro2 glutamate sYnthase activitY 23 Effects of amino acids on glutamate synthase 107 activitY 24 Effects of various metabolites on glutamate 108 synthase activitY 25 Effects of various concentrations of azaserine 109 on glutamate synthase activity 26 Effects of various inhibitors on glutanate 110 synthase activitY xl- LIST OF F]GURES Figure Pase No: 38 1 A.