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A retrospective view of plant lipid research
Prog. Lipid Res. Vol. 33, No. 1/2, pp. I-8, 1994 Copyright © 1994 Elsevier Science Lid Printed in Great Britain. All rights reserved 0163-7827/94/$24.00
Pergamon
A RETROSPECTIVE
VIEW OF PLANT
LIPID
RESEARCH PAUL K. STUMPF Section on Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
CONTENTS I. II. III. IV. V. VI. VII.
INTRODUCTION T ~ SCENE IN 1950 Tim FIRST t4C LIPID EXPERIMENTS DEMONSTRATIONOF THE ANCILLARY ENZYMES Tn~ SITE OF FATTY ACID SYNTHESIS IN PLANTS DESATURATIONAND BETA OXIDATION FINAL THOUGHTS REFERENCES
I. I N T R O D U C T I O N
The thoughts I will relate to the reader are essentially limited to the work that was carried out in my laboratory from 1950 to 1970 by many gifted students and postdoctoral fellows, some of whom are present at this gathering. I apologize to those in my laboratory at that time whose contributions were not examined because of limits of time and space, and to those in other laboratories who made equally important observations to advance the field of plant lipid biochemistry during this period. II.
THE SCENE
I N 1950
So let us begin to describe this period from my own probably biased perspective. In 1950 James Bonner's textbook ~ entitled Plant Biochemistry made its appearance. In 537 pages, Bonner summarized what was then known in this very new area of research. Of those 537 pages, in Part V titled "Secondary Plant Product" a 31 pp. chapter was devoted to "Lipids and Lipid Metabolism". Sixty seven percent of the chapter was on classification, distribution and structure of lipids and the remaining thirty three percent on metabolism of lipids. What was known about lipid synthesis in the late 1940s? In 1949 Stadtman and Barker2 had prepared for the first time a very active cell-free extract from Clostridium kluyveri, which rapidly converted free acetate to butyric and caproic acids in the presence of ATP and suitable reductants. About this same time Rittenberg and Bloch3fed deuterated acetate to rats and recovered long chain deuterated fatty acids from liver lipids. One could therefore construct the equation 2CH3COOH + reductants -~ CH3COCH2COOH --~ CH3CH2CH2COOH, etc. with a beta keto acid as the intermediate. Bonner, on p. 372, continues: "It will be of interest to ascertain whether fatty acid synthesis in the higher plant follows this same pattern of synthesis from a two carbon fragment. The seed tissues of the plant which carry out such extraordinary rapid and extensive fat synthesis should provide excellent material both for in vitro and in vivo studies." As for beta oxidation, Bonner states that "The oxidation of fatty acids in the plant is but little understood and work on this subject is confined to the study of the enzyme
2
P.K. STUMPF
lipoxidase, discovered in 1928 by Bohn and Haas in the soybean . . . " He invokes the 1904 work of Knoop which proposed the concept of beta oxidation. The concept of beta oxidation was important in the late 1940s and early 1950s for two reasons: (1) it explained the degradation of even chain fatty acids by two carbon deletions, and (2) it was theorized that the reverse of beta oxidation was responsible for the synthesis of the even chain fatty acids. This, then, was the status of knowledge of fatty acid oxidation and synthesis in the late 1940s and early 1950s. As for methodology, although solvent extraction of lipids was well developed, the detection and identification of very small amounts of fatty acids were in their infancy. Gas-liquid chromatography in its crudest form was in its early stage of development in the English laboratory of Martin and James, thin-layer chromatography as practiced now did not exist, substrates and cofactors such as ATP, NAD, were available, but of very poor quality. Indeed, NADP was difficult to obtain. Discovered by Otto Warburg and isolated from horse red cells in his laboratory, his published description of its isolation could not be reproduced in the U.S.A. until one of his collaborators, E. Haas, who immigrated from Germany to the U.S.A. after the end of the war, revealed to his mentor, Professor T. R. Hogness, that a crucial step in the isolation of NADP was deliberately omitted for obvious reasons. Coenzyme A was just discovered in the late 1940s by Lipmann and the active C2 unit--the mythical activated acetate unit--was identified by F. Lynch in Germany as acetyl Coenzyme A. 14C substrates of somewhat questionable purity were becoming available at that time. 14CO2was laboriously collected as a barium salt--usually the reactions were run in Warburg manometer cups with KOH in the center well and the CO2, released by the enzymic reaction and trapped as potassium carbonate, was converted to barium carbonate, washed several times with 80% alcohol--and then spread as a thin film on aluminum planchets which were then counted for radioactivity by a thin window Geiger-Muller tube. Non-volatile end products were similarly counted after extraction and removal of contaminating 14C substrates. Liquid scintillation counting equipment made its appearance in the late-1950s. The most common procedure of measuring colorimetric assays was by employing the Klett-Summerson colorimeter with its blue, red and green glass filters. For more sophisticated measurements, the new Beckman spectrophotometer was available, one to a department! Most reactions were run in a 3 ml volume, mainly because that was the standard volume used in Warburg equipment and, of course, glass pipettes were standard delivery systems with a 0.1 ml pipette drawn out to a long tip for more accurate delivery of a solution. III. THE FIRST NC LIPID EXPERIMENTS This, then, was the situation when I decided to explore the field of plant lipid biochemistry. I would now like to describe our first experiments on lipid metabolism. Three factors were important in this decision. First of all, as Bonner so effectively described the area of plant lipid biochemistry in 1950, the field was wide open. The second factor was the arrival of my first postdoctoral fellow, Eldon Newcomb, who had just received his Ph.D. from the University of Wisconsin. And the third was that I, as an Assistant Professor of Plant Nutrition on the Berkeley campus, occupied a small bench-space in H. A. Barker's laboratory. It was in this laboratory that the elegant experiments by Earl Stadtman had been conducted with cell-free extracts of Ciostridium kluyveri. As a result, Barker had available a rare collection of 14C-labeled substrates which he kindly allowed me to use in the first experiments on both fatty acid oxidation and biosynthesis. Two sets of experiments were developed, one exploring fatty acid oxidation and the second fatty acid biosynthesis. Once again, the availability of a set of substrates dictated the success of a series of experiments. Professor I. Chaikoff of the Department of Physiology at the UC Berkeley campus was engaged in a series of experiments requiring palmitic acid labeled in positions 1, 2 and 3. These substrates were chemically synthesized by Dr William Dauben of the Chemistry Department. Since Dr Barker had used some of these substrates in his CI. kluyveri experiments, he made them available to us. We arbitrarily decided to use
Plant lipid research--retrospective view
3
germinating peanuts as a source of enzymes, since these seeds had not only a high lipid content but also they were readily available and could be easily germinated. I still recall the first experiment that Newcomb performed: grinding up germinated cotyledons in buffer, making a crude extract and then pipetting the extract and palmitic acid labeled in the 1 position into a Warburg cup and after a 1 hr incubation assaying for ~4CO2 as previously described. To our surprise, a very significant amount of radioactivity had been released as carbon dioxideJ It was soon discovered that this system did not involve beta-oxidation but, instead, required a microsomal fraction, a supematant fraction and unknown heat-stable cofactor. Over a period of years involving the participation by Thomas Humphreys, Paul Castelfranco, Robert Martin and finally Ward Shine, these results obtained in 1950 were resolved as describing an alpha oxidation system involving a fatty acid peroxidase, a long chain aldehyde dehydrogenase and a H202 generating system which oxidized C,4 to C~s free fatty acids one carbon at a time to the n-1 fatty acid. The unknown cofactor was identified as glycolic acid which served as the substrate for the recently discovered glycolic oxidase, one of the products of the reaction being Hs 02. While the functionality of this system is probably limited in higher plants, it proved to be a great stimulant in moving further into the wide open field of plant lipid biochemistry. The second set of experiments carried out in 1950 by Newcomb 4 involved comparing the capacity of germinated and developing peanut cotyledons to incorporate radioactive compounds into long chain fatty acids. Table 1 summarizes these early data. There was vigorous incorporation of radioactive carbon from both acetate labeled in either the 1 or the 2 position of acetate. Surprisingly while the I-labeled carbon gave rise to higher levels of '4CO2 than did the 2-labeled carbon of acetate, the 2-labeled carbon of acetate consistently showed greater incorporation into fatty acid than did the l-labeled carbon substrate. We were puzzled by these results at that time. Now with the knowledge at hand a probable explanation would be as follows: CH3COCoA + CO2 --* COOHCHeCOCoA _...5. .5. COOHCH2COOH ,5"
.5"
+ CoA ---* COOHCHeCOCoA .5*
~L .5"
CO2 + Cfa
CH3COCoA + COs--* COOHCH2 COCOA--* COOHCH2COOH I*
+ CoA --* COOHCHeCOCoA $ i. COs + Cfa TABLE1. Conversion of t4C-Labeled Substrates to Carbon Dioxide and Long Chain Fatty Acids by Slices of Maturing Cotyledons
Experiment I
2
3 4
Substrate [I J(C]Acetate [2-~4C]Acetate [l-~4C]Butyrate [l-~"C]Hexanoate [1J4C]Acetate [2J4C]Acetate [IJ4C]Butyrate [1J~]Hexanoate t4C-Formate [3-14C]Valerate 14C-evenly-labeled glucose '4C-evenly-labeled fructose
Percentage of supplied radioactivity in respiratory CO:
Percentage of supplied radioactivity recovered in fatty acids
29.0 10.5 20.5 42.0 32.5 13.8 41.0 48.3 31.5 0.6
11.7 13.6 0.8 I. 1 22.0 34.6 2.7 4.3 0.3 0.2
15.9
6.1
17.6
5.8
4
P.K. STUMPF
Indeed, Stumpf and Burris demonstrated in 19815 that free malonic acid was the most prevalent dicarboxylic acid in legumes. They also showed that when either 1-]4C acetate or 2-J4C acetate was fed to soybean seedling root tissues, the newly synthesized ~4C malonic acid had the same labeling pattern as described in the above reactions. These results would suggest that in the plant cell the reactions described above do indeed occur; accordingly, if free malonic acid can move into the cytosolic compartment, there to be reactivated, there would be no need to postulate a cytosolic acetyl CoA carboxylase which has been demonstrated to occur in proplastids and in chloroplasts. Not only the developing but also the germinating peanut cotyledons incorporated radiolabeled acetate into long chain fatty acids. Some years after this paper was published I was asked why we did not identify the individual radioactive fatty acids obtained in these experiments. We certainly would have, but the separation of the individual from the total fatty acids had to wait until gas-liquid chromatography became available! These results were followed up by a series of experiments conducted by George Barber, Craig Squires, Vernon McMahon, Shangfa Yang, Claude Willmot, Peter Overath, Robert Simoni, Clem Hawke, James Brooks, Jan Jaworski, John Ohlrogge, and finally Takashi Shimakata, the results of which advanced considerably the basic knowledge of fatty acid biosynthesis in higher plants. Fine tuning of their results is now occurring in a number of laboratories, among them those of Jan Jaworski, John Ohlrogge, and Chris Somerville. IV. DEMONSTRATION OF THE ANCILLARY ENZYMES A number of key discoveries were made in the late 1950s and early 1960s that markedly accelerated research in this field. These include the demonstration of the role of CO2, of the enzyme acetyl CoA carboxylase, and of the protein acyl carrier protein in plant fatty acid biosynthesis as well as the identification of the plastid as the only site of fatty acid biosynthesis in the plant cell. As we had indicated earlier, in vitro fatty acid biosynthesis in any tissue was more or less at a standstill in the early 1950s. Attempts to develop enzyme extracts from animal tissues were spotty; the only consistent system was developed by Sam Gurin in the early 1950s, but the levels of incorporation of radiolabeled acetate were low. Moreover, the concept of a reversal beta-oxidation system still prevailed; but it was finally put to rest by two important sets of experiments. The first resulted from the isolation and purification of all the beta oxidation enzymes from animal mitochondria by the laboratory of David Green 6 at the University of Wisconsin. Green's group decided to test once and for all the reversal beta-oxidation concept and in an important paper in 19537 Stansley and Beinert were able to synthesize butyric acid from acetyl CoA in the presence of all the beta-oxidation enzymes and suitable reductants. But there was no indication for the synthesis of longer chain fatty acids. These results were more or less in agreement with the earlier Stadtman and Barker experiments with extracts of CI. kluyveri. Even more puzzling were the observations of a postdoctoral fellow in Green's group, Salih Wakil, who was able to demonstrate in 19578 that a clear extract of pigeon liver, free of beta-oxidation enzymes, was able to convert acetyl CoA to long chain fatty acids in the presence of ATP, NADPH and CO 2. In the same year, H. Klein9 also had made the rather strange observation that when carbon dioxide was added to a yeast extract, there was a considerable increase in fatty acid synthesis, but, curiously, when labeled CO2 was added to the reaction mixture, no incorporation into fatty acids was observed. To cut a long story short, in 1958 Wakil ~0identified malonyl CoA as the second substrate and in 1959~ identified the biotin-containing protein as acetyl CoA carboxylase. In 1957~2 Barber, in my laboratory, had been successful in preparing a particulate system from avocado mesocarp tissue which could readily incorporate radiolabeled acetate into long chain fatty acids in the presence of ATP, CoA, NADPH and Mn 2+ . The products were radiolabeled palmitic
Plant lipid research--retrospective view
5
and stearic acids. Squires in 195813 was able to prepare very stable acetone powders of avocado mesocarp and showed a vigorous stimulation of fatty acid synthesis by the further addition of sodium bicarbonate to the reaction mixture. In the summer of 1958, I transferred to the University of California at Davis to organize the new Department of Biochemistry and Biophysics on that campus. Three new postdoctoral fellows joined my group in 1959 (by which time we had moved into new laboratories on the Davis campus) and they were Edward Barron, M.D. (Hal) Hatch and J. Brian Mudd. I assigned Ed the job of sorting out the avocado system, Hal the job of determining the presence of acetyl CoA carboxylase in higher plants and Brian to sort out the problem of oleic acid biosynthesis. Ed further defined the avocado system and also observed that the addition of avidin completely shut down fatty acid synthesis from radioactive acetate, although radiolabeled malonyl CoA incorporation into fatty acids was not affected by avidin. ~4Two puzzling problems were the persistent requirement to work with particle preparations as the starting material rather than supernatant systems as was being described in animal and yeast systems and the loss of activity of the extracts when subjected to ammonium sulfate precipitation. In the meantime, Hal Hatch tackled the acetyl CoA carboxylase system by employing wheat germ as the starting material. He purified the enzyme some 170 fold and described its properties and showed that acetyl CoA was the best substrate with propionoyl and butyroyl CoAs in decreasing activities. Avidin completely inhibited the system. ~5 Unfortunately the enzyme was quite unstable at the 170-fold purification level. Later results would prove that the enzyme does not have the allosteric properties of the animal system in terms of no citrate activation and no phosphorylation/dephosphorylation cycle. Brian Mudd tackled an equally difficult problem, namely the inability to demonstrate an enzyme system capable of desaturating stearic acid to oleic acid. Particulate avocado mesocarp preparations readily converted radiolabeled acetate into high levels of oleic acid under aerobic conditions; however, under anerobic conditions the main product was stearic acid. Addition of stearic acid as a substrate led to no desaturation. Moreover, experiments by Barron also showed that stearoyl CoA was not desaturated by a system known to convert acetate into oleate. We concluded that molecular oxygen was an absolute requirement but that either stearic acid or stearoyl CoA was not a suitable substrate ~6. Later research of course totally explained our lack of success! When Peter Overath joined my laboratory in 1963, he came with a wealth of experience, having received his Ph.D. from F. Lynen in Germany. I suggested that he look into the observation made by Barron that ammonium sulfate fractions of clear extracts of avocado acetone powder gave only enzymically inactive protein fractions. Already in 1963 the laboratory of Vaglos at the NIH had observed that E. coli extracts capable of synthesizing fatty acids from acetyl CoA and suitable cofactors lost activity by fractionation, but that this activity could be restored by the addition of a boiled crude E. coli extract. With these results coming forth, Overath quickly showed that a similar situation existed in the avocado system, in that a concentrated protein extract could be separated into two fractions, the first fraction which came down between 20 and 60% ammonium sulfate was heat labile and the second fraction coming down between 60 and 90% was heat stable. The first fraction was inactive unless the second, heat stable fraction was added. ~7 At this point I travelled to Washington to participate in an NIH panel meeting at Bethesda. During a break I visited Vaglos's laboratory and I was provided with a sample of their heat stable E. coli fraction to test on our avocado fraction. On my return, Overath tested the sample and discovered that it was far more effective than was our heat stable avocado fraction. The difference was probably due to a much higher level of what turned out to be the acyl carrier protein in the Vaglos sample than in our plant sample. When Overath returned to Germany, I suggested to Robert Simoni that he purify and characterize the plant ACP as his doctoral thesis. Bob purified the E. coli, an Arthobacter, the avocado mesocarp and the spinach ACPs, and chemically as well as physically characterized these
6
P.K. STUMPF
proteins. He reported a molecular weight of 10300 for the spinach ACP with total amino acid residues of 88. '8 These results have been further refined by Ohlrogge in his recent publications. The surprising result was that the E. coli ACP was at least three fold more effective than was the spinach ACP in a spinach synthase system. As a result, all our further work with synthase systems employed the E. coli ACP rather than the more difficult to obtain spinach ACP. The most interesting aspect of these results showed that the bacterial and the plant synthase systems resembled each other very closely in that they both were non-associative systems that permitted fractionation of the individual enzymes including their ACPs which could also cross react with the appropriate synthase systems, in sharp contrast to both the yeast and animal synthase systems which were multifunctional polypeptides with their ACPs an integral part of the polypeptide structure. v. THE SITE OF FATTY ACID SYNTHESIS IN PLANTS Evidence was also fast accumulating from several laboratories that the fatty acid systems in plants, unlike those in animal systems, were localized in particles. We had already identified in 1957 that the activity for fatty acid synthesis in the avocado mesocarp was associated with particles) 2 In 1960, Smirnov in Russia ~9had described a system in which isolated spinach chloroplasts incorporated radiolabeled acetate into fatty acids in the presence of ADP, CoA and Mn 2+ and light. Two years later Mudd and McManus2° with isolated spinach chloroplasts and later in the same year Stumpf and James2' with lettuce chloroplasts demonstrated the very active synthesis of palmitic and oleic acids in the presence of ATP, CoA, Mg 2+, CO2 and light. In 1964, Yamada 22 in my laboratory fractionated a crude extract of developing castor bean seeds and showed that the major activity for de novo synthesis of fatty acids was associated with large particles. The significance of these observations were not apparent at that time. Probably some confusion arose by our calling these systems 'particles', 'large particles' and 'mitochondrial'. Fortunately, Zilkey and Canvin23 in Canada carried out a careful sucrose density gradient centrifugation of homogenates of developing castor bean endosperm which converted acetate into oleate and gave these particles the name of oleosomes. Further clarification came from the laboratory of Kekwick in 197524 who showed quite conclusively that large particles isolated from cauliflower buds and avocado mesocarp were identified as proplastids, the sites of synthesis fatty acids in developing seeds and mesocarp tissues. And of course, in leaf tissue, chloroplasts were presumably the site for fatty acid synthesis. To settle finally the problem of the exclusivity of the chloroplast as the only site for fatty acid synthesis in the leaf cell, Ohlrogge, Kuhn and Stumpf in 197925 showed that only in the chloroplast was ACP localized and hence only in the chloroplast did de novo fatty acid synthesis take place. vI. DESATURATION AND BETA OXIDATION Returning to the 1960s, a few comments should be made concerning desaturation and beta-oxidation. During the 1960s, evidence was accumulating that the site of synthesis of oleic acid was associated with plastids. The work of Mudd with avocado particles, and later McManus and Mudd with spinach chloroplasts and by Stumpf and James in lettuce chloroplasts supported those results. Still, the isolation of an enzyme system to duplicate what occurred in the intact plastids was unsuccessful until Nagai and Bloch26 clearly showed in 1968 that the correct substrate for desaturation was stearoyl ACP rather than stearoyl CoA, the substrate in mammalian and yeast systems, and that the enzyme was completely soluble rather than microsomal as in the non-plant systems. This work was then extended by Jaworski 27 in our laboratory in 1974. At that time we were puzzled by the observation that free oleic acid was the product of desaturation rather than oleoyl ACP. Shine and Mancha28examined this problem and discovered the presence of a highly specific oleoyl ACP thioesterase. The importance of this observation is exemplified by the great interest in this type of enzyme in lipid metabolism. The desaturase has now been
Plant lipid research--retrospective view
7
crystallized in Somerville's laboratory. As for the synthesis of linoleic acid, in 1964 McMahon :9 for the first time prepared a particulate fraction from developing safflower seeds and demonstrated the rapid conversion of oleoyl CoA to linoleic acid. This work was re-examined in 1971 by Vijay3° who described a microsomal preparation from developing safflower seeds that rapidly converted oleoyl CoA to linoleic acid (associated with phosphatidylcholine) in the presence of NADH and molecular oxygen. Because we were convinced that an oleoyl thioester was the true substrate viz stearoyl ACP in oleic acid synthesis, we ignored the obvious !ata we had and incorrectly ascribed the substrate as oleoyl CoA. Later other workers clearly showed that the correct substrate was oleoyl phosphatidylcholine and the product linoleoyl phosphatidyl choline. As for beta oxidation, in 1956, Barber 3' prepared a particulate system from germinating peanut seeds which we considered to be mitochondria and which rapidly oxidized short and long chain fatty acids to t4CO2 as well as '4C Krebs cycle acids. We concluded that beta oxidation did indeed occur in plants and that they were localized in mitochondria. Of course now our interpretation would have been that we had a mixture of mitochondria and glyoxysomes. The elegant work of Beevers and his group in the late 1960s32 did much to clarify the true nature of the beta-oxidation cycle. More recent work has also identified leaf peroxisomes as another site of beta oxidationY
VII. F I N A L T H O U G H T S
With the basic aspects of plant lipid biochemistry in place as a result of the work we have described in our laboratory and other important work from the laboratories of Beevers, Roughan and Slack, Harwood, to name a few, this area has now entered the revolution of molecular biology and sophisticated instrumentation, and accordingly, progress is accelerating at a very rapid rate. Thus the work of Somerville, Ohlrogge, Jaworski, Harwood, Styme, Murphy, Heinz, and Joyard and Douce to mention a few of the laboratories, as well as the research groups in a number of companies, most prominent being those in Calgene, promise many new and exciting discoveries in the near future that will make my retrospective thoughts appear almost irrelevant. Nevertheless, for me at least the research developed between 1950 and 1970 did paint the sometime confusing, but certainly challenging, picture that began to emerge by the early 1970s. It will be equally interesting to paint a comparable picture in the year 2025, covering the 1980s and 1990s as, you the current actors in this drama, play out your roles! Hindsight is always so much easier than foresight!
REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
B O ~ R , J. Plant Biochemistry, 537 pp. Academic Press, New York, 1950. STADTMAN,E. R. and BARKER,H. A. J. Biol. Chem. 1 ~ , 1095 (1949). RI~'rENaERG,D. and BLOCH,K. J. Biol. Chem. 160, 471 (1945). NEW¢OMB,E. H. and S~MPF, P. K. In Phosphorus Metabolism, Vol. II, p. 291 (McELROY,W. O. and GLASS, B., eds) The Johns Hopkins Press, Baltimore, MD, 1952. SI~MPF, D. K. and BURRIS,R. H. Plant Physiol. 68, 992 (1981). GRF~N, D. E. Biol. Revs., Cambridge Phil. Soc. 29, 330 (1954). STANSLE¥,P. G. and BEINERT,H. Biochim. Biophys. Aeta 11, 600 (1953). GIBSON,D. M., JACOBS,M. J., PORTER,J. W., TIETZ, A. C. and WAglL, S. J. Biochim. Biophys. Acta 23, 219 (1957). KLEIN, H. P. J. Bacteriol. 73, 530 (1957). WAKIL,S. J. J. Am. Chem. Soe. 80, 6465 0958). WAKIL,S. J., TITCHNER,E. B. and GIBSON,D. M. Biochim. Biophys. Acta 29, 225 0958). STUMPF,P. K. and BARBER,G. A. J. Biol. Chem. 227, 407 (1957). SOUmF.S,C. L., STUMPF,P. K. and SCHMID,C. Plant Physiol. 33, 365 (1958). BARRON,E. J., SQUIRES,C. and STUMPF,P. K. J. Biol. Chem. 236, 2610 (1961). H^xcn, M. D. and STUMPF,P. K. ,I. Biol. Chem. 236, 2879 (1961). MUDD, J. B. and S'rUMPF,P. K. J. Biol. Chem. 236, 2602 (1961). OVE10.TH,P. and S~MPF, P. K. J. Biol. Chem. 239, 4102 (1964). SXMONI,R. D., CRXDDI.E,R. S. and S1-OMI'F,P. K. J. Biol. Chem. 242, 573 (1967). S~aRNOV,B. P. Biokhimia 25, 419 (1960). MUDD, J. B. and McMANus, T. T. J. Biol. Chem. 237, 2057 (1962).
8 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
P.K. STUMPF STUMPF,P. K. and JAMF.S,A. T. Biochim. Biophys. Acta 70, 20 (1962). YAM^DA,M. and STUMPF,P. K. Biochem. Biophys. Res. Commun. 14, 165 (1964). ZILKEYB. F. and CANVIN,D. T. Can. J. Bot. 50, 323 (1971). WF.AIRE,B. J. and KEKWIC[, R. G. O. Biochem. J. 146, 425 (1975). OI-ILROC~E,J. B., KUHN, D. N. and STUMPF,P. K. Proc. Natl. Acad. Sci. U.S.A. 76, 1194. NAGAI,J. and BLOCH,K. J. Biol. Chem. 243, 426 (1968). JAWORSKi,J. G. and STUMPF,P. K. Arch. Biochem. Biophys. 162, 158 (1974). SHINE,W. E., MANCHA,M. and STUMPF,P. K. Arch. Biochem. Biophys. 172, 110 (1976). MCM^HON, V. and STUMPF,P. K. Biochim. Biophys. Acta 84, 359 (1964). VuAY, I. K. and STUMPF,P. K. J. Biol. Chem. 246, 2910 (1971). STUMPF,P. K. and BARBER,G. A. Plant Physiol. 31, 304 (1956). COOPER,T. G. and B ~ , H. J. Biol. Chem. 244, 3507 (1969). GERHARDT,B. Planta 59, 238 (1983).
Pergamon
A RETROSPECTIVE
VIEW OF PLANT
LIPID
RESEARCH PAUL K. STUMPF Section on Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
CONTENTS I. II. III. IV. V. VI. VII.
INTRODUCTION T ~ SCENE IN 1950 Tim FIRST t4C LIPID EXPERIMENTS DEMONSTRATIONOF THE ANCILLARY ENZYMES Tn~ SITE OF FATTY ACID SYNTHESIS IN PLANTS DESATURATIONAND BETA OXIDATION FINAL THOUGHTS REFERENCES
I. I N T R O D U C T I O N
The thoughts I will relate to the reader are essentially limited to the work that was carried out in my laboratory from 1950 to 1970 by many gifted students and postdoctoral fellows, some of whom are present at this gathering. I apologize to those in my laboratory at that time whose contributions were not examined because of limits of time and space, and to those in other laboratories who made equally important observations to advance the field of plant lipid biochemistry during this period. II.
THE SCENE
I N 1950
So let us begin to describe this period from my own probably biased perspective. In 1950 James Bonner's textbook ~ entitled Plant Biochemistry made its appearance. In 537 pages, Bonner summarized what was then known in this very new area of research. Of those 537 pages, in Part V titled "Secondary Plant Product" a 31 pp. chapter was devoted to "Lipids and Lipid Metabolism". Sixty seven percent of the chapter was on classification, distribution and structure of lipids and the remaining thirty three percent on metabolism of lipids. What was known about lipid synthesis in the late 1940s? In 1949 Stadtman and Barker2 had prepared for the first time a very active cell-free extract from Clostridium kluyveri, which rapidly converted free acetate to butyric and caproic acids in the presence of ATP and suitable reductants. About this same time Rittenberg and Bloch3fed deuterated acetate to rats and recovered long chain deuterated fatty acids from liver lipids. One could therefore construct the equation 2CH3COOH + reductants -~ CH3COCH2COOH --~ CH3CH2CH2COOH, etc. with a beta keto acid as the intermediate. Bonner, on p. 372, continues: "It will be of interest to ascertain whether fatty acid synthesis in the higher plant follows this same pattern of synthesis from a two carbon fragment. The seed tissues of the plant which carry out such extraordinary rapid and extensive fat synthesis should provide excellent material both for in vitro and in vivo studies." As for beta oxidation, Bonner states that "The oxidation of fatty acids in the plant is but little understood and work on this subject is confined to the study of the enzyme
2
P.K. STUMPF
lipoxidase, discovered in 1928 by Bohn and Haas in the soybean . . . " He invokes the 1904 work of Knoop which proposed the concept of beta oxidation. The concept of beta oxidation was important in the late 1940s and early 1950s for two reasons: (1) it explained the degradation of even chain fatty acids by two carbon deletions, and (2) it was theorized that the reverse of beta oxidation was responsible for the synthesis of the even chain fatty acids. This, then, was the status of knowledge of fatty acid oxidation and synthesis in the late 1940s and early 1950s. As for methodology, although solvent extraction of lipids was well developed, the detection and identification of very small amounts of fatty acids were in their infancy. Gas-liquid chromatography in its crudest form was in its early stage of development in the English laboratory of Martin and James, thin-layer chromatography as practiced now did not exist, substrates and cofactors such as ATP, NAD, were available, but of very poor quality. Indeed, NADP was difficult to obtain. Discovered by Otto Warburg and isolated from horse red cells in his laboratory, his published description of its isolation could not be reproduced in the U.S.A. until one of his collaborators, E. Haas, who immigrated from Germany to the U.S.A. after the end of the war, revealed to his mentor, Professor T. R. Hogness, that a crucial step in the isolation of NADP was deliberately omitted for obvious reasons. Coenzyme A was just discovered in the late 1940s by Lipmann and the active C2 unit--the mythical activated acetate unit--was identified by F. Lynch in Germany as acetyl Coenzyme A. 14C substrates of somewhat questionable purity were becoming available at that time. 14CO2was laboriously collected as a barium salt--usually the reactions were run in Warburg manometer cups with KOH in the center well and the CO2, released by the enzymic reaction and trapped as potassium carbonate, was converted to barium carbonate, washed several times with 80% alcohol--and then spread as a thin film on aluminum planchets which were then counted for radioactivity by a thin window Geiger-Muller tube. Non-volatile end products were similarly counted after extraction and removal of contaminating 14C substrates. Liquid scintillation counting equipment made its appearance in the late-1950s. The most common procedure of measuring colorimetric assays was by employing the Klett-Summerson colorimeter with its blue, red and green glass filters. For more sophisticated measurements, the new Beckman spectrophotometer was available, one to a department! Most reactions were run in a 3 ml volume, mainly because that was the standard volume used in Warburg equipment and, of course, glass pipettes were standard delivery systems with a 0.1 ml pipette drawn out to a long tip for more accurate delivery of a solution. III. THE FIRST NC LIPID EXPERIMENTS This, then, was the situation when I decided to explore the field of plant lipid biochemistry. I would now like to describe our first experiments on lipid metabolism. Three factors were important in this decision. First of all, as Bonner so effectively described the area of plant lipid biochemistry in 1950, the field was wide open. The second factor was the arrival of my first postdoctoral fellow, Eldon Newcomb, who had just received his Ph.D. from the University of Wisconsin. And the third was that I, as an Assistant Professor of Plant Nutrition on the Berkeley campus, occupied a small bench-space in H. A. Barker's laboratory. It was in this laboratory that the elegant experiments by Earl Stadtman had been conducted with cell-free extracts of Ciostridium kluyveri. As a result, Barker had available a rare collection of 14C-labeled substrates which he kindly allowed me to use in the first experiments on both fatty acid oxidation and biosynthesis. Two sets of experiments were developed, one exploring fatty acid oxidation and the second fatty acid biosynthesis. Once again, the availability of a set of substrates dictated the success of a series of experiments. Professor I. Chaikoff of the Department of Physiology at the UC Berkeley campus was engaged in a series of experiments requiring palmitic acid labeled in positions 1, 2 and 3. These substrates were chemically synthesized by Dr William Dauben of the Chemistry Department. Since Dr Barker had used some of these substrates in his CI. kluyveri experiments, he made them available to us. We arbitrarily decided to use
Plant lipid research--retrospective view
3
germinating peanuts as a source of enzymes, since these seeds had not only a high lipid content but also they were readily available and could be easily germinated. I still recall the first experiment that Newcomb performed: grinding up germinated cotyledons in buffer, making a crude extract and then pipetting the extract and palmitic acid labeled in the 1 position into a Warburg cup and after a 1 hr incubation assaying for ~4CO2 as previously described. To our surprise, a very significant amount of radioactivity had been released as carbon dioxideJ It was soon discovered that this system did not involve beta-oxidation but, instead, required a microsomal fraction, a supematant fraction and unknown heat-stable cofactor. Over a period of years involving the participation by Thomas Humphreys, Paul Castelfranco, Robert Martin and finally Ward Shine, these results obtained in 1950 were resolved as describing an alpha oxidation system involving a fatty acid peroxidase, a long chain aldehyde dehydrogenase and a H202 generating system which oxidized C,4 to C~s free fatty acids one carbon at a time to the n-1 fatty acid. The unknown cofactor was identified as glycolic acid which served as the substrate for the recently discovered glycolic oxidase, one of the products of the reaction being Hs 02. While the functionality of this system is probably limited in higher plants, it proved to be a great stimulant in moving further into the wide open field of plant lipid biochemistry. The second set of experiments carried out in 1950 by Newcomb 4 involved comparing the capacity of germinated and developing peanut cotyledons to incorporate radioactive compounds into long chain fatty acids. Table 1 summarizes these early data. There was vigorous incorporation of radioactive carbon from both acetate labeled in either the 1 or the 2 position of acetate. Surprisingly while the I-labeled carbon gave rise to higher levels of '4CO2 than did the 2-labeled carbon of acetate, the 2-labeled carbon of acetate consistently showed greater incorporation into fatty acid than did the l-labeled carbon substrate. We were puzzled by these results at that time. Now with the knowledge at hand a probable explanation would be as follows: CH3COCoA + CO2 --* COOHCHeCOCoA _...5. .5. COOHCH2COOH ,5"
.5"
+ CoA ---* COOHCHeCOCoA .5*
~L .5"
CO2 + Cfa
CH3COCoA + COs--* COOHCH2 COCOA--* COOHCH2COOH I*
+ CoA --* COOHCHeCOCoA $ i. COs + Cfa TABLE1. Conversion of t4C-Labeled Substrates to Carbon Dioxide and Long Chain Fatty Acids by Slices of Maturing Cotyledons
Experiment I
2
3 4
Substrate [I J(C]Acetate [2-~4C]Acetate [l-~4C]Butyrate [l-~"C]Hexanoate [1J4C]Acetate [2J4C]Acetate [IJ4C]Butyrate [1J~]Hexanoate t4C-Formate [3-14C]Valerate 14C-evenly-labeled glucose '4C-evenly-labeled fructose
Percentage of supplied radioactivity in respiratory CO:
Percentage of supplied radioactivity recovered in fatty acids
29.0 10.5 20.5 42.0 32.5 13.8 41.0 48.3 31.5 0.6
11.7 13.6 0.8 I. 1 22.0 34.6 2.7 4.3 0.3 0.2
15.9
6.1
17.6
5.8
4
P.K. STUMPF
Indeed, Stumpf and Burris demonstrated in 19815 that free malonic acid was the most prevalent dicarboxylic acid in legumes. They also showed that when either 1-]4C acetate or 2-J4C acetate was fed to soybean seedling root tissues, the newly synthesized ~4C malonic acid had the same labeling pattern as described in the above reactions. These results would suggest that in the plant cell the reactions described above do indeed occur; accordingly, if free malonic acid can move into the cytosolic compartment, there to be reactivated, there would be no need to postulate a cytosolic acetyl CoA carboxylase which has been demonstrated to occur in proplastids and in chloroplasts. Not only the developing but also the germinating peanut cotyledons incorporated radiolabeled acetate into long chain fatty acids. Some years after this paper was published I was asked why we did not identify the individual radioactive fatty acids obtained in these experiments. We certainly would have, but the separation of the individual from the total fatty acids had to wait until gas-liquid chromatography became available! These results were followed up by a series of experiments conducted by George Barber, Craig Squires, Vernon McMahon, Shangfa Yang, Claude Willmot, Peter Overath, Robert Simoni, Clem Hawke, James Brooks, Jan Jaworski, John Ohlrogge, and finally Takashi Shimakata, the results of which advanced considerably the basic knowledge of fatty acid biosynthesis in higher plants. Fine tuning of their results is now occurring in a number of laboratories, among them those of Jan Jaworski, John Ohlrogge, and Chris Somerville. IV. DEMONSTRATION OF THE ANCILLARY ENZYMES A number of key discoveries were made in the late 1950s and early 1960s that markedly accelerated research in this field. These include the demonstration of the role of CO2, of the enzyme acetyl CoA carboxylase, and of the protein acyl carrier protein in plant fatty acid biosynthesis as well as the identification of the plastid as the only site of fatty acid biosynthesis in the plant cell. As we had indicated earlier, in vitro fatty acid biosynthesis in any tissue was more or less at a standstill in the early 1950s. Attempts to develop enzyme extracts from animal tissues were spotty; the only consistent system was developed by Sam Gurin in the early 1950s, but the levels of incorporation of radiolabeled acetate were low. Moreover, the concept of a reversal beta-oxidation system still prevailed; but it was finally put to rest by two important sets of experiments. The first resulted from the isolation and purification of all the beta oxidation enzymes from animal mitochondria by the laboratory of David Green 6 at the University of Wisconsin. Green's group decided to test once and for all the reversal beta-oxidation concept and in an important paper in 19537 Stansley and Beinert were able to synthesize butyric acid from acetyl CoA in the presence of all the beta-oxidation enzymes and suitable reductants. But there was no indication for the synthesis of longer chain fatty acids. These results were more or less in agreement with the earlier Stadtman and Barker experiments with extracts of CI. kluyveri. Even more puzzling were the observations of a postdoctoral fellow in Green's group, Salih Wakil, who was able to demonstrate in 19578 that a clear extract of pigeon liver, free of beta-oxidation enzymes, was able to convert acetyl CoA to long chain fatty acids in the presence of ATP, NADPH and CO 2. In the same year, H. Klein9 also had made the rather strange observation that when carbon dioxide was added to a yeast extract, there was a considerable increase in fatty acid synthesis, but, curiously, when labeled CO2 was added to the reaction mixture, no incorporation into fatty acids was observed. To cut a long story short, in 1958 Wakil ~0identified malonyl CoA as the second substrate and in 1959~ identified the biotin-containing protein as acetyl CoA carboxylase. In 1957~2 Barber, in my laboratory, had been successful in preparing a particulate system from avocado mesocarp tissue which could readily incorporate radiolabeled acetate into long chain fatty acids in the presence of ATP, CoA, NADPH and Mn 2+ . The products were radiolabeled palmitic
Plant lipid research--retrospective view
5
and stearic acids. Squires in 195813 was able to prepare very stable acetone powders of avocado mesocarp and showed a vigorous stimulation of fatty acid synthesis by the further addition of sodium bicarbonate to the reaction mixture. In the summer of 1958, I transferred to the University of California at Davis to organize the new Department of Biochemistry and Biophysics on that campus. Three new postdoctoral fellows joined my group in 1959 (by which time we had moved into new laboratories on the Davis campus) and they were Edward Barron, M.D. (Hal) Hatch and J. Brian Mudd. I assigned Ed the job of sorting out the avocado system, Hal the job of determining the presence of acetyl CoA carboxylase in higher plants and Brian to sort out the problem of oleic acid biosynthesis. Ed further defined the avocado system and also observed that the addition of avidin completely shut down fatty acid synthesis from radioactive acetate, although radiolabeled malonyl CoA incorporation into fatty acids was not affected by avidin. ~4Two puzzling problems were the persistent requirement to work with particle preparations as the starting material rather than supernatant systems as was being described in animal and yeast systems and the loss of activity of the extracts when subjected to ammonium sulfate precipitation. In the meantime, Hal Hatch tackled the acetyl CoA carboxylase system by employing wheat germ as the starting material. He purified the enzyme some 170 fold and described its properties and showed that acetyl CoA was the best substrate with propionoyl and butyroyl CoAs in decreasing activities. Avidin completely inhibited the system. ~5 Unfortunately the enzyme was quite unstable at the 170-fold purification level. Later results would prove that the enzyme does not have the allosteric properties of the animal system in terms of no citrate activation and no phosphorylation/dephosphorylation cycle. Brian Mudd tackled an equally difficult problem, namely the inability to demonstrate an enzyme system capable of desaturating stearic acid to oleic acid. Particulate avocado mesocarp preparations readily converted radiolabeled acetate into high levels of oleic acid under aerobic conditions; however, under anerobic conditions the main product was stearic acid. Addition of stearic acid as a substrate led to no desaturation. Moreover, experiments by Barron also showed that stearoyl CoA was not desaturated by a system known to convert acetate into oleate. We concluded that molecular oxygen was an absolute requirement but that either stearic acid or stearoyl CoA was not a suitable substrate ~6. Later research of course totally explained our lack of success! When Peter Overath joined my laboratory in 1963, he came with a wealth of experience, having received his Ph.D. from F. Lynen in Germany. I suggested that he look into the observation made by Barron that ammonium sulfate fractions of clear extracts of avocado acetone powder gave only enzymically inactive protein fractions. Already in 1963 the laboratory of Vaglos at the NIH had observed that E. coli extracts capable of synthesizing fatty acids from acetyl CoA and suitable cofactors lost activity by fractionation, but that this activity could be restored by the addition of a boiled crude E. coli extract. With these results coming forth, Overath quickly showed that a similar situation existed in the avocado system, in that a concentrated protein extract could be separated into two fractions, the first fraction which came down between 20 and 60% ammonium sulfate was heat labile and the second fraction coming down between 60 and 90% was heat stable. The first fraction was inactive unless the second, heat stable fraction was added. ~7 At this point I travelled to Washington to participate in an NIH panel meeting at Bethesda. During a break I visited Vaglos's laboratory and I was provided with a sample of their heat stable E. coli fraction to test on our avocado fraction. On my return, Overath tested the sample and discovered that it was far more effective than was our heat stable avocado fraction. The difference was probably due to a much higher level of what turned out to be the acyl carrier protein in the Vaglos sample than in our plant sample. When Overath returned to Germany, I suggested to Robert Simoni that he purify and characterize the plant ACP as his doctoral thesis. Bob purified the E. coli, an Arthobacter, the avocado mesocarp and the spinach ACPs, and chemically as well as physically characterized these
6
P.K. STUMPF
proteins. He reported a molecular weight of 10300 for the spinach ACP with total amino acid residues of 88. '8 These results have been further refined by Ohlrogge in his recent publications. The surprising result was that the E. coli ACP was at least three fold more effective than was the spinach ACP in a spinach synthase system. As a result, all our further work with synthase systems employed the E. coli ACP rather than the more difficult to obtain spinach ACP. The most interesting aspect of these results showed that the bacterial and the plant synthase systems resembled each other very closely in that they both were non-associative systems that permitted fractionation of the individual enzymes including their ACPs which could also cross react with the appropriate synthase systems, in sharp contrast to both the yeast and animal synthase systems which were multifunctional polypeptides with their ACPs an integral part of the polypeptide structure. v. THE SITE OF FATTY ACID SYNTHESIS IN PLANTS Evidence was also fast accumulating from several laboratories that the fatty acid systems in plants, unlike those in animal systems, were localized in particles. We had already identified in 1957 that the activity for fatty acid synthesis in the avocado mesocarp was associated with particles) 2 In 1960, Smirnov in Russia ~9had described a system in which isolated spinach chloroplasts incorporated radiolabeled acetate into fatty acids in the presence of ADP, CoA and Mn 2+ and light. Two years later Mudd and McManus2° with isolated spinach chloroplasts and later in the same year Stumpf and James2' with lettuce chloroplasts demonstrated the very active synthesis of palmitic and oleic acids in the presence of ATP, CoA, Mg 2+, CO2 and light. In 1964, Yamada 22 in my laboratory fractionated a crude extract of developing castor bean seeds and showed that the major activity for de novo synthesis of fatty acids was associated with large particles. The significance of these observations were not apparent at that time. Probably some confusion arose by our calling these systems 'particles', 'large particles' and 'mitochondrial'. Fortunately, Zilkey and Canvin23 in Canada carried out a careful sucrose density gradient centrifugation of homogenates of developing castor bean endosperm which converted acetate into oleate and gave these particles the name of oleosomes. Further clarification came from the laboratory of Kekwick in 197524 who showed quite conclusively that large particles isolated from cauliflower buds and avocado mesocarp were identified as proplastids, the sites of synthesis fatty acids in developing seeds and mesocarp tissues. And of course, in leaf tissue, chloroplasts were presumably the site for fatty acid synthesis. To settle finally the problem of the exclusivity of the chloroplast as the only site for fatty acid synthesis in the leaf cell, Ohlrogge, Kuhn and Stumpf in 197925 showed that only in the chloroplast was ACP localized and hence only in the chloroplast did de novo fatty acid synthesis take place. vI. DESATURATION AND BETA OXIDATION Returning to the 1960s, a few comments should be made concerning desaturation and beta-oxidation. During the 1960s, evidence was accumulating that the site of synthesis of oleic acid was associated with plastids. The work of Mudd with avocado particles, and later McManus and Mudd with spinach chloroplasts and by Stumpf and James in lettuce chloroplasts supported those results. Still, the isolation of an enzyme system to duplicate what occurred in the intact plastids was unsuccessful until Nagai and Bloch26 clearly showed in 1968 that the correct substrate for desaturation was stearoyl ACP rather than stearoyl CoA, the substrate in mammalian and yeast systems, and that the enzyme was completely soluble rather than microsomal as in the non-plant systems. This work was then extended by Jaworski 27 in our laboratory in 1974. At that time we were puzzled by the observation that free oleic acid was the product of desaturation rather than oleoyl ACP. Shine and Mancha28examined this problem and discovered the presence of a highly specific oleoyl ACP thioesterase. The importance of this observation is exemplified by the great interest in this type of enzyme in lipid metabolism. The desaturase has now been
Plant lipid research--retrospective view
7
crystallized in Somerville's laboratory. As for the synthesis of linoleic acid, in 1964 McMahon :9 for the first time prepared a particulate fraction from developing safflower seeds and demonstrated the rapid conversion of oleoyl CoA to linoleic acid. This work was re-examined in 1971 by Vijay3° who described a microsomal preparation from developing safflower seeds that rapidly converted oleoyl CoA to linoleic acid (associated with phosphatidylcholine) in the presence of NADH and molecular oxygen. Because we were convinced that an oleoyl thioester was the true substrate viz stearoyl ACP in oleic acid synthesis, we ignored the obvious !ata we had and incorrectly ascribed the substrate as oleoyl CoA. Later other workers clearly showed that the correct substrate was oleoyl phosphatidylcholine and the product linoleoyl phosphatidyl choline. As for beta oxidation, in 1956, Barber 3' prepared a particulate system from germinating peanut seeds which we considered to be mitochondria and which rapidly oxidized short and long chain fatty acids to t4CO2 as well as '4C Krebs cycle acids. We concluded that beta oxidation did indeed occur in plants and that they were localized in mitochondria. Of course now our interpretation would have been that we had a mixture of mitochondria and glyoxysomes. The elegant work of Beevers and his group in the late 1960s32 did much to clarify the true nature of the beta-oxidation cycle. More recent work has also identified leaf peroxisomes as another site of beta oxidationY
VII. F I N A L T H O U G H T S
With the basic aspects of plant lipid biochemistry in place as a result of the work we have described in our laboratory and other important work from the laboratories of Beevers, Roughan and Slack, Harwood, to name a few, this area has now entered the revolution of molecular biology and sophisticated instrumentation, and accordingly, progress is accelerating at a very rapid rate. Thus the work of Somerville, Ohlrogge, Jaworski, Harwood, Styme, Murphy, Heinz, and Joyard and Douce to mention a few of the laboratories, as well as the research groups in a number of companies, most prominent being those in Calgene, promise many new and exciting discoveries in the near future that will make my retrospective thoughts appear almost irrelevant. Nevertheless, for me at least the research developed between 1950 and 1970 did paint the sometime confusing, but certainly challenging, picture that began to emerge by the early 1970s. It will be equally interesting to paint a comparable picture in the year 2025, covering the 1980s and 1990s as, you the current actors in this drama, play out your roles! Hindsight is always so much easier than foresight!
REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
B O ~ R , J. Plant Biochemistry, 537 pp. Academic Press, New York, 1950. STADTMAN,E. R. and BARKER,H. A. J. Biol. Chem. 1 ~ , 1095 (1949). RI~'rENaERG,D. and BLOCH,K. J. Biol. Chem. 160, 471 (1945). NEW¢OMB,E. H. and S~MPF, P. K. In Phosphorus Metabolism, Vol. II, p. 291 (McELROY,W. O. and GLASS, B., eds) The Johns Hopkins Press, Baltimore, MD, 1952. SI~MPF, D. K. and BURRIS,R. H. Plant Physiol. 68, 992 (1981). GRF~N, D. E. Biol. Revs., Cambridge Phil. Soc. 29, 330 (1954). STANSLE¥,P. G. and BEINERT,H. Biochim. Biophys. Aeta 11, 600 (1953). GIBSON,D. M., JACOBS,M. J., PORTER,J. W., TIETZ, A. C. and WAglL, S. J. Biochim. Biophys. Acta 23, 219 (1957). KLEIN, H. P. J. Bacteriol. 73, 530 (1957). WAKIL,S. J. J. Am. Chem. Soe. 80, 6465 0958). WAKIL,S. J., TITCHNER,E. B. and GIBSON,D. M. Biochim. Biophys. Acta 29, 225 0958). STUMPF,P. K. and BARBER,G. A. J. Biol. Chem. 227, 407 (1957). SOUmF.S,C. L., STUMPF,P. K. and SCHMID,C. Plant Physiol. 33, 365 (1958). BARRON,E. J., SQUIRES,C. and STUMPF,P. K. J. Biol. Chem. 236, 2610 (1961). H^xcn, M. D. and STUMPF,P. K. ,I. Biol. Chem. 236, 2879 (1961). MUDD, J. B. and S'rUMPF,P. K. J. Biol. Chem. 236, 2602 (1961). OVE10.TH,P. and S~MPF, P. K. J. Biol. Chem. 239, 4102 (1964). SXMONI,R. D., CRXDDI.E,R. S. and S1-OMI'F,P. K. J. Biol. Chem. 242, 573 (1967). S~aRNOV,B. P. Biokhimia 25, 419 (1960). MUDD, J. B. and McMANus, T. T. J. Biol. Chem. 237, 2057 (1962).
8 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
P.K. STUMPF STUMPF,P. K. and JAMF.S,A. T. Biochim. Biophys. Acta 70, 20 (1962). YAM^DA,M. and STUMPF,P. K. Biochem. Biophys. Res. Commun. 14, 165 (1964). ZILKEYB. F. and CANVIN,D. T. Can. J. Bot. 50, 323 (1971). WF.AIRE,B. J. and KEKWIC[, R. G. O. Biochem. J. 146, 425 (1975). OI-ILROC~E,J. B., KUHN, D. N. and STUMPF,P. K. Proc. Natl. Acad. Sci. U.S.A. 76, 1194. NAGAI,J. and BLOCH,K. J. Biol. Chem. 243, 426 (1968). JAWORSKi,J. G. and STUMPF,P. K. Arch. Biochem. Biophys. 162, 158 (1974). SHINE,W. E., MANCHA,M. and STUMPF,P. K. Arch. Biochem. Biophys. 172, 110 (1976). MCM^HON, V. and STUMPF,P. K. Biochim. Biophys. Acta 84, 359 (1964). VuAY, I. K. and STUMPF,P. K. J. Biol. Chem. 246, 2910 (1971). STUMPF,P. K. and BARBER,G. A. Plant Physiol. 31, 304 (1956). COOPER,T. G. and B ~ , H. J. Biol. Chem. 244, 3507 (1969). GERHARDT,B. Planta 59, 238 (1983).