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A STUDY OF THE LACTIC AND MALIC OXIDATIVE ENZYMES OF TOBACCO SEEDLINGS GROWN IN ASEPTIC CULTURE PDF

72 Pages·02.729 MB·English
by  RIDENJOSEPH R.JR
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Preview A STUDY OF THE LACTIC AND MALIC OXIDATIVE ENZYMES OF TOBACCO SEEDLINGS GROWN IN ASEPTIC CULTURE

DOCTORAL DISSERTATION SERIES m* / m i tt m m e m t o u t m m mms of mao xtmci s u m m t o m e e m m ___________________________________ ikttHt I MttN^L AUTHOR _ _ _ _ _ _ _ _ _ _ NUN. STATi COil /fS! UNIVERSITY , DATE JdW /% P. DESREE PUBLICATION NO. 41199999998 I UNIVERSITY MICROFILMS » ANN A I I O I • MICHIGAN The Pennsylvania State College The Graduate School Department of Agricultural and Biological Chemistry A Study of the Lactic and Malic Oxidative Enzymes of Tobacco Seedlings Grown in Aseptic Culture A Dissertation by Joseph R. Riden, Jr. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 19^1 Approved: ^ ^ PrnfesS'orf of Phytochemistry Head, Department of Agri- cultural and Biological Chemistry AC KN 0 VVLEDGEMENT The writer wishes to express his appreciation to Dr. C. 0. Jensen for having suggested this problem and for his advice and criticism during the course of this thesis and its preparation; and to Dr. R. A. Steinberg, Department of Agriculture, Beltsville, Maryland, for demonstrating the technique of growing tobacco plants in aseptic culture. TABLE OF CONTENTS Page I INTRODUCTION ...................................... 1 II REVIEW OF LITERATURE..................... 3 A. Plant Oxidative Enzymes ...................... 3 B. Methods for the Determination of Oxidative Systems ........................... 10 III EXPERIMENTAL A. Development of Method 1. Sterile Culture of Tobacco Plants . . 13 2. Preparation of Plant Tissue for Enzyme Studies................« . . . 15 3. Details of Methods Used to Determine Oxidative Enzymes .......... 21 B. Measurement of Plant Enzymes 1. The Lactic Enzyme a. Cofactors Necessary for Activation................... 21j. b. pH S t u d i e s ................... 26 c. Determination of Carbon Dioxide Produced..................... 28 d. Cyanide Inhibition Study • . • • 29 e. Optimum Substrate Studies • • . • 32 f. Triphenyltetrazolium Chloride Studies . . • • • • • 32 g. Methylene Blue Studies on Acetone Extracted Tissue . . . . 36 h. The Effect of Sucrose Upon Lactic Oxidase ................ 38 Page i. Lactic Oxidase Content of Etiolated Tissue ............... 39 j. Purification of Lactic Oxidase . I4J. k. Identification of an End Product L^2 2. The Malic Enzyme...................... 2+3 a. Cofactors Necessary for Activation...................... lj.3 b. pH S t u d i e s ...................... 2+7 c. Determination of Carbon Dioxide Produced............... 2+7 d. Triphenyltetrazolium Chloride Studies.......................... 5>0 e. Methylene Blue Studies on Acetone Extracted T i s s u e ............. . 51 IV DISCUSSION OP R E S U L T S .......................... $$ V SUMMARY AND CONCLUSIONS ........................ 60 VI BIBLIOGRAPHY..................................... 62 A I INTRODUCTION "When distressed by the thought that in your own particular branch of biochemistry progress is slow, significant facts are few, and confusion is rampant, you will be consoled to learn that some other fields of biochemistry are in an even more primitive state of development*" " .... In view of the present state of knowledge of plant respiratory enzymes it would be unwarranted to accept such an assumption without proof." The first statement was made by Lardy (27) in 1951 while reviewing the latest in a series of volumes on enzymology. Maxwell (36) was speaking about the presence of cytochrome oxidase in corn embryos when he published the latter statement in 19^0. This thesis was undertaken in hopes of making a contribution to the now meager knowledge of plant enzymes and the methods used to study them. When a method appeared in the literature for growing tobacco plants in aseptic culture, the technique was adapted for this study of tobacco enzymes. By using sterile tobacco plants, the contaminating enzymes of bacteria, molds, fungi and other living matter found on tobacco grown under field conditions can be eliminated. By the proper handling of sterile plant tissue, we may be certain that we are studying only the enzymes which are 2 found in the green tobacco plant. Cigar leaf tobacco is an important crop in Pennsylvania agriculture. The tobacco must be cured or fermented before it is usable, such processes probably being caused by enzymes. These enzymes may be of bacterial or plant origin. Jensen and Parmele (23) have studied the fermentation of cigar-type tobacco and have isolated and identified seven species of bacteria from fermenting tobacco. Reid, McKinstry, and Haley (5l, 5>2) have also made a study of the bacterial flora of fermenting tobacco, and have shown that more than one billion bacteria per gram may exist in sweating tobacco. The contributions of bacterial enzymes to the changes taking place in fermenting tobacco are undoubtedly important. However there is not sufficient evidence to indicate that plant enzymes play no part in the process. The relation of enzymes of the tobacco plant and enzymes of microorganisms to the curing of tobacco is largely unknown. Before such knowledge is produced more information concerning the enzymes of tobacco itself and a study of the microflora must be available. 3 II REVIEW OP THE LITERATURE A. Plant Oxidative Enzymes. The oxidative enzymes of higher plants have not had the thorough investigation that has been given to the oxidative systems of animals and bacteria. A partial explanation for this may be the lack of a common oxidative system such as that found in all animals. The literature on plant enzymes is composed of many small pieces of information on the variation of oxidative systems between species of plants between differ­ ent parts of the same plant, and even between the same plant at various stages of growth. For example, Tolbert and Burris (61) reported that the leaves, but not the roots, of the mature tobacco plant contain glycolic oxidase, which is not present in etiolated tissue and which is not stable in young plants. Also, Albaum and Eichel (2) found that respiration in oat seedlings is mediated by the cytochrome system for the first 72 hours, but after that period is not inhibited by any of the cytochrome system inhibitors. As a result of this variation in the enzyme composition of plants, a review of the literature provides few general­ izations and many inconclusive statements regarding plant oxidative enzymes. The oxidative enzymes in plants may be classified either as dehydrogenases, which remove hydrogen atoms from substrates and pass them along a chain of carriers to atmospheric oxygen, or as oxidases which directly unite the 1* substrate's hydrogen or the substrate itself with oxygen. A terminal oxidase is an oxidase which accepts electrons from the dehydrogenase carrier chain, and reduces molecular oxygen, thereby completing the system. In a study of the electron carriers of plant dehydrogenase systems, Whatley (68) found only traces of diphosphopyridine nucleotide (DPN, Coenzyme I) in green leaves, although 20 ;ug. of triphos- phopyridine nucleotide (TPN, Coenzyme II) per gram of dry tissue weight was present. Lockhart (31) found flavoprotein in peas, beans and white potatoes, although it was stable only four days. Bach (4) reported that garden vegetables were rich in dehydrogenases, but contained very little DPN. In studying the dehydrogenases of plant tissues, it was necessary to add DPN to activate the dehydrogenases. Berger and Avery (7) have made a study of malic dehydrogenase in the avena coleoptile, using Thunberg techniques with thionin as the hydrogen acceptor. The enzyme was purified by ammonium sulfate fractionation and activity was obtained only after DPN and flavoprotein were added to the system. An accumulation of the end-product, oxaloacetic acid, inhibited the system, but could be removed by binding it with cyanide. The addition of various non-heavy metallic ions had no effect upon the system. Thionin was used in this study and prevented gaining any knowledge of the nature of the terminal oxidase. I'alic dehydrogenase has been found in corn embryos (2lj.), spinach leaves (69), pollen (5»5>) and parsley roots (65). $ Succinic dehydrogenase has been round in wheat term by Goddard (20) who reported that it was destroyed by acetone precipitation of the tissue. In spinach leaves, Bonner and Wildman (11) found it rapidly destroyed after injury to the leaves. This instability of succinic dehydro­ genase may well be the reason that Berger and Avery (7) were not able to detect it in oat seedlings. This dehydro­ genase has been found in corn embryos by Jensen, Sacks and Baldauski (2lj.). A detailed discussion of this enzyme has been given by Stauffer (55). Mathews and Vennesland (33) examined the action of formic dehydrogenase which they isolated from green pea seeds. They found DPN was necessary for the reaction and followed its reduction spectcphotometrically. The formic dehydrogenase reduced the DPN, while another enzyme catalyzed the oxidation of DPN by methylene blue. The plant formic dehydrogenase received no direct stimulation from the addition of the adenosine phosphates; this being in contrast to animal formic dehydrogenase. Formic dehydrogenase has been found in many plant seeds. Eerger and Avery (8) have studied the glutamic dehydrogenase of avena coleoptiles. They have shown that it requires DPN for its action and had an optimum pH from 6.5 to 8.7. The enzyme was inhibited by 0.001 molar copper - and cobalt solutions. Glutamic dehydrogenase also appears in spinach leaves (11). Isocitric dehydrogenase has been found in the avena

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