MALIC ENZYME IN POME FRUITS; SOME PROPERTIES AND RELATION TO FRUIT RIPENING By Isaac Klein AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 ABSTRACT MALIC ENZYME IN POME FRUITS; SOME PROPERTIES AND RELATION TO FRUIT RIPENING By Isaac Klein Changes in protein content and malic enzyme activity were studies in relation to protein synthesis during maturation and ripening of the apple (Pyrus Malus, L.) and the pear (Pyrus communis, L. cv. Bartlett), Protein N content of acetone dried powders prepared from cv. McIntosh and Wealthy apples decreased sharply at the end of the cell division stage and again prior to ripening. An increase in protein N con— tent of acetone dried powders prepared from several apple varieties and McIntosh apples collected at various locations was observed during ripening. Specific activity of malic enzyme in acetone dried powders was found to increase during the early stage of cell growth and during fruit ripening. Synthesis of malic enzyme during fruit ripening was investigated in detail. A vacuum infiltration procedure was developed to infuse carrier solutions containing radioactive amino acids into intact pome fruits. Infiltrated intact fruits were incubated 6 to 12 hours before analysis. Measurements of 14C L-phenylalanine incorporated into malic enzyme which was purified electrophoretically, indicated that the enzyme was preferentially synthesized during fruit ripening. Incorporation of the radioactive label into malic enzyme exceeded the overall incorpora— tion rate into all other proteins examined. Furthermore, experiments Isaac Klein in which endogenous ethylene was continuously withdrawn from the fruit to maintain a sub—catalytic concentration showed that induction and en— hanced synthesis of malic enzyme during fruit ripening requires the presence of ethylene. ElectrOphoretic separtion of pome fruit proteins revealed the presence of several uniformly spaced bands exhibiting malic enzyme activity. One band was invariably the most prominent as judged by protein stain and enzyme assay in the polyacrylamide gel. Fractionation of the enzyme on Sephadex G-200 gel and repeated electrophoresis of the prominent band indicated that all the other bands exhibiting malic enzyme activity were merely aggregates of the enzyme and not true malic enzymes. Two malic enzymes having pI of 4.55 and 5.45 were isolated from pear fruit which had been in storage. The two enzymes had equal electrophoretic mobilities in 5 to 7% acrylamide gel at pH 8.3 but could be separated by isoelectric focusing in natural pH gradients in a sucrose gradient and in acrylamide° The amino acid composition of the two enzymes was found to be similar with the noteworthy absence of threonine and reduced aspartic acid and lysine content in the pI 4.55 enzyme. Isoelectric focusing fractionation of Wealthy apple proteins in poly— acrylamide disc gels indicated the existence of the two malic enzymes from an early stage of development and a possible shift in the relative quantities of the two enzymes during fruit ripening. Improved preparative procedures developed during the course of the work such as extraction of malic enzyme and electrophoresis of proteins from ripening pome fruits and purification of malic enzyme by means of ammonium sulfate fractionation at the pI of the enzyme are presented. MALIC ENZYME IN POME FRUITS; SOME PROPERTIES AND RELATION TO FRUIT RIPENING By Isaac Klein A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 DEDICATION to my children, Ofra and Michal ii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Professor David R. Dilley for his guidance and encouragement through- out the course of this work and preparation of this manuscript. The author is also indebted to Doctors J. E. Varner, A. A. De Hertogh, M. J. Bukovac and J. R. Brunner, who served on the guidance committee and reviewed the manuscript. Thanks are extended to Dr. S. K. Ries who provided the facility for amino acid analysis. The author gratefully acknowledges the financial support of the National Science Foundation (GB-4656) which helped make this study possible. The patience and understanding of the author's wife, Shoshana, throughout the course of this graduate program is gratefully acknowledged. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ...... i.....;....x LIST OF TABLES ...:....,........- LIST OF FIGURES .....ro.....,...,.y INTRODUCTION ‘i'OOOO’JiCDV‘VDOOOOOO‘ REVIEW OF LITERATURE ... ~........ Nitrogen Metabolism of Pome Fruits. 0 OOO"0°0 Distribution and PrOperties of Malic Enzyme.. Malic Enzyme in Pome Fruitso. Intracellular Localization.. Function of Malic Enzyme in Function of Malic Enzyme in MATERIALS AND METHODS .. Enzyme and Protein Source... CIOCCOCQCOK‘. 0000000003 00000. 10.05006000000000 Tissue Other Than ,3 Pome Fruits....... i‘ "'3 0 Protein and Enzyme Extraction.. CaClz Treatment............. Electrophoresis.............. Isoelectric Focusing........ Amino Acid Analysis......... Malic Enzyme Assay.......... 0005'; ’3 fl 0‘). CO" Nitrogen and Protein Determinat Radioactive Measurement..... RESULTS AND DISCUSSION ............ 0905000 6 0 0 0001‘ ICU ) Amino Acid Incorporation Studies... Notes on Technique Employed....... Protein Determination.. 00000-3 O 0 “00000: 0 O h 000C600 0006500 9 O O 0 O 0 on O 0 D O C 000000 Fruits. 00000 05‘ 0(.-O O O C O C Protein and Malic Enzyme Recoveries..............o. Localization of Malic Enzyme' pyruvate + C02 1All enzyme classification in this thesis are according to the re- commendation (1964) of the International Union of Biochemistry in: Enzyme Nomenclature. Elsevier, 1965. l It was firmly established that apple peel discs, whether taken at different stages of ripening and analyzed immediately or taken from preclimacteric fruits and allowed to age up to 48 hours, develop the capacity to decar- boxylate malate (Neal and Hulme, 1958; Flood et al., 1960; Dilley, 1962; Hulme and Wooltorton, 1962; Rhodes et al., 1968a, 1968b; Hulme et al., 1968). The increase in malic enzyme and pyruvate decarboxylase (2 oxoacid carboxy~lyase 4.1.1.) activity was thought to be responsible for this phenomenon which was termed the 'malate effect.‘ The production of C02 without concomitant utilization of 02 by these two enzymes can account for the increase in R.Q. of peel discs taken from fruit at different stages of maturity (Neal and Hulme, 1958; Dilley, 1962), and presumably for the increase in R.Q. of the intact fruit as well (Hulme and Neal, 1957). Malate is abundant and is metabolized, as it will be outlined later, in many climacteric type fruits during ripening. Malic enzyme and NADP, are present although in variable quantity. Dilley (1962) considered the availability of NADP rather than malic enzyme content to be the regulating factor in malate metabolism in the mature preclimacteric apple. This hypothesis was supported by a report (Rhodes and Wooltorton, 1968) of increased NADP and NADPH content of apple and pear cortex during the climacteric rise. Participation of malic enzyme in the ripening pro— cess is therefore we11.established. Activities of several other enzymes, as will be outlined in the literature review of this thesis, were reported to increase or decrease throughout the development and ripening of fruits. Changes in enzyme activity can be the result of several factors: enzyme synthesis, change in rate of degradation, activation, removal of inhibition, or simply an artifact of preparative procedure. The last point applies particularly to enzyme preparation from fruits undergoing chemical and physical changes. A notable example was provided when earlier measurement of increased aldolase activity in ripening banana (Tager and Biale, 1957) was later shown to be due to inhibition of enzyme activity in the green fruit (Young, 1965). Definitive proof for dg_ngyg_synthesis of inducible enzymes can be obtained by one of two ways (Filner et al., 1969): 1) Induction of the enzyme in the presence of radio- active amino acid and purification and measurement of specific radioactivity throughout the newly synthesized polypeptide; 2) Induction of enzyme in the presence of pre—existing protein or free amino acid which has been density labeled with a stable isotope. Old and new proteins can be separated under these conditions on basis of density difference. A relatively rapid incorporation into the inducible enzyme compared to other proteins has to be demonstrated under inducible conditions. Fur- thermore, the possibility of decreased rate of degradation has to be ruled out if possible (Filner et al., 1969). The experimental approach to protein and enzyme synthesis by posr- harvest physiologists in recent years, tends to meet such criteria. In— corporation of radioactive amino acids into proteins of avocado (Richmond and Biale, 1966a, 1966b), pear (Frenkel et al., 1968), apple (Hulme et al., 1968), and banana (Smillie et al., 1969), were carried out success- fully, supporting the hypothesis (Richmond and Biale, 1966b) that en- zymes induced during the early phase of ripening catalyze the climacteric process. Direct evidence for d§_ngyg_synthesis of a specific protein, namely malic enzyme, was obtained in the work outlined in this thesis, part of which was reported previously (Frenkel et al., 1968). The role of malic enzyme in the physiology of the fruit is only partially understood. Regulation of malate or oxaloacetate metabolism, generation of reducing power, or C02 fixation under certain conditions are possible functions of the enzyme. It is feasible that the enzyme operates in more than one capacity and possibly in opposite direction (i.e., oxidative decarboxylation of L-malate and reductive carboxylation of pyruvate). Any attempt to assign a role to the enzyme has to take into account the fact that it is present in considerable quantity even before the onset of fruit ripening. Synthesis of a malic enzyme which operates in a different capacity or located in a distinct subparticle of the cell during the ripening of the fruit could provide some rationale for the synthesis of the enzyme during ripening. An investigation of the enzyme content and its molecular forms throughout the ontogeny of the fruit, as well as during ripening, was therefore carried out. REVIEW OF LITERATURE Nitrogen Metabolism of Pome Fruits Pome fruits were found to contain all the known protein amino acids (Hulme, 1958). Notable changes were found in asparagine and glutamine con- tent of apples during development. Asparagine content of apple cortex, but not that of the peel, rose sharply after full bloom, constituting 80% and 50% of the total soluble N during cell enlargement and maturation, respectively (Griffiths et al., 1950). Relatively high concentrations of glutamine were found in very young, and overmature apples (McKee and Urbach, 1953). The total N content of pome fruits was found to be in the limits of 0.02 - 0.35% of fresh weight for apples of different varieties and stage of development. Slightly higher values were found in pears (Hulme, 1958; Biale, 1964; Hansen, 1969). Changes in total N, protein N and soluble N in the apple fruit were studied by Hulme (1936, 1954), Robertson and Turner (1951), and Pearson and Robertson (1953). The total nitrogen content of an apple was found to increase during development, closely following the growth curve of the fruit. Protein N expressed as % of total N dropped sharply from approximately 80% at the time of full bloom to 45% at maturity. Protein N rose,-and soluble N declined during the climacteric rise in detached fruits (Hulme, 1936, 1954). Robertson and Turner (1951) obtained similar results which were expressed per cell basis rather than on a whole apple basis. In Granny Smith apples attached to the tree, the rise in protein N per cell during the climacteric was preceded by a sharp increase of soluble N (Pearson and Robertson, 1953). The climacteric rise in respiration could be delayed by high C02 concentration (Hulme, 1949) and removal of ethylene (Burg and Burg, 1966), or brought about prematurely by ethylene treatment (Hulme, 1948; Hansen, 1967). In all three cases the change in protein content promptly followed the respiration climacteric. Frenkel et a1. (1968), however, could dis— sociate protein synthesis softening and chlorophyll degradation in Bartlett pears from an apparent respiratory climacteric. Lewis and Martin (1965) reported the occurrence of at least one addi— tional peak in protein content of several apple varieties during senescence. The increase in protein content during senescence was greater than the in— crease during the climacteric. Li and Hansen (1964) studied changes in protein content and ripening capacity of Anjou and Bartlett pears during and after removal from storage. Protein content of fruits held in conventional storage (-1°C, 21% 02) in— creased, and the magnitude of protein synthesis associated with the cli- macteric after removal from storage decreased gradually. Controlled at— mosphere storage (-l°C, 2t0 2.5% 02, 2t0 3% C02) suppressed the rate of protein accumulation, and fruit ripened upon removal from storage showed a high net protein synthesis. The loss of ripening capacity of Bartlett pears, and abnormal ripening of Anjou pears after prolonged conventional storage was associated with loss of protein synthesis capacity (Li and Hansen, 1964). The presence and changes in content of enzymes found in climacteric type fruits was recently reviewed (Dilley, 1969), and only a few examples from pome fruits are presented here. The possible connection between respiration, catalase and 'oxidase' activity was investigated by Ezell and Gerhardt (1938, 1942). Catalase activity of Bartlett pears followed the respiration curve of the fruit, decreasing from a high value in June to a minimum in August. The data for ripening pears, attached or detached from the tree suggest that catalase activity increased again during the climacteric minimum. No correlation was found between catalase activity and respiration during ripening (Ezell and Gerhardt, 1938). Catalase activity of several apple varieties, in contrast, was low in July and increased steadily as long as the fruit was left on the tree whether a climacteric rise occurred or not (Ezell and Gerhardt, 1942). Activity of a—and s—amylase in 3 pear varieties was found to increase during development of the fruit. The highest activity was found in mature fruit, at a time when most of the starch disappeared from the fruit. Activity of a-amylase in detached fruit decreased during ripening and in- creased abnormally during cold storage, while that of B—amylase varied irregularly. The loss in ripening capacity after prolonged cold storage was correlated to the abnormally high activity of a-amylase (Maris McArthur- Hespe, 1956). Attempts were made to correlate changes in activities of enzymes involved in cell wall metabolism to the observed physical and chemical changes taking place in fruits. Characteristic chemical changes in pectins are demethylation of esterified carboxyl groups, and hydrolysis of the polygalacturonic chain (McCready and McComb, 1954). Weurman (1954a) ob— served that after an initial rise, the activity of pectin methyl esterase decreased during the development and ripening of pears on the tree. Har- vesting the fruit at maturity seemed to stabilize the enzyme level from a further drop in activity observed in fruits attached to the tree. Nagel and Patterson (1967) found a decrease in specific activity of pectin methyl esterase during growth and maturation of Bartlett pears. Although a small peak in specific activity of pectin methyl esterase was observed in fruits attached to the tree at a stage corresponding to the pre-climac- teric minimum, it was considered to be too small“toraccount for the marked deesterification taking place during ripening. A consistent change in polygalacturonase activity was observed only in 'ideal ripe' pears (Weurman, 1954b), after provision was taken to remove a thermolabile inhibitor pre— . sent in the fruit (Weurman, 1953). Participation of the pentose phosphate shunt in the metabolism of pome fruits during maturation and the early climacteric is evident from the studies of Faust (1965) and Meynhardt et al. (1965). Glucose-l 140 decarboxylation by McIntosh apple peel increased from 4 to 47% during the last two months prior to harvest, the time interval when anthocyanins are accumulating in the apple (Faust, 1965). DecarBoxylation of glucose-6 14C did not change during the same time interval. Inhibition of the TCA cycle by respiratory inhibitors increased simultaneously the activity of the pentose phosphate shunt and anthocyanin formation, indicating that the enhanced anthocyanin formation normally observed prior to maturation is a result of increase in the phosphate pentose shunt (Faust, 1965). Meynhardt et a1. (1965) concluded on basis of C1/C6 ratio studies and enzyme assays that the activity of the phosphate pentose shunt decreased during ripening and senescence of Bartlett pears. Activities of the following enzymes were also reported to increase during the climacteric rise in respiration: malic enzyme (Dilley, 1962; Hulme and Wooltorton, 1962), pyruvate decarboxylase (Hulme and Wooltorton, 1962), lipoxidase (Linoleate: oxygen oxidoreductase 1.13.1.13) (Looney and Patterson, 1967; Rhodes and Wooltorton, 1967), Ribonuclease (ribo- nucleate nucleotido-2'—transferase cyclizing 2.7.7.17), lipase (glycerol- ester hydrolase 3.1.1.3) and acid phosphatase (orthOphosphoric monoester phosphohydrolase) (Rhodes and Wooltorton, 1967). The increase in lipoxi- dase and chlorOphylase activities were found to slightly precede, and that of pyruvate carboxylase to extend beyond the respiration climacteric. In- creases in activities of all other enzymes coincided with the respiration climacteric. With the exception of malic enzyme (Frenkel et al., 1968), definite proof of enzyme synthesis as a cause of enhanced enzyme activity is lacking. Positive correlation between enzyme activities and chemical changes, however, are well documented. Hulme et a1. (1965) and Jones et al. (1965a) observed a relative in- crease in activities of succinic dehydrogenase, malate dehydrogenase, cytochrome reductase,diaphorase, and glutamate-oxaloacetate transaminase in mitochondria prepared from apple peel and pulp during the climacteric. The increase in activities of mitochondrial enzymes can be considered only tentative since it was impossible to arrive at a valid estimate of mito— chondrial protein, and the increase in activities were expressed on the basis of a constant weight of the original tissue from which the mito- chondria was prepared (Hulme, 1963). In contrast, Romani and Fisher (1966) obtained evidence indicating a decline in 140 leucine incorpora- tion into mitochondrial protein during the climacteric rise. Distribution and Properties of Malic Enzyme Malic enzyme was reported first by Ochoa et a1. (1947) in pigeon liver. Subsequently, in a series of reports, Ochoa and co-workers 10 l separated the enzyme from other activities, identified the substrates and products, and established the stoichiometry of the reaction (Ochoa et al., 1947; Mehler et al., 1948; Ochoa et al., 1948; Veiga Salles et al., 1950; Ochoa et al., 1950; Ochoa et al., 1951; and Harary et al., 1953). Malic enzyme from pigeon liver catalyzed the reversible oxidative decarboxylation of L-malate to pyruvate as well as decarboxylation of oxaloacetate and had pH optima of 7.4 and 4.5, respectively. NADP was exclusively essential in the oxidative decarboxylation of L—malate, and enhanced oxaloacetate decarboxylation. Mn++ , or Mg++ was also required. Oxaloacetate was thought to be an enzyme-bound intermediate. Purifica— tion studies by means of ammonium sulfate, or alcohol fractionation, ad- sorbtion on calcium phosphate or alumina C gamma gels failed to separate the two activities, indicating that both reactions are carried out by the same protein. The equilibrium constant of the wheat germ malic enzyme reaction was K = [L-malate] [NADP] / [pyruvate] [C02] [NADPH] = 19.6 liter x mole-l at pH 7.4 and 22.25°c. Korkes et al. (1950) and Kaufman et al. (1951) reported the exis- tence and prOperties of an adaptive malic enzyme in Lactobacilus arabinosus which was similar to that of pigeon liver, with the exception of the absolute requirement for NAD rather than NADP. Van Heyningen and Pirie (1953) assayed a reaction in cattle lens where NADP formed by malic enzyme was coupled to reduction of gluthione. The reaction proceeded at pH optimum of 7.4 and 8.2 with low and high concentrations of L-malate, respectively. A high pH optimum malic enzyme (8.5t0 9.0) was also assayed in the cell—free blood of the silk worm, where the enzyme was thought to 11 be involved in the control of redox potential and maintenance of ionic balance (Faulkner, 1956). The silk worm enzyme was inhibited by oxaloacetate, p—chloromercuribenzoate and certain sugar phosphates. Still another malic enzyme exists in Ascaris lumbricoides which is active only to the extent of 30% with NADP compared with NAD, and does not decarboxylate oxaloacetate (Saz and Hubbard, 1957). The properties of purified pigeon liver malic enzyme were reported by Rutter and Lardy (1958), who confirmed the association of oxidative de— carboxylation of malate and decarboxylation of oxaloacetate reactions, and the requirement of NADP for both activities. A similarity to the enzyme in cattle lens with respect to the dependency of the pH optimum on malate concentration, and inhibition of the enzyme by sulfhydryl inhibitors was also demonstrated. Location of a sulfhydryl group at the catalytic site was deduced from the protection afforded, either by Mn++ + malatecu'NADP + malate, against sulfhydryl inhibition. Crystallization and physical characterization of pigeon liver malic enzyme was successfully carried out by Hsu and Lardy (1967a, 1967b). The crystallized enzyme had a sedimentation coefficient of 10.0 and molecular weight of 2.8 x 105. Fluorometric binding tests with Mn++ AND NADPH showed that one mole enzyme binds four moles of coenzyme, indicating four subunits in the protein. Aged crystallized preparation lost activity gradually, and reactivation with dithiothreitol did not change the ratio of malate to oxaloacetate decarboxylation. Kinetic studies of the crystallized pigeon liver malic enzyme (Hsu et al., 1967c) showed an ordered mechanism of sub— strates binding and release of products, all of which inhibit enzyme activity. A reaction mechanism involving two conformational states of 12 the enzyme was postulated (Hsu et al., 1967c). An extensive study of partially purified malic enzyme of §;_ ggli (Sanwal and Smando, 1969a, 1969b) indicated an allosteric be- havior in the presence of allosteric inhibitors (oxaloacetate, Acetyl-CoA, NADH and NADPH). Ammonium and potassium ions and glyéhxgat high concentration desensitized the enzyme. The complex control of malic enzyme activity in §;_ggli_was thought to be nec- essary to coordinate channeling of metabolites in absence of rigid compartmentation (Sanwal and Smando, 1969a, 1969b). Malic enzyme is widely distributed in higher plants. The enzyme was assayed in 11 different families (Conn et al., 1949; Anderson et al., 1952), apples (Dilley, 1962; Hulme and Wooltorton, 1962), several succulent plants (Coles and Waygood, 1957; Walker, 1960; Mukerji and Ting, 1968), and trepical grasses (Slack and Hatch, 1967). Properties of the enzyme from wheat germ (Conn et al., 1949, Kraemer et al., 1951), apple (Dilley, 1966a) and Kalanchoe crenata (Walker, 1960) were studied in detail and found to be similar to those of the pigeon liver enzyme with some minor exceptions. Apple malic enzyme did not require NADP for oxaloacetate decarboxylation and that from wheat germ was strongly inhibited by the cofactor (70% inhibition with 4 x 10’5 DINADP). D-malate activated apple malic enzyme at rate limiting concentration of L-malate (Dilley, 1966a). Malic enzyme was also isolated, partially purified and charac- terized from the stem rust fungi Uromyces phaseoli (pers.). The peculiar observation was made that the rust fungi caused an increase in specific activity of malic enzyme in the infected leaf tissue (Rick and Mirocha, 1968). l3 Malic Enzyme in Pome Fruits The discovery that apple peel discs develop a capacity to decar— boxylate added malate during ripening (Neal and Hulme, 1958) focused the attention of post harvest physiologists on malic enzyme. Injection of malate into branches of apple trees caused an earlier climacteric and higher CO production in the fruit (Hulme and Neal, 1957). Hulme 2 (1961) suggested that malic enzyme may be involved in malate decarboxyla- tion in peel discs and in the development of the climacteric in the whole fruit. Dilley (1962) assayed for malic enzyme in the soluble fraction of protein extracts from McIntosh apples, and found an increase in spec— ific activity of the enzyme in post-climacteric fruit. Simultaneously, Hulme and Wooltorton (1962) obtained a mitochondrial preparation from the peel and cortex of Coxs Orange Pipin apple which contained malic enzyme and pyruvate decarboxylase. Association of malic enzyme with the mitochondria was discounted in all subsequent work by Hulme and associates on grounds of contamination. Thus, in a detailed study of malic enzyme activity, only a small portion of the total enzyme content of the tissue was found in the mitochondrial fraction (Hulme et al., 1963). The tendency of this fraction to increase after the climacteric was discounted. The potential activity of mitochondria increased dur- ing the climacteric, particularly in the pell tissue, and the increase started before any noticeable rise in CO2 evolution from the whole fruit. Malic enzyme activity in the soluble fraction of the cell rose steeply and coincided with the rise in respiration. Activities of mitochondria and malic enzyme in the pulp lagged behind those in the 14 peel. It was concluded that increased mitochondrial activity releases energy for malic enzyme and pyruvic decarboxylase synthesis, which in turn are responsible for the increased 002 production associated with the climacteric. Carbon dioxide production of fruits attached to the tree was one-third higher than that of detached fruits and the fruit was more watery when ripened (Hulme et al., 1963, 1965). Jones et al. (1965a) could not account for the extra C02 produced in fruit attached to the tree in terms of increased malic enzyme or pyruvate decarboxylase activity since activities of these enzymes in fruit picked at different stages along the climacteric and allowed to ripen at 12°C increased only so long as the activities of the enzymes on the tree increased. Respiration of fruit on the tree, however, continued to rise above and beyond the increase in malic enzyme content. The conclusion was made that an increase in activity of malic enzyme and pyruvic decarboxylase is largely responsible for the climacteric rise in respiration up to a maximum rate attained in detached fruit and that the excess respira— tion above this value on the tree stems from other sources (Jones et al., 1965a). Development of the malate effect in a model system of preclimacteric aged peel discs could be prevented by inhibitors of pro- tein and RNA synthesis, and by low oxygen tension which inhibits ethyl- ene synthesis. Peel discs developed the capacity to synthesize ethylene during aging, and application of ethylene shortened the time required for the development of the malate effect but did not change the magnitude of carbon dioxide production (Rhodes et al., 1968b). Rhodes et al. (1968b) and Galliard et al. (1968b, 1968c) also investigated the time course of events in peel disc taken from fruit 15 at the preclimacteric minimum. The first noticeable change was enhanced l4C acetate incorporation into fatty acids (2 to 3 hours of aging), fol- lowed by ethylene synthesis (6 to 8 hours). Incorporation of 14C uri— 14C valine peaked next (8 hours), and finally the development dine and of the malate effect reached its maximum (16 to 24 hours). The same sequence of events was found in discs sampled from whole fruits at suc- cessive stages of ripening (Hulme et al., 1968). The development of y the malate effect along the climacteric rise in respiration depends therefore on protein synthesis, and presumably it is the result of malic enzyme induction at an earlier stage. Similar but more conclusive re- sults were obtained by Frenkel et al. (1968) who studied protein synthe- 14C sis in the intact pear fruit. Infiltration of whole fruits with L—phenylalanine resulted in sufficient labeling of individual proteins for detection. Electrophoretic separation of the labeled proteins at different stages of ripening revealed that 14C amino acid was prefer- entially incorporated into malic enzyme during the early to mid- climacteric stage. Intracellular Localization Malic enzyme is considered to be a cytoplasmic enzyme (Kun, 1963). Several investigators, however, attempted to associate it with mito- chondria, chloroplasts, or both. Brandon (1967) claimed to assay malic enzyme in mitochondrial preparations from Bryophyllum tubiflorum Harv. Careful evaluation of malic enzyme assays and the method used does not support his claim (Brandon, 1967, see Figures 3 and 4. Also, Procedure 11 of "mitochondrial" preparation in section of Materials and Methods). l6 Mukerji and Ting (1968) isolated mitochondria and chloroplasts in aqueous and non-aqueous media from phylloclads of the cactus Opuntia ficus Indica Mill. Malic enzyme and other enzymes associated with CO 2 metabolism were found both in mitochondria and chlorOplasts after low speed gradient centrifugation. Malic enzyme was also located in chlor— ophyll-rich layers after gradient centrifugation of chloroplasts pre- pared from trOpical grasses in non-aqueous media (Slack and Hatch, 1967). Sonicated 'heavy' mitochondria prepared from bovine adrenal cortex re— leased an isozyme of malic enzyme which was shown to be distinct from the cytOplasmic enzyme as indicated by a different elution pattern from a DEAE-cellulose column and also by its kinetic properties. The mito- chondrial isozyme which constituted about 10% of the total activity was inhibited by the respiratory uncouplers dicumarol and dinitrOphenol (Simpson and Estabrook, 1969). The few reports listed above on parti- culate localization of malic enzyme are hard to assess since the rela- tive amounts of enzyme in the cytoplasmic and the particulate fraction were rarely presented, and insufficient purification of mitochondria or chloroplasts on gradient centrifugation can be a common cause of artifacts. Function of Malic Enzyme in Tissue Other Than Fruits The direction in which malic enzyme Operates in zixg has been the subject of considerable investigation. Utter (1959) and Krebs (1954) suggested originally that the enzyme participates in glucogenesis by converting pyruvate to malate which is then oxidized to oxaloacetate and phosphoenolpyruvate. Several lines of evidence led later to the 17 widely accepted hypothesis that malic enzyme in certain animal tissue supports lipogenesis by supplying NADPH rather than participating in glucogenesis. Conditions known to increase lipogenesis were found to promote an increase in malic enzyme content of liver and adipose tissue in rats (i.e., 1% thyroid or insulin injection, high glucose diet, or restoration of normal diet after starvation). In contrast, conditions promoting gluconeogenesis (i.e., diabetes, starvation) or utilization of fats, decrease malic enzyme content of the tissue (Young et al., 1964; Wise and Ball, 1964; Lardy et al., 1964). Young et a1. (1964) preposed a mechanism of trans—hydrogenation from NADH to NADP mediated by the coupled reaction of malate dehydrogenase and malic enzyme (oxa- loacetate-9 malate-9 pyruvate + C02). Wise and Ball (1964) suggested that oxaloacetate for the above scheme is generated by pyruvic carboxylase (pyruvate + C02—9 oxaloacetate-+ L malate-+ pyruvate + C02). Consider- I ation of potential activities, levels of substrate required, and tissue and intracellular disfgibution indicate that CO2 fixation proceeds through pyruvate carboxylase rather than malic enzyme (Wood and Utter, 1965). However, in view of the absence of any other enzyme that can fix C02 in skeletal and cardiac muscle, Wood and Utter (1965) postulated that malic enzyme is responsible for the maintenance of oxaloacetate levels '-9 L-malate-* oxaloacetate). Wood and 2 Utter (1965) also suggested a hypothetical role for malic enzyme in for the TCA cycle (pyruvate + CO hydrogen transport. Accordingly, NADPH generated by the pentose phos- phase cycle drives the malic enzyme reaction in the direction of reduc- tive carboxylation, and the malate formed then serves as a precursor of oxaloacetate in the mitochondria. Recently it was proposed that steroid hydroxylation in bovine adrenal cortex mitochondria is carried out by NADPH generated by mito- chondrial malic enzyme (Simpson and Estabrook, 1969). Hydrogen transfer from cytoplasm to mitochondria occurs supposedly by way of a malate shuttle. Cytoplasmic malic enzyme accordingly operates in the direction of'reductive carboxylation and that of the mitochondria in oxidative decarboxylation. Ting and Dugger (1965, 1967) presented evidence for a hydrogen transport system in corn root tips mediated by coupling several enzymes. l4C labeled sodium bicarbonate resulted Incubation of corn root tips in in considerable incorporation of the label into malic acid, or pools directly connected to malic acid. Radioactive 14CO2 with a 3 hour half— life was subsequently released from the tissue. Crude homogenates of corn root tips contained C0 metabolizing enzymes that when coupled 2 could fix and release C02. The kinetics of CO2 fixation and release suggested a coupled cycle (phosphoenolpyruvate + CO2—~ oxaloacetate-s L-malate-‘pyruvate + C02) in which C0 fixation is carried out by 2 phosphoenolpyruvate carboxylase and decarboxylation by malic enzyme (Ting and Dugger, 1965, 1967). Regulation of the hydrogen transport system in corn roots was carried out by feed back non-competitive in— hibition of phosphoenolpyruvate carboxylase with malic acid (Ting, 1968). Indirect evidence indicates that in crassulacean acid metabolism malic enzyme is involved in acid depletion rather than C02 fixation (Walker, 1962). Two of the many arguments put forward involved in_yi££2_ studies. Acid synthesis, either by whole leaves or in the coupled re- action of phosphoenolpyruvate carboxylase - malic dehydrogenase l9 (phosphoenolpyruvateee oxaloacetate-e malate) was found to be progres- sively inhibited by increasing concentrations of carbon dioxide. Malate synthesis by malic enzyme on the other hand increased (Walker, 1960). Similarly, Brandon (1967) showed that malate decarboxylation by malic enzyme increased linearly when the temperature was raised, without reaching a peak value even at 530C. Malate synthesis by phosphoenol- pyruvate carboxylase - malate dehydrogenase, however, increased up to 359C and then decreased. The net result, after appropriate correction for NADH oxidase, indicated that temperatures higher than 15tx)200C favor acid consumption which is in agreement with results obtained in whole leaves. Thermodynamic consideration also indicate that CO2 fix— ation proceeds by way of phosphoenolpyruvate carboxylase - malate de- hydrogenase, whenever these enzymes are present in the tissue, rather than by way of malic enzyme (Walker, 1962). Function of Malic Enzyme in Pome Fruits / l4C CO2 incorporation capacity of McIntosh apples was found to increase five-fold during the last month prior to harvest (Allentoff, 1954b). The rate of incorporation was maintained at a high level during _ ._ 14 storage. Two thirds of the C02 incorporated was found in malic acid (Allentoff et al., 1954a). The high C02 concentration found in apple fruits under certain conditions and the lack of information, or inabil- ity to demonstrate presence of any other enzyme involved in CO2 fixation points to the possibility that malic enzyme may participate in this process. The extensive studies on malate decarboxylation, however, indicate a role of oxidative decarboxylation for the enzyme, at least 20 during the later part of the climacteric. During the early climacteric,malic enzyme along with glucose-6— phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, may supply NADPH for fatty acid synthesis. However, both fatty acid synthesis (Galliard et al., 1968b) and the activity of the pentose phosphate shunt (Meynhardt et al., 1965) were reported to diminish during ripening. In contrast, activity of malic enzyme increased up to the climacteric peak in respiration (Rhodes et al., 1968a, 1968b; Hulme et al., 1968). The observed induction of malic enzyme synthesis (Frenkel et al., 1968), therefore, can not be explained as needed to support active fatty acid synthesis. Of interest in this connection is the quantitative analysis of lipids in apple fruits which showed that although nearly equal amounts of total lipids are present in pre— and post—climacteric fruit, a marked change in composition does occur during ripening (Galliard, 1968a). Lipids containing linolenic acid disappeared and sterols had been synthesized. A number of enzymes involved in sterol synthesis and hydroxylation require NADPH (Mahler and Cordes, 1966). Apart from its contribution to C02 and NADPH production, malic enzyme undoubtedly has a role in the regulation of mitochondrial activ— ity. Walker (1962) pointed out that if malate is an intermediary storage product accumulating in the vacuole, then malic enzyme provides the mechanism for its mobilization for mitochondrial oxidation under. conditions where levels of C4 dicarboxylic acids are raised and pyruvate is in short supply. It can be stated on the basis of available information that malic acid is an intermediary storage product in the fruit. Malic acid was 21 found to be the major acid in mature apples (Hulme and Wooltorton, 1957). Malate was also found to be the main acid lost after prolonged air stor- age of Bartlett and Anjou pears, although the Bartlett pear contained measurable quantities of other acids (Li and Hansen, 1964). Respiration studies of apples indicate that the respirable substrates are carbohy— drates (Fidler, 1948, 1951), and organic acids (Kidd et al., 1950). Fidler (1951) demonstrated that the amount of carbohydrate + acid lost in respiring apples was equal to CO2 + ethanol + acetaldehyde formed. Acid loss was equal in aerobic and anaerobic conditions, but carbohydrate breakdown was spared in the presence of oxygen. Consid- erable amounts of ethanol and acetaldehyde accumulated in anoerobic conditions (Fidler, 1951). Acetaldehyde was also found to accumulate in apple peel discs in amounts close to the theoretical value of 1:2:1 ratios of malate:C02: acetaldehyde and 1:1:1 of pyruvate:C02:acetaldehyde when incubated in malate and pyruvate, respectively (Neal and Hulme, 1958). The fate of acetaldehyde in the fruit is unknown. Only small quantities of acetal- dehyde were found normally in apples and acetaldehyde added to the air surrounding the fruit was taken up and part of it reduced to ethanol, the remainder oxidized to C02. Carbon dioxide evolution from the fruit declined after an initial rise and carbohydrate oxidation was spared (Fidler, 1968). Thus, it appears from the above information that one possible path is successive decarboxylation of malate by malic enzyme and pyruvate decarboxylase to yield acetaldehyde which becomes oxi— dized eventually under aerobic conditions or reduced to ethanol anaerobically. An alternate path of malate utilization can be the 22 successive decarboxylation to yield acetyl-CoA for mitochondrial oxida- tion. Evidence accumulated in recent years indicates that the potential activity of mitochondria isolated from apples during development and ripening follow closely the respiration curve of the fruit, decreasing during growth and maturation, and increasing during the climacteric (Hulme et al., 1963; Hulme et al., 1965; Jones et al., 1965a). The reasonable assumption can be made that in aerobic conditions and the presence of more active mitochondria close to the climacteric peak, a large portion of the pyruvate formed by malic enzyme will be utilized directly in the TCA cycle, rather than decarboxylated to acetaldehyde. Without regard to the path in_yiyg,malate mobilization by malic enzyme contributes to the respiration of the fruit beyond the readily detectable 002 production attributed to it. It can be concluded that malate stored in the vacuole is mobilized by malic enzyme in increasing rates during the climacteric rise in respiration. The immediate con- sequence is an upsurge in C02 production which is readily detectable (the 'malate effect'). Mobilization of malate by malic enzyme is likely to serve a dual function: a) to supply reduced triphosphopyridine nucleotide for synthetic processes (i.e., fatty acid synthesis, sterol synthesis and hydroxylation) and b) to increase the supply of respir- able substrate for enhanced mitochondrial activity during the climac- teric rise. MATERIALS AND METHODS Enzyme and Protein Source Apples (Pyruslialus L): All studies on apples were conducted using acetone dried powders prepared from the following varieties and stages of development: 1) McIntosh and Wealthy apples at 25, 46, 58, 76, 88, 102, 117 and 131 days from full bloom; 2) McIntosh, Wealthy, Northern Spy, Jonathan, Red Delicious, Golden Delicious, Grimes Golden, Wagner, Fameuse and R. I. Greening at harvest maturity and after 12 days of ripening at 20°C; 3) McIntosh picked at harvest maturity from several locations throughout Michigan, and after 12 days ripening at 20°C. . Pears (Pyrus communis 1:): Studies on Bartlett pears were con- ducted using either fresh fruit or acetone powders prepared from the fruit. Fresh fruit for ripening studies were picked at harvest maturity and stored 0°C in air up to 2 months prior to use. Fruit employed up to 3t0 4 months were stored 0°C, enclosed in polyethylene bags contain- ing hydrated lime. Malic enzyme purification studies were conducted on fruit stored 0°C in air or in polyethylene bags containing hydrated lime for up to 8 months. Acetone dried powders of Bartlett pears were prepared after fruit was removed from storage and left to ripen up to 10 days at 20°C. All studies were conducted on the cortex of the fruit, except where specified otherwise. Whole fruits were used at 25 days after full bloom. 23 24 Acetone dried powders were prepared according to the low tem- perature procedure of Clements (1965) and stored in flasks with a tube of phosphorus pentoxide, at —250C under vacuum. Protein and Enzyme Extraction Acetone powders were extracted in open tubes or centrifugal fil— ters (Gelman Instruments, Ann Arbor, Michigan). Standard extraction solution contained 0.1 M Tris-triCine, 0.35 M mannitol, and 5 x 10’4 M EDTA at pH 8.5‘K>9.5. Successive extractions with 8, 6 and 6 ml per 500 mg acetone dried powder were carried out to obtain quantitative measurement of protein and malic enzyme content of the powders. The first of the three extractions was carried out for 1 hour, and the sub- sequent two for 1/2 hour, with occasional mixing, at O'mo 4°C. At the end of the extraction period, solutions were separated from residue by centrifugation at 2000 x g (10 minutes). Pectic substances extracted with the proteins from acetone dried powders prepared from ripe fruit clogged the centrifugal filters. To circumvent the problem Open tubes were used in conjunction with high speed centrifugation (100,000 x g, 15 minutes). The protein complement of successive extracts was the same, but protein concentration drOpped exponentially (Frenkel et al., 1969). To avoid concentration steps, only the first extract was used for electrophoretic studies. Fresh fruits were cut into small pieces (ca. 1 cm square) and vac- uum infiltrated with a buffer solution (ratio of tissue to solution was 1:2) containing 0.1to 0.3 M Tris, 0.3 M mannitol, and 0.05tD'0.1 M CaClz (pH unadjusted). Occasionally, as indicated in the text, CaClz , 25 was omitted and 0.1to 0.2 M K2C03 and 4 x 10—3 M diethyldithiocarbomic acid was used rather than Tris buffer. Excess infiltration buffer (that not taken up by the tissue) was decanted. The fruit tissue was ground in an electric mortar and pestle, squeezed through a nylon cloth and centrifuged at 15,000 x g (10 min- utes). The pH of the supernatant solution, which contained the malic enzyme activity was between 8.5t0 9.5. Fresh fruit extracts were stirred with 2 to 10% insoluble poly- vinylpyrrolidone (Polyclar AT powder, General Aniline and Film Corpora- tion, New York, N. Y.) for 15 to 30 minutes, according to Jones et al. (1965b) and Klepper and Hageman (1969). Polyclar AT was removed by centrifugation at 15,000 x g (10 minutes). Treatment with Polyclar AT, when employed, was carried out either after extracts were squeezed through the nylon cloth or after the extract was centrifuged. In purification studies of malic enzyme, two successive treatments with Polyclar AT, at pH 9.5 or 7.5 and 5.5 were employed. CaCl2 Treatment Acetone powder or fresh fruit extracts (when prepared without CaCl2 in the extraction buffer) were adjusted to pH 7.3t0 7.8 with 25% acetic acid, and the CaCl concentration was brought to 0.02 M with the 2 addition of 0.5 M CaClZ. Vigorous mixing (without foaming) and quick addition of acetic acid and CaCl2 was required to ensure effective mixing since extracts containing pectic substances gelled instantane- ously. Insufficient mixing resulted in greater protein and enzyme loss. The CaClZ treated extracts were centrifuged immediately at 100,000 x g 26 (15 to 30 minutes) to precipitate the Ca—pectate gel. Electrophoresis Polyacrylamide gel was used as the supporting media for disc gel or vertical slab electrOphoreSis. Current was maintained at 8.8 mA per cm2 cross section for disc electrophoresis at 0°C, and at 7.7 to 10.0 mA per cm2 for vertical slab electrophoresis at 10 u>15°c. Acrylamide running gels (5 to 7%) and spacer gel for disc electrophoresis were prepared according to Davis (1964). Vertical slab gels were prepared by mixing the appropriate quantities of acrylamide, N,N-methylenebis— acrylamide, Tris, HCl and water (in the same proportion as used in disc electrophoresis) to prepare 100 m1 of 6 or 7% gel. Gelling was affected by the addition of 0.1 ml of N, N,Nl, Nl-tetramethylethylenediamine and 200 mg ammonium persulfate immediately before‘pouring the solution into the gel chamber. Protein extracts were treated with CaCl2 and centri- fuged at 100,000 x g (15 H330 minutes) before electrophoresis. Up to 0-2 mg and 5t0 10 mg protein was loaded per disc column or slab gel, respectively. Electrophoresis was conducted for 1 to 4 hours, until the bromophenol blue tracking dye migrated 3 to 5 cm in the running gel. At the end of the electrophoresis gels were stained for 15 minutes with 0.1% aniline blue black dissolved in H20: ethanol: glacial acetic acid in the ratio of 5:5:1, and destained electrically in 7.5% acetic acid. Malic enzyme was located in the polyacrylamide gel following electrophoresis by incubating gels in a solution containing 100 umoles glycylglycine, 50 umoles L-malate, 10 umoles MnSO 0.37 umoles 4’ NADP, 0.8 mg m-nitroblue tetrazolium chloride (Nitro BT) and 0.14 mg 27 phenazine methosulfate perl.ml at pH 7.3. The gels were covered with approximately two volumes of the incubation solution and left to develop at 20°C. Precipitation of the purple diformazan at the site of the enzyme occurred within two hours. Pictures of the gels were taken with a Polaroid MP-3 industrial View land camera (Polaroid Corporation, Cambridge, Mass.) with incan— descent light illuminating the gels from below. Isoelectric Focusing Isoelectric focusing in natural pH gradient was performed according to Haglund (1967). Carrier ampholine and electrofocusing columns were purchased from LKB-Productor AB, Bromma, Sweden. Linear sucrose gradients of 110 ml volume containing 1% carrier ampholine pH 3 to 10 or 3 to 6 were used. Temperature was maintained at 0: 0.050C at 700 volts potential. Samples containing up to 20 mg protein were incorporated into the gradient, or layered as a distinct band in a pOSitlon that ultimately had an iso- electric pH of 6.0 to 8.0. Electrofocusing was terminated several hours after the conductivity of the column was stabilized (total of 36 to 48 hours), and 2 to 3 ml fractions were subsequently collected. Absorbancy at 280 nanameters was monitored continuously With flow through cells in a Beckman D.U. spectrOphotometer equipped with a Gilford instruments absorbence in- dicator and automatic cuvette positioner, or measured after fractions were collected. pH of the fractions were measured on a Photovolt pH meter (t 0.02 pH unit). Enzyme activity could be assayed without interference from the carrier ampholine. Amino acid analysis of malic enzyme was performed following isoelectric 28 focusing after proteins were precipitated and washed with ammonium sulfate to remove carrier ampholine. ElectrOphoresis of fractions collected from isoelectric focuSing was conducted without remoVing carrier ampholine. Subsequently, however, carrier ampholine had to be removed from the acryla- mide gel before proteins could be Stained (see below). IsoeleCtric fotuSing in polyacrylamide gels containing 2% carrier ampholine were made up as follows: 1.4 ml of 28% - 0 735% acrylamide—N, N, methylene Bis acrylamide 0.4 ml of 40% carrier ampholine pH.3 K)10 or 3t0 6 2.0 ml of 40% sucrose 1.0 ml of 0.14% ammonium persulfate l,Nl—tetramethyiethylenediamine 1.0 ml of 1.0% N,N,N 2.2 ml of H20 Water and carrier ampholine volumes were adjusted appropriately when 8% carrier ampholine was used. Solution to make up 12 gels was mixed, degassed, and poured into the glass tubes capped at the bottom. The upper surface w W layered with H20 and the tubes left to gel for 30 minutes. The water was removed after the gels polymerized and 0.1 U30 2 ml or the protein sample in 10% sucrose was layered on the top of the gel. The protein sample was layered with 0.05 ml of 5% sucrose, and the rest of the space in the tube filled with 2% ethanolamine. Tubes were placed in position and the upper eleCUIde compartment was filled with 2% ethanolamine, and the lower electrode compartment with 1% phosphoric acid. Electrofocusing was conducted at 0 to 4°C for 14 to 16 hours at 2.5 V per disc gel 5 cm long. Malic enzyme was localized in the gel by the previously described procedure without interference 29 from the carrier ampholine. Protein stain with aniline blue black could be obtained only after :emovai of the carrier ampholine. A satisfactory procedure consisted of fixrng the protein in the gel for 1 hour with 10% TCA, and removing carrier ampholine by eleCtric charge (2 to 3 hours 'destaining'). Subsequently, gels could be stained and destained by the usual rocedure em lo ed after electro horeSis. P Amino Acid Analysrs Purified malic enzyme was hydrolyzed under vacuum in 6 N HCl for 22 hours at 110°C. At the end of the hydrolysis HCl was removed under vacuum and the residue was taken up With sodium citrate - sodium hydroxide buffer adjusted to pH 2.88 with 6 N HCl. Amino aC1d analysis was carried out using Technicon Auto Analyzer. A single glass column, (6 x 140 mm) packed with Chromosorb Type B was used for amino acid chromatography, which lasted 19 hours. Amino acids were eluted with sodium citrate butter (increasing pH and salt concentration) at 453C for 2 hours, and 603C for 17 hours. Malic Enzyme Assay Oxidative decarboxylation of L-malate was measured spectrophorometriciy at 340 nanameters. The standard assay consisted of 300 moles glycylgiyci:a, 3 umoles MnSO4, 0.5 u moles NADP, 36 u moles L-malate, and up to 20 enzyme units in a final volume of 3 m1, pH 7.3 and 20°C. One enzyme unit is defined after Ochoa (1948) as the amount of enzyme that causes 0.01 0.D. unit change Per minute. Oxaloacetate decarboxylation was assayed in the Warburg respirometer. 30 “he standard assay contained 300 g moles sodium acetate — acetic acid ouffer, 3 u moles MnSO4, 30 u moles oxaloacetate (freshly prepared), 1 mg bov1ne serum albumin and up to 100 enzyme units in a final volume if 3.2 ml, at pH 5.1 and 250C. Warburg vessels were equilibrated for 15 minutes before the reaction was started by tipping the oxaloacetate from the side arm. Readings were taken at 5 or 10 minute intervals for 1 hour. Jitrogen and Protein Determination Total and 80% ethanol insoluble nitrogen content of acetone powders was determined by titration following distillation of a micro-Kjeldahl digest consisting of 3 ml H2804 containing 0.3 m moles CuSO4. Protein content of fresh fruit or acetone powder extracts and that of various protein preparations was estimated by the Biuret (Gornal et al., 1949) or Lowry (Lowry et al., 1951) methods on aliquots precipitated and washed twice with 80% ethanol or 10% TCA. imino Acid Incorporation Studies In order to determine amino acid incorporation into specific protein: during the respiratory climacteric and fruit ripening, a vacuum infiltra— tion procedure (Figure l) was developed to introduce controlled amounts of solutions containing radioactive amino acid into the fruit. Intact fruits were employed with relatively long incubation times in order to facilitate de— tection of radioactive label in small quantities of purified proteins. A dypodermic needle (containing a cleanout wire to prevent clogging) was Inserted from the calyx end into the central cavity region of the fruit. 31 Figure 1. A setup for vacuum infiltration of intact fruits. 32 ~«— Syringe 1....1 1 V V __1 1‘ Solution ocuum +~____ . ‘ stopper 11111111 1 ml +— Glass jar Hypodermic needle Fruit 33 After removal of the wire, the fruit with needle in place was attached to a syringe fitted into a rubber stopper and positioned in a large mouth glass chamber. The stem end of the fruit was brought to rest on the bottom of the glass chamber by adjusting the level of the syringe. Solution was introduced into the fruit through the syringe as the chamber was evacuated at approximately 100 mm Hg. This procedure provided for rather uniform distribution (as judged by dye distribution) of solution. The volume of solution administered to each fruit was controlled to pro- vide 0.9to 1.1 m1 of the infiltration solution per 10 g fresh weight. Twenty to forty minutes were required to infiltrate a 125to 150 g pear fruit. The infiltrating solution consisted of 0.35 M mannitol and 1 x 10‘“ M 120 L-phenylalanine containing 0.05 p C uniformly labeled 14 C L-phenyla- lanine (specific activity 336 millicuri/millimole) per ml. During the ex- periment carried out in the fall of 1967 fruits were infiltrated at 0, 2, and 4 days after removal from storage and incorporation of 14C L-phenyl— alanine was allowed to proceed for 6, 12, and 24 hours before the fruits were analyzed. At the appropriate time part of each fruit was used to measure 14C L—phenylalanine incorporation into the TCA precipitable frac— tion (Frenkel et al., 1968). Two replicates of acetone powders were pre— pared from the rest of the fruit. A composite sample for each replicate was taken from 4 fruits. In the experiment carried out in the summer of 1968, fruit were picked at harvest maturity and placed immediately in desiccators (12 liter volume) under reduced atmospheric pressure according to the method of Burg and Burg (1965b). Desiccators were kept at room temperature at 150 mm Hg. A flow rate of 4.2 liter per hour of oxygen or oxygen containing 34 6000 ppm ethylene was maintained through the desiccator from compressed oxygen cylinder. Mixture of oxygen-ethylene was prepared by injecting ethylene into an evacuated cylinder followed by equilibration with a full compressed oxygen cylinder tank. Provision was taken to humidify the atmosphere inside the desiccators. Fruits were removed from the desiccators at 22, 44, 88, and 110 hours, vacuum infiltrated with the standard infiltration solution and returned to the disiccators to allow l4C L-phenylalanine incorporation for 6 and 12 hours. At the end of the incorporation time 2 replicates of acetone powders were prepared, each from 4 fruits. At the end of the experiment (110 hours) the remaining fruits were placed in an automated gas analysis system employing an infra— red C02 analyzer (Dilley, 1966b) to measure the respiratory status of the fruit. Incorporation of 14C L—phenylalanine into malic enzyme during the climacteric was measured after the enzyme was separated from other proteins by electrophoresis (disc or slab gel). Malic enzyme was localized in the disc gels and a narrow band containing the enzyme was cut out. Duplicate samples were placed in scintillation vials containing 0.4 ml of 30% H202, sealed and heated until the gel was solublized (3 H14 hours at 60°C). Scintillation mixture was added to the vials for radioactive counting. Results are expressed as cpm per enzyme unit applied on the gel. Two alternate procedures were employed to analyze results of electrophoresis on slab acrylamide gels: l) The r.f. value of each protein band (visualized by protein stain) was predetermined for each sample to be analyzed. Subsequently, at the end of electrophoretic separations each band was cut out according to its r.f.. The gel pieces 35 were left over night in 0.1 M.phosphate buffer pH.9.5 to allow diffusion of proteins from gel into buffer. Radioactive counts and estimate of mg protein applied on each slab gel yielded a specific radioactivity value (dpm per band per 2.5 mg protein applied on the slab gel). 2) Alternatively, malic enzyme was localized on a piece cut out from the edge of the slab gel. A band corres- ponding to the position of the enzyme was cut from the remaining gel, macerated with a pestle and extracted three times successively with 0.05 M KCl. Recovery of 80 to 90% of the enzyme activity applied on the gel was obtained. Malic enzyme assay and radioactive count (corrected for quenching) yielded a specific radioactivity value (dpm per enzyme unit). Radioactive Measurement All radioactive measurements were made with a Packard liquid scintillation spectrometer Model-b. The scintillation mixture consisted of: 4.5 g BBOT [2,S-bis-(2-(5-tert-buty1benzoxazolye))-theophene], 80 g naphthalene, 385 ml xylene, 385 ml p-dioxane, 231 m1 ethanol, and 37 g Cab-o-sil (thixotrOpic gel powder obtained from Cabot Corporation, Boston, Mass.). Counting efficiency of 75 to 81% was obtained with up to 1 ml of the various aqueous solutions or suspensions mixed with 15 m1 of the scintillatiOn solution. RESULTS AND DISCUSSION Notes on Techniques Employed Acrylamide gel electrophoresis (Davis, 1964) offered the best available means to separate large numbers of crude protein extracts into highly purified proteins. Methods introduced to localize enzymes, section and solublize the polyacrylamide gel made the technique applicable to the study of amino acid incorporation into proteins. Early attempts to separate apple and pear proteins, extracted from acetone dried powder or fresh fruit during the climacteric rise failed. Typical resu1ts of electrOphoresis of protein extracts obtained from acetone dried powders prepared from Bartlett pears at successive stages during ripening are demonstrated in Figure 2A (protein stain)and Figure 2B (malic enzyme assay). Large portions of the applied protein sample (including malic enzyme) were retained on the top of the Spacer gel and deformed it. Longitudinal streaking and irregular patterns rather than horizontal bands were seen on the gels (Figure 2). Electrophoresis could be performed satisfactorily after treatment of protein extracts with low concentration of Ca012 (see Material and Methods). Calcium chloride treatment of protein extracts often resulted in instantaneous formation of a gel that could be removed by centrifugation. Although equally good electrophoretic separa- tion was obtained after the gel formed was removed by 15,000 x g (30 minutes) or 100,000 x g (30 minutes) centrifugation (Figure 2A); the latter was used routinely in order to compress the gel and recover the original volume of the protein extract. 36 Figure 2. 37 Electrophoretic separation of protein extracts before and after CaClz treatment. Electrophoresis of acetone dried powder extract pre- pared from ripening Bartlett pear is shown. A. Protein stain. From left to right (repeated three times): 0, 2.5, 5, and 7 days ripening. Left: Before CaC12 treatment. Middle: After CaC12 treatment and 20,000 x g centrifugation (30 minutes). Right: After CaClz treatment and 100,000 x g centrifugation (30 minutes). Malic enzyme assay. From left to right (repeated two times): 0, 2.5, 5, 7, and 9 days ripening. Left: Before CaClz treatment. Right: After CaClz treatment and 100,000 x g centrifugation (15 minutes). 38 t; .2... ‘. v Q” - 39 It was observed that the viscosity of protein extracts prepared from fruits at successive stages during ripening increased and then de- creased gradually. The volume of gel obtained after CaClz treatments and high speed centrifugation, as judged by visual observation, was preportional to the viscosity of the protein extracts. Calcium chloride was used previously (Haller, 1929) to precipitate soluble pectins extracted from apples. Weurman (1954a) measured an increase in viscosity of solu— tions containing pectins and pectin methyl esterase when CaC12 was added and concluded that in addition to activating the enzyme, ca++ ions also had an effect on the pectin itSelf. On the basis of this information, it was concluded that protein extracts from ripening fruits contained in- creasing amounts of soluble pectins capable of adsorbing proteins, and that the protein-pectin complex was retained on top of the spacer gels during electrophoresis. Precipitation of soluble pectin in the-form of Ca-pectate facilitated electrophoretic separation of fruit proteins during ripening (Figure 2). r.f. Measurement The presence of the tracking bands (bromOphenol blue dye) close to the bottom of the gels can be seen in Figure 2 and all subsequent figures of gels which were assayed to localize malic enzyme. Tracking bands in gels stained with aniline blue black disappeared during the destaining process. r.f. values of 0.1 to 0.4 were observed for malic enzyme. A reasonably reproducible r.f. value was obtained only when a particular protein extract was repeatedly separated electrophoreticly. Factors affecting the r.f. value of malic enzyme were not investigated. It was 40 noted that electrophoresis under standard conditions (% acrylamide, gel length, buffer composition and current) varied between 3/4 to 3 hours and that the variation was mainly due to the composition of the sample applied. Excess salts, proteins or phenols tended to extend the duration of electrophoresis. It is to be expected that slight variation in ionic strength and pH ofcrude protein extracts will affect the mobility of pro- teins to a larger extent than that of bromophenol blue, resulting in vari- ability in r.f. values. Protein Determination Presence of soluble nitrogenous compounds in protein extracts, in- cluding those obtained from acetone powders, required the precipitation and washing of samples prior to the biuret or Lowry protein determination. Precipitates of pectin-protein complexes, only partially soluble in l N NaOH, were obtained particularly with protein extracts from ripe fruit. The soluble protein-pectin residue had to be removed by centrifugation be- fore the appropriate spectrOphotometric measurement could be made thus introducing an error in the quantitative estimation of proteins. This problem will be referred to during subsequent interpretation of the data. Protein and Malic Engyme Recoveries The preliminary results of electrophoresis (Figure 2) and the quick loss of malic enzyme activity observed in crude extracts made it highly desirable to find appropriate conditions under which soluble pectin could be eliminated without great loss of enzyme and protein content. The effect 0f CaC12 concentration at 3 pH levels on % recovery of protein and malic 41 enzyme in potassium carbonate extracts of green and ripe Bartlett pears is shown in Table 1. Better recoveries of protein and malic enzyme were ob— tained when precipitation was carried out at higher pH values. Protein loss at any Ca++ concentration or pH tested was greater in ripe fruit, pre— sumably due to the presence of soluble pectin. The effect of 0.02 M CaClZ treatment (pH 7.5) on protein and malic enzyme recoveries in protein extracts of acetone dried powders prepared from Bartlett pears at successive stages of ripening (Table 2) showed a close correlation between the presence of soluble pectins and loss of protein. The viscosity of the extracts and protein loss during CaClz precipitation increased at 2 1/2 days of ripening and then gradually decreased. This trend was qualitatively reflected in disc gel electrophoresis of the corresponding protein extracts (Figure 2) where greatest interference (before Ca012 precipitation) was obtained at 2 1/2 days. Recoveries of malic enzyme after Ca012 treatment could be only broadly correlated to the presence of soluble pectins. It should be pointed out that up to 60 to 70% loss of malic enzyme activity was observed at 0 to 4°C over a period of 24 hours in crude protein extracts of fresh fruits or acetone powder. Following CaC12 treatment, however, only 10% loss in activity occurred at 20 to 25°C over a period of 2 weeks. The presence of phenols in fresh fruit extracts or the absence of them in acetone powder extracts did not have great effect on malic enzyme loss during storage. Jones (1965b) reported the use of polyvinylpynxflidone to overcome inhibition of several enzymes, including malic enzyme, by'phenol and phenolase present during the extraction period. This mechanism, however, cannot account for loss of malic enzyme in protein extracts obtained from acetone powders devoid of phenols. A somewhat different mechanism of 42 Table l. The Effect of CaC12 Treatment at Different pH Values on Protein and Malic Enzyme Recoveries in Extracts of Bartlett Pears. mg Protein and Units of M.E. per 10 ml Extractl Green fruit Ripe fruit CaC12 . % % % % pH (M)‘ Protein recovery M.E. recovery Protein recovery M.E. recovery 9.5 - 9.35 100 2020 100 11.16 100 2130 100 5.5 0.01 4.48 48 1530 70 1.65 14 1410 66 0.02 5.32 57 1580 78 1.98 18 1540 72 0.04 4.99 53 1730 86 1.25 11 1270 60 6.5 0.01 6.07 65 1600 79 2.79 25 1740 82 0.02 6.42 59 1630 81 3.60 31 1800 84 0.04 4.67 50 2060 102 2.94 26 1970 94 7.5 0.01 8.61 92 1930 96 3.02 27 1930 91 0.02 8.05 86 2070 102 4.36 39 1760 83 0.04 2.71 29 2110 104 3.80 34 2160 101 1Green and ripe fruits were infiltrated and extracted with buffer containing 0.2 M K2C03, 0.35 M mannitol, 5 x 10"3 DIECA pH 10.5 10 m1 aliquots adjusted to pH and CaClz concentration as indicated. Protein was estimated according to the biuret method on aliquots precipitated with 10% TCA. 43 imble 2. The Effect of CaC12 Treatment on Protein and Malic Enzyme Recoveries in Protein Extracts of Acetone Dried Powder Prepared from Bartlett Pear. mg Protein2 and Units of M.E. per 500 mg Acetone Dried Powder Before CaC12 After extract treated Flesh treatment with 0.02 M CaClZ Days firmness % % ripened lbs. Protein M.E. Protein recovery M.E. recovery 0 16.4 6.86 1755 3.03 44 1072 63 2.5 6.0 10.82 2002 3.55 33 1592 80 5. <3 7.33 1400 3.91 53 931 66 7 <3 8.94 2052 12.28 137 1890 92 9 <3 9.80 1820 10.11 104 1323 73 lAcetone dried powder was prepared from fruit removed after 3 months from CA storage, and held at 20°C for ripening. 500 mg acetone dried powder was extracted with 8 ml of buffer containing 0.1 M Tris—tricine, 0.35 M mannitol and 5 x 10-4 M EDTA. pH 8.8. 2Protein was estimated according to the biuret method on aliquots precipitated with 80% ethanol. 44 enzyme inhibition and precipitation was reported by Goldstein and Swain (1965) and Young (1965). Alcohol dehydrogenase (S-glucosidase, lactic dehydrogenase and catalase (commercial preparation) formed complexes and lost activity when mixed with taninic acid or tannins. Non-ionic and cationic detergents could effectively reverse the inhibition caused by taninic acid. The use of tannins resulted in a greater loss and poorer recovery of activity when detergents were employed. It was concluded that hydrogen bonds as well as other types of bonds participate in enzyme- tannin complex formation (Goldstein and Swain, 1965). Young (1965) reported the release of a leucoanthocyanin type tannin from the latex cells of pre- climacteric banana that adsorbed aldolase in the presence of cell wall debries. Aldolase adsorbtion could be prevented by 7% carbowax, or 2% caffein or nicotine. A similar mechanism of tannin participation in malic enzyme adsorption to soluble pectins cannot be discounted. Detailed investigation of the problem, beyond the statement that malic enzyme can adsorb to soluble pectins, is beyond the scope of this work. It was pertinent, however, to eliminate soluble pectins whenever possible. A satisfactory procedure for fresh fruit extraction included vacuum infiltration of the tissue with 0.1 to 0.3 M Tris (pH 10, to neutra— lize high cell sap acidity) containing 0.05 to 0.1 M CaC12 (Table 3). Better recoveries of malic enzyme were noted with an optimal concentration of CaClz. Protein content of extracts decreased from 70 to 37 mg per 100 g fresh weight when 0.02 M CaC12 was infiltrated into the fruit. A ten-fold increase in CaClz concentration caused only a small additional decrease in Pretein recovery. When protein extracts were treated with 0.02 M CaClz (Table 3) 10 to 60% of protein and malic enzyme was precipitated, unless 45 Table 3. The Effect of CaC12 on Protein and Malic Enzyme Recovery During and After Extraction from Bartlett Pears. 1i mgiProtein and Units of Malic Enzyme (M.E.) per 100 g fresh wt.l After vacuum After extract treated infiltration with 0.02 M CaC12_ CaClz M.E. units % % (M) Protein M.E. mg protein Protein recovered M.E. recovered 0 70 8,740 125 31 44 5,400 62 0.02 37 11,960 323 38 102 10,580 88 0.05 35 13,455 384 41 118 13,168 98 0.10 29 11,500 396 33 114 11,385 99 0.20 30 11,270 376 30 100 10,120 90 10.2 M Tris buffer at pH 10 containing 5 x 10"”3 M DIECA was used as the infiltration and extracting solution. Fruits ripened 4 days were in— filtrated with the indicated concentrations of CaClz and homogenized. A11 extracts then received an additional treatment with CaClz. 46 fruits had been infiltrated previously with CaClZ. Apparently, a protein-like malic enzyme which is localized in the cytoplasm can be precipitated with insoluble cell wall material during extraction or adsorbed to soluble pectins following extraction. Low concentrations of CaClz (0.01 M) precipitated soluble pectins in protein extracts as effectively as higher concentrations (1.0 M). When vacuum infil~ trated into the fruit, the optimum concentration of CaC12 shifted slightly from 0.02 M to 0.075 M during ripening. Higher concentrations (0.075 to 0.1 M) were also used with fruit kept in storage for periods in excess of 3 months. Localization of Malic Enzyme on Polyacrylamide Gels The color intensity of the reduced tetrazolium dye precipitated at the site of the enzyme was found to be preportional to the enzyme activity (enzyme concentration) and incubation time at constant temperature. After prolonged incubation of polyacrylamide gels in malic enzyme assay mixture, a fraction of an enzyme unit, not detectable by aniline blue black stain, could be readily localized (Figure 3, 4). Up to 6 uniformly spaced bands (designated 1 through 6, counting from the bottom to the top of the gel, or from anode to cathode, respectively) exhibiting malic enzyme activity were seen on polyacrylamide gels. Band 2 was invariably the most prominent, as judged by protein stain or malic enzyme assay. This pattern (Figure 3) was reproducible in 5 to 7% polyacrylamide gels without regard to the actual r.f. of the enzyme. 4 When band 2 was extracted from slab acrylamide gel and rerun on disc gels, all bands reappeared, indicating that the different bands were merely 47 Figure 3. Malic enzyme assay in polyacrylamide gel. From left to right (alternately): assay and protein stain of partially purified malic enzyme. Electrophoresis of malic enzyme eluted from Sephadex G-200 gel. Figure 4. A. Protein stain. Left to right; fractions 14 to 25. B. Malic enzyme assay. Left to right: fractions 15 to 25. 48 I} J Up 1; 1"] U‘beL’ IIIIIIP- ~"j1- 49 aggregates and not true isozymes. This conclusion was substantiated by fractionation of the enzyme on Sephadex G-200 (Figure 4). Fractionation was carried out using a 46 x 2.5 cm long column according to Andrews (1965). The column was equilibrated and eluted (4.14 ml per cm2 per hour) with 0.05111KH" Malic enzyme was eluted from the Sephadex G-200 column in fractions 12 to 22 (V0 = fractions 11 to 15). Electrophoresis was carried out on fractions 14 to 25 (Figure 4A, protein stain) and 15 to 25 (Figure 4B, malic enzyme assay). Malic enzyme assay on acrylamide gel (Figure 4B) clearly indicates that the slower moving bands are aggregates of larger molecular weight molecules that had been eluted from the Sephadex G-200 gel in front of the malic enzyme peak. Changes in Protein Content and Malic Enzyme Activity During Fruit Ontogeny Acetone dried powders prepared at successive stages during fruit development were conveniently employed to study changes in protein content and malic enzyme activity. Kjeldahl analysis of total N and 80% ethanol insoluble N and buffer extractable protein (biuret) and malic enzyme activity were performed. A fraction of the total N in acetone dried powders was found to be soluble in 80% ethanol, although the powders were washed exhaustively with acetone and acetone-ether during preparation (Figure 5, 6). Protein content of acetone dried powders prepared from McIntosh and Wealthy apples during development decreased from 60 to 80 mg per 500 mg powder during cell division (25 days from full bloom) to 20 to 40 mg per 500 mg powder during cell enlargement (46 to 117 days from full bloom). Immediately before ripening (131 days from full bloom), protein content further decreased to 10 to 20 mg per 500 mg powder (Figure 5, 6). 50 Figure 5. Total N and 80% ethanol insoluble N of acetone dried powders prepared from McIntosh apples at successive stages of development. 51 100 I TOTAL N 15.0 x 80% ETHANOL INSOLUBLE N 33 212.305 .9: 5 O 5 7 5 2 o n n. 5 «330m waEU< .9: 0032 .29: I 105 65 45 85 DAYS FROM FULL BLOOM Isl IIIIIII _5 O 52 Figure 6. Total N and 80% ethanol insoluble N of acetone dried powders prepared from Wealthy apples at successive stages of develOpment. 53 in”? z Vz.pr¢m.mE 5 7 O m _N E IL 3 U L O S m .I__ O N um MU mm T8 I m m u . awn—30m wZOp O 5 5 2 _. I45 I25 5 w l 5 O 7. 5 H3 .9: 83 5 2 Z .9: 0 DAYS FROM FULL BLOOM 54 Protein content of acetone dried powders prepared from different apple varieties (Figure 7), or from McIntosh apple collected at different locations (Figure 8), showed considerable variability. However, an in- crease in protein content was observed during ripening (Figure 7, 8). Activity of malic enzyme in protein extracts of acetone dried powders of McIntosh and Wealthy apples was first detected at 46 days after full bloom (Figure 9, 10). Specific activity of the enzyme increased up to 76 days and again during maturation and ripening. Increase in specific activity of malic enzyme during ripening was evident in all apple varieties tested (Figure 11, 12) and McIntosh apples collected at different locations (Figure 13, 14). In view od the difficulties encountered with the biuret protein determination in ripe fruit extracts (see page 40), the specific activi- ties (enzyme units per mg buffer extractable protein) for ripe fruits in Figures 9, 11 and 13 can be considered as maximal values. The corresponding specific activities (enzyme units per mg Kjeldahl N protein from acetone powder) in Figures 10, 12 and 14 has to be considered as minimal values since only 2 to 40% of the total protein content of acetone powders could be extracted with buffer (Table 4) (Frenkel et al., 1969). The increase in specific activity of malic enzyme during fruit on— togeny (Figure 9 to 14) cannot be explained solely on the basis of the in— crease in the percent of total protein N that could be extracted from acetone powders (Table 4). For example, the specific activity of the enzyme increased between 46 to 58 days without change in the percent of protein extracted from the acetone powders. Likewise, the increase in specific activity of malic enzyme during ripening is considerably greater 55 Figure 7. 80% ethanol insoluble N of acetone dried powders prepared,from various apple varieties at the pre- and post-climacteric stages. 56 mg.N/500 m9 ACETONE POWDER 9 - N u a u: l l l I l — HSOlNl’W IIIIII AHDVJM I I I . I Ads N I I I I I I a V a V’ '7‘ Nvmvmr . ., 7‘ n ,o r: i if SOODFBO 038 I I o R n _‘ -4 m m :o ’2 SOODIUG 9 I I a a n K430105) S3Wl89 - UJNEDVM I I I I I 3SO3WV1 DNINSJUS) I I I I I I I 8 O _ .. l I .~ u. f: M U! mg. PROTEIN 1 N . 6.25 a 57 Figure 8. 80% ethanol insoluble N of acetone dried powders prepared from McIntosh apples (pre-and post-climac- teric) collected at different locations. 5w Mn (N 2N w ’H L‘N a" MHZ!" N NOIIVDO‘I Olw “N 58 mg. N_/soomg. ACUONE POWDER u u 1 .I..' L. - ; é a; g E l l ....-. g ; éfi u u a u . PROTEIN (IN-6.25) 59 Figure 9. Specific activities of malic enzyme (enzyme units per mg buffer extractable protein) in extracts of acetone dried powders prepared from McIntosh and Wealthy apples at successive stages of development. SPECIFIC ICIIVIFY 0F IAUC EIZVIE ltfiha HKHuu 160 140 I20 100 40 20 25 60 I ncmosu +12 nus-O . n 20’: . mum I 45 65 85 105 ‘25 DAYS FROM [UN 8100'! 61 Figure 10. Specific activities of malic enzyme (enzyme units per mg Kjeldahl N protein) in acetone dried pow- ders prepared from McIntosh and Wealthy apples at successive stages of development. E.U./mg. KJELDAHL N PROTEIN from ACETONE POWDER 7o 62 I +12 DAYS At 20°C — Mc INTOSH \ W WEALTHY 65 .5 125 DAYS FROM FULL BLOOM ' ummmmm HM‘MWI. I45 63 Figure 11. Specific activities of malic enzyme (enzyme units per mg extractable protein) in extracts of acetone dried powders prepared from various apple var- ieties at the pre- and post-climacteric stages. 64 ODD-III. R' ammo OIOIIIOOIII. FAMEUSE mama CRIMES GOLDEN G. DELICIOUS RED DELICIOUS IIIIIII JONAIHAN <-_-> 8 a: 3‘. ._ .— IIIrIIIIIII 3 g ' [SPY r7: '1? a: g IIIIIII WEALIHY a. «L I i E IIIIIIIII NINIDSH C l l 1 l l 1 o O a so § 3 3 . u 0" N MAIN] 311mm “uum OIJIIJldS 65 Figure 12. Specific activities of malic enzyme (enzyme units per mg Kjeldahl N protein) in acetone dried pow- ders prepared from various apple varieties at the pre- and post-climacteric stages. 66 I PRE-CLIMACTERIC 555555. a . . . 0:32.20 .2222: _______________________ . _ : (>222 .=_=======33"3333"3333333” Q;& 0082. _z:3:._:====:=_::===z:_=====E====== . _ _ 1 __ __ $85905 . -u“““”--—-..- __ __.______.. __________________ tr: . 1:... 5.3:: ”.8P6_8m ~3§$$$e$$$$$==== . _ _ _ _ 822:): .2::==._=====' ._ : _ .3 _ __ _ _ __ _ z. MV< e... £955 POST-CLIMACTERIC 160— “ _ w — s............:............_:: _ _ _ 322402.. «330.. ”2058 52. 290.... z «.295 .23” 67 Figure 13. Specific activities of malic enzyme (enzyme units per mg buffer extractable protein) in extracts of acetone dried powders prepared from McIntosh ap- ples (pre- and post-climacteric) collected at different locations. W013i" NI Ham on: o-w o-w nu v-w 54v y-w t-N MN PH “'70 68 <~= - Acuw ormuc , «w: norm» al....I-I-III-IIIIII ‘9 38 mommy - 69 Figure 14. Specific activities of malic enzyme (enzyme units per mg Kjeldahl N protein) in acetone dried pow- ders prepared from McIntosh apples (pre- and post-climacteric) collected at different locations. NV9IHDIW NI NOLlVDO'l 70 E-U.lmK.£LDAHL N PROTEIN Irom ACETONE POWDER 0‘8 8 3 Li ° 81 8 i2 8 (IN 6"! B'N L'W 9‘" SW "W C‘N C'W AIW ‘llll+[lll “IIIIIIIIIOl"MOMNMHIMHHM. " moulmum:numummmmm: ““mummummmImmune: ................... DIIJDVWID-lSOd um DIUSIDVWHD-SN - .- Sll mmmmmmnmv 71 fable 4. Total and Buffer Extractable Protein Content of Acetone Dried Powder Prepared from McIntosh and Wealthy Apples. mg protein per 500 mg acetone dried powder McIntosh Wealthy Days from Buffer Buffer full bloom Total extractable % Total extractable ' % 25 74.6 1.9 2.5 62.9 5.9 9.3 46 30.3 5.6 18.5 27.6 4.8 17.3 58 34.8 6.7 19.2 - 30.5 :5.5 18.1 76 23.2 6.4 27.6 32.0 7.0 21.9 88 23.5 6.1 26.0 35.1 10.7 30.4 102 20.9 6.0 28.7 . 38.4 9.9 25.8 117 21.5 7.0 32.6 ‘I37.7 11.1 29.5 131 10.2 4.0 39.2 21.0 8.1 38.7 131 + 123 20.0 7.1 35.6 27.6 8.5 32.5 . lKjeldahl (N x 6.25) N protein of 80% ethanol insoluble residue of acetone dried powder. 2Protein estimated by the biuret method on aliquots precipitated with 80% ethanol. 3Acetone powder prepared from fruit picked at 131 days and left to ripen for 12 days at 20°C. 72 than the increase in percent protein extracted from the acetone dried powder. Electrophoretic separation of the various proteins extracted from acetone dried powders is shown in Figures 15 to 17. Protein stain and a corresponding assay to localize malic enzyme on disc acrylamide gels is reproduced for each sample. A sequence of 4 pictures taken from the same gels at 45, 80, 100 and 120 minutes (Figure 15B, C, D, E) clearly demonstrate the sensitivity of the assay employed to localize malic — enzyme, as well as the need of long incubation times for detection of all traces of the enzyme in the gel. Zero, 2, 5, and 10 enzyme units were applied on gels l, 2, 3, and 4 to 9, respectively (Figure 15). Incu- bation was~carried out at 20°C. Given sufficient time (120 minutes), a trace of an enzyme unit, undetectable by the spectrophotometric assay, could be localized (Figure 15E, sample of 25 days from full bloom). It is also evident that as little as 2 enzyme units (sample of 46 days from full bloom) could be localized before any one of the enzyme aggregates. It can be estimated therefore that under the preparative procedure em- ployed, the aggregates of the enzyme constituted an equivalent of 1 tofi2 enzyme units, or approximately 10 to 20% of the total enzyme activity. Details of enzyme units applied on each disc gel and incubation times are specified in legends of Figure 16 for protein extracts of acetone powders prepared from McIntosh apples during development, and in Figure 17 for several pre-and post-climacteric apple varieties. Electrophoretic separation of apple malic enzyme during development and ripening of the fruit (Figure 151317) was remarkably similar. Disregarding the presence of aggregates, malic enzyme activity at any stage of fruit development 73 Figure 15. Electrophoresis of Wealthy apple proteins from fruits at successive stages of development. Details of enzyme units loaded are specified in the text. A. Protein stain. B, C, D and E are incubation times of malic enzyme assay in the disc gels for 45, 80, 100 and 120 minutes respectively. A 74 WEALTH Y (Acetone Powder) 1:5 I i‘ L31 I I 25 46 v «50 16 BB 102 117 131 :3: DAYS F OM FULL BLOOM , ' PL. 211.: WEN-"'7 (Acoflno Pond,” 25 46 58 76 88 102 111 131 131 +124 ‘ DAYS FROM FULL BLOOM “2011.: 25 25 Q .—~ —-—.‘---1—M.s. .% 46 58 16 88 102 DAYS FROM FULL BLOOM “ . ~50 -' 4‘ 46 58 76 88 102 DAYS FROM FULL BLOOM ’J‘ 117 117 --Ig!ii *I". - u “1.55 25 46 58 76 88 102 DAYS FROM FULL BLOOM "7 - «‘w 131 131 4‘2 days II 2! 'c J -.I ~ . 131 131 +12 days II 21 ‘c ‘C I 131 I31 +12 days a! 21 ‘c Figure 16. 76 Electrophoresis of McIntosh apple proteins from fruits at successive stages of development. The following enzyme units were applied per disc gel: 25 day - 0, 46 day - 2, 58 day - 5, 76 to 131 + 12 days - 8. A. Protein stain, and B. malic enzyme assay incu- bated for 100 minutes. Cd 77 Me INIOSH (Acflqno Powder) 25, 46 58 76 88'- 102 DAYS FROM FULL BLOOM McINTOSH (Acotono Powder) » —- " V Q“In!" 25 46 \- ._—' fi ““ " ’ ‘ ‘nnr ' 'ifld“ 58 76 88 102 DAYS F ROM F U L L BLOOM 117 131 131 +12 days 01 21 'c ha) 117 131 131 +12 days a! 21 'c Figure 17. 78 Electrophoresis of proteins of pre— and post- climacteric apples of several varieties. 10 enzyme units were applied to each disc gel. A) and B) protein stain. C) and D) malic enzyme assay incubated for 60 minutes. 79 (Aceione Powder) APPLE VARIEIIES' R— PosIcIimoCIeric G- PreclimocIeric a 3.91.123 Ibo-.20.; uoao.rea boae.rea win won (Io—2; ieo...‘ 12:3... Igloo... (Acetone Powder) APPL E VARIEIIES u f I - c I m c - S O P R G- Preclimocieric IorOI max-20 I... OIIMZiQ mgr—Mm m>ZmCmm (Balms (BMZMI 023nm OOPOMZ Quinn 890ml 98:99.3 puntfiOCm 80 Anti VARIEIIES (Acevone Powder) a 1. 5- .3 . .. .... U; “'2. .‘U; . a a 81.12: .V 6 ”be—.26.: 1 9 I 532...... .6 .—o..e.-.Ia AMI. m: cg. .. .. a «3 L - E (09...: v.1 O ‘21:: m .I 123...... L 0 123.3: (Acetone Powder) R— Pout Hmonerlc I APPLE VARIFHES G- Preclimocteric I._.0Imm1_10 G) I-rOImm1.ZO 1>XnCmm 9'3. 0 «)3mcmm d I (>095: 0 (>033: OEIMM OOPOMZ 6) 021mm OOPOMZ O.°m§h.OCm 5“ £590.; \..\ 81 and ripening was associated with a single band separated during electro— phoresis. Similar results were obtained with malic enzyme extracted from the cortex (Figure 18A) or peel (Figure 183) of ripening Bartlett pears. The presence of a band located in front of the bromophenol blue tracking dye was detected during electrophoresis of crude protein extracts prepared from the cortex of fresh fruits (Figure 18A). The intensity of the band increased markedly during ripening. A corresponding band was absent altogether in extracts of peel from fresh fruits (Figure 188) or in acetone powder extracts (Figure 2B). This fast running band (bottom of Figure 18A) was identified as NADPH by comparing its mobility to that of authentic NADPH. The identification is substantiated by fluorometric estimations of changes in NADPH concentrations in cortex and peel tissue during ripening (Rhodes and Wooltorton, 1968). Amino Acid Incorporation Into Malic Enzyme The foregoing results and discussion amply demonstrate the diffi— culties encountered with preparative procedures during developmental studies on enzymes in pome fruits. No apparent inhibition or activatiov of malic enzyme during fruit development and ripening was evident. Ex— tractability of the enzyme from fresh fruit or acetone dried powders, however, is an unknown quantity considering the presence of tannins and phenols and cell wall residue undergoing changes during ripening. In- crease in specific activity of malic enzyme (Dilley, 1962) can be con- sidered therefore, only circumstantial evidence of enzyme synthesis. The more direct approach of using incorporation of radioactive amino acid (Filner et al., 1969) was employed to ascertain synthesis of malic enzyme during the climacteric rise in respiration. Infiltration of Figure 18. 82 Assay of Bartlett pear malic enzyme in polyacryla- mide disc gels at successive stages of ripening. Gels were incubated for 10 minutes. A. Malic enzyme from the cortex of the fruit. Left to right: 0, 1.5, 3.5, 5.5, 8.5 and 10.5 days of ripening. Forty enzyme units in volumes of 0.17, 0.14, 0.16, 0.24, 0.20 and 0.23 ml of extracts were applied, respectively, on each disc gel. Malic enzyme from the peel of the fruit. Left to right: 0, 2.5, 4.5, 6.5, 9.5, and 11.5 days of ripening. Forty enzyme units in volumes of 0.21, 0.17, 0.14, 0.17, 0.24, and 0.26 ml of extracts were applied, respectively, on each disc gel. 83 K.- “- l-I I- I W :H .2 H b-I «I In. 4"? 84 14C L—phenylalanine into intact fruit, rather than into tissue slices, offered a bacterial-free system for protein synthesis studies. Under such a system, long incubation periods could be employed which in turn facilitated detection of radioactive label in small amounts of specific proteins. Although several ripening parameters were reported to be slightly modified, the overall ripening behavior of the fruits after vacuum in- filtration with solution containing 0.3 to 0.4 M mannitol was found to be close to normal (Frenkel et al., 1969). Incorporation of 14C L-phenylalanine into specific proteins of Bartlett pears at the early- and mid— climacteric stage is given in Figure 19. Results are expressed as dpm per protein band per 2.5 mg protein applied on the slab gel. The percent of total counts recovered from each slab acrylamide gel by diffusion into 0.1 M phosphate buffer (pH 9.6) was 33.6 i 4.2. _Data in Figure 19 was corrected to show an exact 32% recovery. Incorporation of 14C L-phenylalanine into malic enzyme (band 3, Figure 19) increased approximately three—fold between the early- and mid-climacteric stage. The rate of incorporation into malic enzyme leveled off after 12 hours incubation at the mid-climac— teric stage. At this time, only 49% of the total counts infiltrated into the fruits were found in a TCA precipitable fraction compared to 65 to 70% at longer incubation times (Frenkel et al., 1968). The value of incorporation obtained at 12 hour incubation at the mid-climacteric stage (Figure 19) may represent an equilibrium condition where enzyme. synthesis and degradation proceeds at an approximately equal rate. This however would imply an extremely quick turnover rate. Alternately, under 85 Incorporation of 14C L-phenylalanine into specific proteins of Bartlett pears at early- and mid-cli- macteric stages. Two and one-half mgs of protein was separated on each of 6% polyacrylamide slab gel. Figure 19. 86 11150590911109 or “c-PIIEIIYLALANINE 11110 smmc mmus or 51111111 mos 11 111111-1110 1a1o-c111uc1u1c 511555 um mo uooos nouns 6 I2 24 — 6 I2 24 (on/mum mo) ”—1 (on/mm: mo) 335 445 510 1—4 I 129 121:1 1459 4:15 1250 1219 1— 2 553 951 915 251 no 1440 H 39.1.9o1o35 1043 252 503 1594 11.— 4 1osI 913 929 mm 2120 2450 :1 5 1055 1205 1329 922 1310 2238 b 950 1119 1435 445 1005 1503 7 149 310 1005 733 930 1351 '~— 8 555 115 595 502 123 1354 PM 9 555 512 910 m 1355 1254 urn-10 192 1355 1904 570 1113 1525 Y 515 950 1110 87 conditions of slow turnover rate the decline in incorporation may in- dicate a decline in the rate of enzyme synthesis. It is unlikely that the observed decrease in 14C L-phenylalanine incorporation as ripening progressed resulted from dilution with endo- genous amino acids. The 14C L-phenylalanine was administered with 1 x "10'”4 M 120 L-phenylalanine. Concentration studies indicated no dilution of label by endogenous amino acid occurred above 1 x 10"5 M L-phenylalanine (Frenkel et al., 1968). 14 Results of C L-phenylalanine incorporation (6 hour incubation time) into malic enzyme expressed as dpm per enzyme unit is presented in Table 5. 14 C L-phenylalanine incorporation into all proteins in relation to malic enzyme activity increased by less than two-fold at the mid- climacteric stage (column 1, Table 5). In comparison, specific radio— activity of electrophoreticly separated malic enzyme increased three-fold, indicating preferential synthesis of the enzyme at the early- to mid- climacteric stage. The effect of ethylene on ripening and 14 C L-phenylalanine incor- poration into Bartlett pear proteins and malic enzyme under reduced at- mospheric pressure is presented in Figure 20 and Table 6. Under condi- tions where ethylene was removed continuously fruit ripening was pre- vented. Respiration was maintained at a pre-climacteric level for as long as 15 days (Figure 20). The fruit remained green and softening was arrested (Table 6). When ethylene was administered under reduced pressure, the fruit exhibited a normal respiratory climacteric (Figure 20), degreened, and softened (Table 6). The fruit was vacuum infiltrated at the successive stages as indicated in Figure 20, and incubated for 88 table 5. Incorporation of 14C-Phenylalanine into Malic Enzyme as Com- pared to Incorporation into Other Proteins During Ripening of Bartlett Pears. Specific activity (dpm/enzyme unit) Stage of ripening Crude extract1 After electrophoresis2 Early-climacteric 32.4 5.4 Mid-climacteric 57.4 15.3 1 Radioactivity in acetone powder extract was determined following precipitation of proteins with TCA. 2Malic enzyme was extracted from the acrylamide gel by homogena- tion with 0.05 M KCl. Pectic substances in acetone powder extract were precipitated with 0.02 M CaCl2 prior to electrophoresis. Figure 20. 89 The effect of ethylene on respiration rate of Bartlett pears under reduced atmospheric pressure. Fruit was maintained at 150 mm Hg under contin- uous flow of oxygen and oxygen containing 6000 ppm ethylene. Respiration was measured after removal of the fruit from reduced pressure and extrapolated to the pre-climacteric minimum 90 not- 21 up... no .. d d A O‘ u. N Respiralion Ra'e mcoz/xc—un ol- dg + M§<—Oz 5|. .3 I 5.4.2.. H P by h P b h a & .u x .o 8 8 8 m. ”to Do: .91 I“! «gauI-i m.m o.oN o.mm one mm mm qw.sm om saw.NH NH 111 1111 1111 111 11 11 1111 11 1111 o.oa o OHH N.m N.HN N.mm1 moo mm cm mm.om NN mmm.- NH N.~ o.qs o.aH mam «5 mm «H.HN mm oqm.ma m.ma o «s «was 1 H.~H N.Hm m.om on HNH «m Nqomm NN saw.om NH 111 1111 1111 1111 111 11 1111 11 1111 o.o o OHH m.m m.HN m.s~ OHHH HOH mm Nm.oN mm st.NN NH s.q m.ma m.a~ Hues No om Hm.mN mN oos.m~ w.mH 0 mm m.o 0.0H «.mm «Non om mm om.oN ow oq~.m~ NH com N.mH mnsm can mm mm ON.HN Hm N¢¢.¢H m.mH o sq o.m N.HH N.¢H oqma HHH ms 0H.na cm omo.aH NH o.N N.OH o.oH sees as me Ne.ma mm omm.oa 0 ON N.m m.HH o.wH wmm mm mm osoma am oma.aa NH w.H w.o N.OH wwa mm ms mq.ms Hm oofl.oa a o «:NU.T AS x EQE sombxo BE: BE: 530.3 mg @338 ME 38:8 Emu Away 35 35 mflmouonn 25.3 .m .E\EQU .m .2 Emu ORV 539a ob mmofibfim oEU 083 1850? Hobgom coin oaouoo,~ ‘ C 511 I 59 77.- 58 76 88 102 117 131 131 +12 do 3 DAYS FROM FULL BLOOM a! 2"" \ ' u} ‘ I In, L I 109 buffer in the extraction solution (pH ca. 10.5) was adjusted to obtain a final pH between 8.5 to 9.5 after grinding. Incorporation of 0.02 to 0.2 M CaClz into the infiltration solution increased the specific activity of malic enzyme in the crude extract three-fold (Table 3), and eliminated a step required (Dilley, 1966a) to remove gelling substances from the crude extract. Incorporation of CaC12 into the infiltration solution required | the use of Tris rather than K2C03 buffer (to avoid CaC03 precipitation), although better recoveries of malic enzyme were reported using K2C03 (Dilley, 1966a). Malic enzyme could be extracted from apple fruit, after prolonged storage, using K2C03 but not Tris buffer. Tris buffer, however, could ex— tract enzyme from Bartlett pears, although enzyme recovery from fruit following prolonged storage (partially deteriorated fruit) decreased by approximately 50%. Two to three times more malic enzyme could be extracted from Bartlett pears than from apples when either Tris buffer or K2C03 were used. Ammonium sulfate fractionation (50 to 60% of saturation at 0°C) of the crude protein extract (Tables) indicated that some purification could be achieved. The low enzyme yield, however, did not warrant collection of narrow fractions and in all subsequent purifications the protein precipi- tated between 45 to 65% saturation was collected. The initial specific activity and extent of purification was greater when the enzyme was purified from fresh fruit (Table 8). Furthermore, electrOphoresis of fresh fruit extracts following prolonged storage showed that malic enzyme became the dominant band. Thus, the initial specific activity of malic enzyme in fruits increased during storage. 110 Table 8. Purification of Bartlett Pear Malic Enzyme by Ammonium Sul- phate Fractionation and Calcium Phosphate Gel Adsorbtion. Acetone powder extract Fresh fruit extract Calcium phgsphate Ammonium sulfate Ammonium sulfate gel Ammonium sulfate Specific % Specific % Specific % fraction activity recovery activity recovery activity recovery 1 Crude extract 166 581 45 - 50 110 6 -- -- -- -- 50 - 55 661 40 448 15 1167 51 55 - 60 372 23 1397 45 1969 37 60 - 65 246 14 228 5 412 40 lAcetone dried powder was extracted with Tris-tricine pH 9.0, and pectic substances precipitated with 0.02 M CaCl . Crude extract of fresh fruit was obtaine from the cortex of the fruit previously vacuum infiltrated with 0.2 M Tris and 0.05 M CaClz. 2Positive calcium phosphate gel adsorbtion (6 mg solid gel per mg protein) was carried out after ammonium sulfate fractionation. Percent recovery of malic enzyme from calcium phosphate gel refers to a single extraction with 0.1 M phosphate pH 7.5. 111 Approximately two-fold purification was obtained when calcium phos- phate adsorption (Dilley, 1966a) was performed (Table 8). Malic enzyme adsorbtion on calcium phosphate gel after ammonium sulfate fractionation (undialyzed sample) required the use of 6 mg gel solids per mg of protein. Thus, negative followed by positive calcium phosphate adsorption (Colowik, 1955) was often carried out; one and two mg increments of calcium phosphate gel were mixed with the protein solution, centrifuged and discarded before malic enzyme was adsorbed. Recovery of malic enzyme adsorbed on calcium phosphate gel was low (Table 8) unless successive extractions were made. The extent of malic enzyme purification from acetone dried powder extracts after ammonium sulfate fractionation (40 to 60% saturation) and calcium phosphate adsorbtion is shown in Figure 26. The use of saturated ammonium sulfate solutions in place of solid ammonium sulfate was employed in other purification studies. Concentrations of ammonium sulfate (final molarity) were adjusted according to Pogell and McGilvery (1954). Seventy to eighty percent of malic enzyme activity was recovered in the fraction precipitated between 2.2 to 2.6 M. The extent of purification was not improved, however, browning of the protein extracts of fresh fruits or fruits from storage up to 4 months could be arrested by quick addition of the ammonium sulfate solution. In contrast, an instan- taneous browning accurred in protein extracts obtained from fruit stored longer than 5 to 6 months when ammonium sulfate was added. Attempts to treat crude protein extracts with sodium meta-bisulfite or insoluble poly— vinylpyrrolidone (Jones et al., 1965b and Klepper and Hageman, 1969) at pH 9.5 or 7.5 and 5.5 did not prevent the instantaneous browning during ammonium sulfate fractionation of protein extracts obtained from fruit stored for long periods. Figure 26. Figure 27. Figure 28. 112 ElectrOphoresis of Pear malic enzyme purified by ammonium sulfate fractionation and calcium phosphate gel adsorbtion. Left to right: 22, 45, 41, 68, 27 and 45 enzyme units applied per disc gel. ElectrOphoresis of pear malic enzyme purified by ammonium sulfate fractionation at the pI of the enzyme. Protein stain and malic enzyme assay alternate from left to right. Left: crude extract. Middle: protein and malic enzyme precipitated at 1.8 M ammonium sulfate at pH 5.5. Right: protein and malic enzyme precipitated at 1.8 M ammonium sulfate at pH 4.5. Electrophoresis of pear malic enzyme purified by heat treatment. Malic enzyme precipitated between 55 to 60% ammonium sulfate saturation was heated for 10 minutes at the indicated temperature. Thirty enzyme units (assayed following heat treatment) were loaded on each disc gel. 113 _" ‘9 h‘ .., l “4 ; ~ - - ‘ _ x— v 1H .11 Crude Extract Ammonium Salk". 410-60 + 5'39; _’__ - 114 It was observed that malic enzyme precipitation with ammonium sulfate was dependent on the pH during fractionation. As noted previously, malic enzyme was precipitated at 2.6 M ammonium sulfate over a wide pH range (5.5 to 10.0). Close to the pI of the enzyme, precipitation occurred at 1.8 to 2.0 M. Thus, considerable purification of malic enzyme from crude extracts could be obtained (Figure 27) by raising the ammonium sulfate concentration to 1.8 M at pH 5.5, and subsequently lowering the pH carefully to 4.5. Up to 10% of the original activity was recovered in the fraction precipitated at pH 5.5 and between 40 to 60% in the fraction precipitated during one hour at pH 4.5. Oxaloacetate decarboxylase activity of malic enzyme precipitated at 1.8 M ammonium sulfate at pH 4.5 and 5.5 was found to be inhibited differentially by NADP (Table 9). The enzyme precipitated at pH 4.5 was inhibited 50% and 82% with 4.7 x 10"6 M and 1.6 x 10'5 M NADP, respectively. Considerably less inhibition was found with the enzyme precipitated at pH 5.5 (Table 9). Oxalo- acetate decarboxylase activity of malic enzyme purified from pigeon liver (Rutter and Lardy, 1958), wheat germ (Kramer et al., 1951) and apple (Dilley, 1966a) was reported to be enhanced, inhibited and neither enhanced or inhibited, respectively by NADP. The differential inhibitiontnr NADP of the oxaloacetate decarboxylase activity of malic enzyme precipitated at pH 4.5 and 5.5 (Table 9) suggests that activity resides in two enzymes (Figure 22), which can be pre- cipitated by ammonium sulfate at their respective isoelectric points. Fractionation with ammonium sulfate at the pI of the enzyme was employed only to test oxaloacetate decarboxylase activity of the enzyme.. The results (Figure 27), however, indicate that considerable purification can be achieved employing this procedure. 115 .vouaomoum mw ++az mo ouaommum onu GH aowumahxonumo 1mm oumuoomoamxo moooamucomm now wouoouuou mouoofla om was 0H cmosuon ammoaou N00 «0 mummN ..mwo£uoz was mamfiuoumz. aw woNMHoomm mum mdoaufivaoo hmmm9 wouchHuomum waxwam oHHmz “mom mo >ufl>fiuo< ommahxonumomm oumuoomoamxo mo coaufinwch mn