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Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings
ARCHIVES
OF BIOCHEMISTRY
AND
Properties Coenzyme
167, 723-729
BIOPHYSICS
of Microsomal A Reductase
JOHN Department
3-Hydroxy-3-Methylglutaryl From Pisum
D. BROOKER2 of
Biochemistry, Received
(1975)
AND
University February
sativum
Seedlings1
DAVID
W. RUSSELL
of
Dunedin,
Otago,
New
Zealand
12, 1974
The properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the microsomal fraction of Pisum sat&m seedlings have been described. The enzyme requires NADPH for activity and NADH does not support the reaction. The presence of a thiol compound such as dithiothreitol, is required for activity and a concentration of 10 mM is optimal. The pH optimum is 6.8 and the K, (apparent) for DL-3-hydroxy-3-methylglutaryl coenzyme A is about 100 pM. Activity of the enzyme is not affected by mevalonic acid at the concentrations tested (up to 1.0 mM). 3-Hydroxy-3-methylglutaric acid and free CoA cause substantial inhibition, whereas gibberellic acid has no effect. The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase is twice as high in etiolated seedlings as in green seedlings. In green seedlings activity is highest in the apical bud, declines sharply in semimature leaves, and there is almost no activity in mature leaves.
The reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA)~ to mevalonic acid (MVA) (reaction 1) is catalysed by HMG-CoA reductase [mevalonate: NADP oxidoreductase (acylating CoA). EC 1:1.1.34] and is an irreversible reaction occurring early in the biosynthetic pathway of isoprenoid compounds. HMG-CoA
+ 2NADPH + MVA
+ 2H+ + 2 NADP+
+ CoASH
(1)
The enzyme has been described and purified from both yeast and mammalian tissue (l-4) and subsequent tracer and feeding experiments with mammalian systems (5, 6) have shown that it is probably a 1 Supported in part by grants from the University of Otago andi the Department of Scientific and Industrial Research to D. W. Russell. * Supported by a Post Graduate Scholarship from the University Grants Committee. 3The abbreviations used are: HMG-CoA, 3hydroxy-3-methylglutaryl coenzyme A; MVA, mevaionic acid; GA,, gibberellic acid; HMG; 3-hydroxy-3methylglutaric acid; DBED, dibenzylethylenediamine. 723 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
major control point in the isoprenoid pathway. In higher plants the products of this pathway comprise a wide range of compounds including carotenoids (7), sterols, isoprenoid quinones, and three major plant growth regulators, gibberellic acid (8), abscisic acid (9), and some naturally occurring cytokinins (10). However there have been very few studies of the enzymes which catalyse the early reactions up to farnesyl pyrophosphate and almost nothing is known about the regulation of the pathway. Previous tracer studies with intact tissues from higher plants, which showed incorporation of acetate (ll), HMG-CoA (12), or MVA (13), provide evidence that the isoprenoid pathway in plants is similar to that described for animals and microorganisms. Further work by Hepper and Audley (12) on the cell free synthesis of rubber from HMG has added support to the in vivo tracer work and has provided some evidence of cofactor requirements for the conversion of acetate to polyisoprenoids. However enzyme studies are re-
724
BROOKER
quired to conclusively establish the precise pathway and are essential for analysis of the regulation of biosynthetic activity. In the present work the occurrence of HMG-CoA reductase in a higher plant (Pisum satiuum) has been demonstrated for the first time and the properties of the enzyme are described. EXPERIMENTAL
PROCEDURES
Materials. Sources of chemicals were as follows: HMG, CoA (free acid), MVA (DBED salt), MVA-lactone, NJ’-dicyclohexylcarbodiimide, NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, dithiothreitol, and 2-mercaptoethanol from Sigma; [3-“C]HMG from International Chemical and Nuclear Corporation; [2-“C]MVA-1actone from Amersham; and insoluble polyvinylpyrrolidone (Polyclar AT) from General Aniline & Film Corporation. Preparation of substrate. [3-“C]HMG anhydride was synthesized from [3-“C]HMG by the dicyclohexylcarbodiimide method of Goldfarb and Pitot (14). HMG-CoA was prepared from the anhydride as described by Hilz et al. (15) and was chromatographitally purified on paper using n-butanol:acetic acid: water (5:2:3) as the developing solvent (16). HMGCoA solutions were stored in 0.01 M sodium phosphate buffer (pH 5.8) at -15°C for several months without significant degradation. Plants. Pea seeds, variety Alaska (Dalgety N.Z. Limited, Timaru, New Zealand), were soaked in water for about 6 h and planted either in trays of moist washed vermiculite for etiolated peas, or in a standard potting mixture for light grown peas. Light-grown pea seedlings were maintained under long-day conditions at 23°C * 2” with a 7 h dark period. The plants were grown under 80 W fluorescent tubes and 60 W incandescent bulbs in the ratio of 2:l. Tissues for assay were harvested from plants 15-25 cm high. Etiolated peas were grown in darkness at 27°C i 1’. Illumination for inspection and watering was provided by a green safelight consisting of a 46 cm green fluorescent tube fitted with a green filter with maximum transmission at 520 nm and a band width at half peak height of 20 nm. Seedlings were harvested at the third internode stage. Enzyme extraction. Apical buds were removed from the seedlings and homogenized in a cold mortar in 5 vol of homogenizing medium and 10% w/w prewashed (17) polyclar AT. The homogenizing medium contained 10 mM Tris-HCl (pH 7.0); 0.35 M sucrose; 0.03 M EDTA; and 10 mM mercaptoethanol (added fresh). The slurry was squeezed through two layers of muslin and the crude homogenate centrifuged at 12,OOOg for 15 min to sediment cell debris, nuclei, chloroplasts, and mitochondria. The superna-
AND
RUSSELL
tant was recentrifuged at 18,OOOg to remove any remaining mitochondria and then centrifuged at 50, OOOg for 1 h to sediment the microsomal fraction which contained the enzyme. The pellet contained 95% of the activity associated with the total microsomal fraction sedimented at 105,OOOg for 1 h. The 50,OOOg pellet was suspended in 0.1 M potassium phosphate buffer (pH 6.8; 0.3 ml/g fresh weight of tissue). An aliquot was removed for estimation of protein and the remainder was made to 10 mM dithiothreitol. Each assay contained 0.05 ml of this preparation. Protein was estimated by the Lowry method (18) using bovine serum albumin as a standard. HMG-CoA reductase assay. Two standard procedures were used: (a) a radiochemical assay employing thin layer chromatography (tic); (b) a spectrophotometric assay. Analyses were carried out in duplicate or triplicate. (a) Radiochemical assay. Each incubation mixture (final volume 0.1 ml) contained the following: 2.0 pmol potassium phosphate buffer (pH 6.81; 1.0 rmol dithiothreitol; 2.0 pmol glucose-6-phosphate; 0.1 unit glucose-6-phosphate dehydrogenase; 0.2 pmol NADP+; 60.0 nmol DL-[3-*“C]HMG-CoA (0.83 &i/ rmol); and the enzyme suspension, not exceeding 0.8 mg protein. Where necessary the NADPH generating system was replaced by at least 0.2 rmol NADPH. The reaction was started by the addition of HMG-CoA, incubated at 30°C for up to 20 min and stopped by the addition of 0.01 ml 6 M HCl and 10 pmol of carrier MVA. The solution was allowed to stand at room temperature for at least 1 h to ensure maximum conversion of MVA to its la&one, then centrifuged at half speed in a bench centrifuge to sediment the precipitated protein. Half of the supernatant was then streaked directly onto a 5 x 20 cm activated silica gel thin layer plate. The chromatogram was developed (16 cm) in chloroform:acetone (2:1, v/v). (The chloroform contained 1% w/w ethanol as a preservative.) The thin layer plates were scanned by a Packard radiochromatogram scanner and the peak corresponding to MVA-lactone scraped directly into a counting vial. A neighbouring background area was also counted. Samples were counted in toluene scintillation fluid (efficiency 80%) in a Packard Tricarb liquid scintillation counter. Recovery of MVA-lactone from the thin layer plates was 98%. The enzyme activity was expressed as nmoles MVA/mg protein/h or enzyme units/g fresh wt of tissue. One enzyme unit is defined as the amount of enzyme required to catalyze the formation of 1 nmol of MVA per min under the assay conditions described. (bl Spectrophotometric assay. The incubation mixture contained in a volume of 1.0 ml: 90 Fmol potassium phosphate buffer (pH 6.8); 10 rmol dithiothreitol; 0.15 wmol DL-HMG-CoA; 0.3 pmol NADPH,
HMG-CoA
REDUCTASE
and the enzyme preparation not exceeding 1.0 mg of protein. The reaction was carried out at 30°C in a Pye Unicam SP18C0 spectrophotometer (double beam) with a chart recorder and the enzyme activity was calculated from the change in absorbance at 340 nm. Using either of these assay procedures, the measured rate of MVA synthesis was linear with enzyme concentration up to at least 0.8 mg protein and linear with time for up to 20 min. Under all assay conditions NADPH concentration was nonlimiting. The radiochemical assay was used more frequently than the spectrophometric assay because of high absorbing material which masked smaller A,,, changes, especially in extracts of green tissue. Effect of storage conditions on enzyme activity. Microsomal HMG-CoA reductase from pea seedlings was unstable in the absence of a low molecular weight thiol compouncl such as 2-mercaptoethanol or dithiothreitol, but could be stored for up to a week at - 15°C in the presence of 10 mM dithiothreitol and 10% (v/v) glycerol with B 28% loss in activity. Storage under these conditions for 1 month resulted in a 52% loss in activity. Continual freezing and thawing of an enzyme preparation over a period of 1 week resulted in a 40% loss of enzyme activity. Storage in the presence or absence of a thiol compound at 0°C resulted in a 60-70% loss of activity in 24 h. Storage for 24 h at -15°C in the presence or absence of 10% glycerol (without dithiothreitol) caused a 70-80% loss in activity, but in the presence of both dithiothreitol (15 mM) and glycerol (10% v/v) only 13% of the activity was lost. RESULTS
Product identification. When DL- [3“C]HMG-CoA was incubated in a reaction mixture containing substrate, cofactors and the enzyme preparation, a radioactive product was formed which had a mobility on tic similar to MVA-lactone. Control assays omitting either enzyme, HMG-CoA, or NADPH gave no product. The radioactive product was positively identified as MVA by thin layer and paper chromatography, derivative formation, and recrystallization without loss of specific radioactivity. Thin layer chromatography. Standards chromatographed on activated silica gel thin layer plates in chloroform:acetone (2:1, v/v) were located and their R,‘s calculated (Table I). MVA-lactone had an R, between 0.65 and 0 .70 . Paper chromatography. The radioactive product isolated by tic was eluted with acetone, reduced to a small volume, and
FROM
PEA
725
SEEDLINGS TABLE tic
Compound
R,
MVA-lactone
0.65
MVA HMG CoA HMG-CoA
0.036 0.68 0.00 0.018
Acetoacetate
0.98
Acetate
0.45
I
STANDARDS~ Means
of detection
Radioactivity ylamine-FeCl, Radioactivity Radioactivity uv quenching Radioactivity quenching Semicarbazide action Radioactivity
and hydroxtest
and
uv
colour
re-
o Standards were spotted on activated silica thin layer plates (5 x 20 cm) and developed chloroform:acetone (2:l) at 20°C.
gel in
rechromatographed on Whatman No. 1 paper in a solvent of tertiary butanol:formic acid:water (20:5:8). The developed strip was scanned by a Packard radiochromatogram scanner. Only one radioactive peak was apparent with an R, identical to standard MVA-lactone. Derivative formation. The product was eluted from a thin layer plate as above, diluted with unlabelled MVA-lactone, and converted to the benzhydrylamide derivative by the method of Wolf et al. (19). The product formed was recrystallized five times from hot benzene/petroleum ether. The specific radioactivity was calculated for each crystallization (Table II) and found to be constant. These results identify the reaction product as MVA and establish the validity of the assay. Properties of HMG-CoA reductase from pea seedlings. The effect of pH on the activity of the microsomal enzyme is shown in Fig. 1. Optimum activity was observed at pH 6.8 with a marked decline above and below this figure. Activity at pH 5.5 and pH 7.5 was 40% of that at pH 6.8. Examination of pyridine nucleotide specificity showed that the enzyme has an absolute and specific requirement for NADPH (Table III). When saturating concentrations of NADPH were either added directly to a reaction mixture or produced by an NADPH generating system, good activity was obtained. By contrast virtu-
726
BROOKER TABLE
IDENTIFICATION OF THE
OF RADIOACTIVE BENZHYDRYLAMIDE
AND
II
step
Specific radioactivity (dpmk)
17500
Initial value Crystallization Crystallization Crystallization Crystallization Crystallization
No. No. No. No. No.
1 2 3 4 5
17980 17858
17604 18410
17848
a Carrier MVA-lactone was added to the radioactive product and the benzhydrylamide was prepared by the method of Wolf et al. (19). The derivative was recrystallized from hot benzene/petroleum ether. Crystals recovered were weighed and a small sample counted in toluene scintillation fluid. 55
7:
had no effect upon reductase activity, suggesting that the enzyme did not require free (or loosely bound) metal cations for activity. Comparative studies on the effectiveness of different thiol compounds showed that dithiothreitol was about 20% more effective than 2-mercaptoethanol or glutathione at the concentration tested (Table IV). The optimal concentration of dithiothreitol was 10 mM (Fig. 2); optimal concentrations of 2-mercaptoethanol and glutathione were not determined. In the absence of any thiol compound enzyme activity was about 50% less than optimum. Preincubation with the thiol compound was not necessary for its effect. Studies on the relationship between velocity and substrate concentration showed that the enzyme is saturated with DLHMG-CoA at a concentration of about 0.2
M
PRODUCT BY SYNTHESIS DERIVATIVE AND
RECRYSTALLIZATION”
Crystallization
RUSSELL
50
TABLE ABILITY
45
OF PYRIDINE
HMG-CoA
III
NUCLEOTIDES REDUCTASE
TO SUPPORT ACTIVITY”
40
Pyridfme3nucl)eotide m
35
30
0.35 42.75
NADH NADPH
25
a NADH or NADPH in an aqueous solution (pH 7.01, were added to the reaction mixture containing a microsomal preparation from etiolated pea seedlings and the enzyme activity determined spectrophotometrically.
20
15
10
" ? z c
HMG-CoA reductase activity (nmol/mg protein/h)
TABLE
5
THE
EFFECT
OF VARIOUS
HMG-CoA Thiol
FIG. 1. Effect
of
pH on enzyme activity. microsomal enzyme was assayed over a pH range 5.5 to 7.5 and the specific activity calculated. following buffers (0.1 M) were used: citrate, pH sodium phosphate, pH 6.0; potassium phosphate,
The from The 5.5; pH
6.5-7.5.
ally no activity was obtained with NADH at a similar concentration (Table III). The effect of EDTA on the activity of the microsomal enzyme was examined in order to determine possible metal ion requirements. Concentrations of EDTA up to 0.1
compound
(4.0
rnM)
2-Mercaptoethanol Glutathione Dithiothreitol
IV THIOL
REDUCTASE
COMPOUNDS
ON
ACTIVITY’
HMG-CoA reductase activity (nmolhg protein/h) 37.9 37.1
48.4
“The reaction mixture contained the microsomal fractjon, prepared in the absence of any thiol compound, from etiolated pea seedlings. Various tbiol compounds were added to the reaction mixture and the reaction started immediately by addition of substrate. Preincubation with thiol compound was not necessary for its stimulatory effect.
HMG-CoA
V
m 0
eg
9
c
16.
REDUCTASE
FROM
PEA
727
SEEDLINGS
.:
2 0
2
4 DTT
6
6 contentr~ltmn
10
12 Cm
14
16
16
20
M)
FIG. 2. Effect of dithiothreitol (DTI’) on HMG-CoA reductase activity. A microsomal enzyme extract was prepared in the absence of any thiol compound and aliquots were added to incubation mixtures containing increasing concentrations of DTT. The specific enzyme activity was calculated for each concentration.
mM and no inhibition occurred at substrate concentrations up to 0.6 mM. From a double reciprocal plot (Fig. 3) the apparent K, for DL-HMG-CoA is about 100 PM. Under the assay conditions described, NADPH was always present in saturating concentrations and the affinity of the enzyme for NADPH was not examined. The possibility of product inhibition was examined by incubating the reaction mixture in the presence of a range of concentrations of MVA (Table V). Concentrations up to 1 mM MVA had no effect on the reductase activity. The presence of either HMG or free CoA in the reaction mixture was examined for any effect on the reductase activity. Both HMG and CoA were inhibitory although CoA had the greatest effect (Table V). At a concentration of 0.5 mM, CoA inhibited enzyme activity by about 50%, whereas a concentration of 3.0 mM HMG was required for the same effect. The effect of gibberellic acid (GA,), on enzyme activity was examined with a view to testing for possible feed-back inhibition by this isoprenoid plant hormone. GAB, up to a concentration of 0.2 mM, was included in the reaction mixture and the results of these assays showed that GA, had no effect on reductase activity. There is considerable variation in the level of reductase activity in different tissues. Microsomal reductase activity in apical buds of etiolated seedlings is more than 2-fold higher than in apical bud tissues of green seedlings (Table VI). In green seedlings, assays of microsomal fractions from apical buds, immature and mature leaves
FIG. 3. Double reciprocal plot of velocity against HMG-CoA concentration. A microsomal enzyme preparation was assayed in the presence of varying amounts of DL-HMG-CoA. The enzyme activity was calculated and results are shown as a typical Lineweaver-Burk plot. The apparent Km obtained from this plot is approx 100 pM.
showed that apical buds contain about 0.6 units/g, whereas semimature leaves contain 66% of this value and fully mature leaves contain only 7% of the apical bud activity (Table VI). Thus there are striking differences in microsomal reductase activity between tissues of different developmental age and the results show that high levels of microsomal HMG-CoA reductase activity are associated only with young and developing tissues. DISCUSSION
A rapid assay technique for HMG-CoA reductase from pea seedlings has been described and the properties of the micro-
728
BROOKER TABLE
EFFECT
OF HMG,
HMG-CoA Compound
V
CoA,
AND SOME PRODUCTS REDUCTASE ACTIVITYO
Concentration
HMG-CoA reductase activity (nmol/mg protein/h)
MVA HMG
O-l.0 0.15
ON
mM rnM
34.0 38.2
% of control 100
96.5
1.0 rnM 10.0 rnM
30.4 5.4
76.8 13.6
CoA
0.025 mM 0.5 rnM
26.8 20.8
67.0 52.0
GA,
o-o.2
42.5
rnM
100
“The reaction mixture contained the microsomal preparation from etiolated pea seedlings. The various compounds were added to the reaction mixture and the reaction started by addition of substrate. TABLE LEVEL
OF HMG-CoA
VI
REDUCTASE
IN DIFFERENT
TISSUES~
Tissue
Etiolated seedlings Apical bud Green seedlings Apical bud 1st leaf (semimature) 2nd leaf (mature)
Relative HMG-CoA reductase levels
225
100 66 7
“The microsomal fraction was prepared from tissues of different ages obtained from the same green pea seedlings. Reductase activity was assayed by the standard procedure described under Methods.
somal enzyme have been determined. The reaction product has been identified as MVA by chromatography and benzhydrylamide derivative formation. Results show that the microsomal reductase has an absolute requirement for NADPH which can be satisfied either by an NADPH generating system or added NADPH. Almost no reaction occurs when saturating levels of NADPH are substituted by similar concentrations of NADH. The presence of a low molecular weight thiol compound is required for optimal activity and experiments with EDTA indicate that the enzyme does not require free metal cations for
AND
RUSSELL
activity. Optimum activity occurs at pH 6.8 and the apparent K, for DL-HMG-CoA is about 100 PM. The microsomal HMG-CoA reductase from pea seedlings is similar to the yeast and rat liver enzymes (1, 4, 6) in its pH optimum, specificity for NADPH, and requirement for a protective thiol compound. The inhibition studies indicate that the microsomal HMG-CoA reductase from pea seedlings is not regulated by product inhibition since concentrations of MVA up to 1.0 mM had no effect on activity. However enzyme activity is affected by both free CoA and HMG. This has also been reported in mammalian systems (4) and Beg and Lupien (20) have suggested that HMG may be a regulator of cholesterogenesis in rat liver. However it is doubtful whether the inhibitory effect of HMG has any significance in vivo since HMG-CoA has been shown to arise from the condensation of acetoacetyl-CoA and acetyl-CoA in animal systems (21), and thus free HMG is not an intermediate. Hence high levels of HMG are likely to arise only from the cleavage of HMG-CoA. But the affinity of the reductase for its substrate suggests that significant cleavage of HMG-CoA would occur only if the reductase is inhibited. Thus HMG effects on reductase activity in vivo are likely to be secondary. The hormone GA,, one of the products of the isoprenoid pathway in higher plants, had no effect on the enzyme activity within the concentrations tested (O-O.2 mM). The normal in uiuo concentration of this hormone is not definitely known but a concentration of 1 PM is generally regarded as close to the physiological optimum. Although the presence of GA, in pea tissue has not been conclusively demonstrated, there is evidence for the existence of GA, (22) which differs from GA, only in the absence of the A1 double bond. In addition GA, is known to be biologically active in pea seedlings. Undoubtedly other gibberellins must be tested before it can be definitely concluded that none of these hormonal end products exert an inhibitory effect upon the reductase. Consideration of the probable pathway of biosynthesis also raises the possibility that branch point
HMG-CoA
REDUCTASE
intermediates such as gibberellin A,z, geranylgeranyl pyrophosphate and farnesyl pyrophosphate may exert a regulatory effect. Tissues at different developmental stages show large differences in enzyme activity. Of particular interest is the high activity found in etiolated seedlings compared with the somewhat lower activity in green plants. Microsomal reductase activity in the apical buds of green seedlings was about 50% less than in apical buds of etiolated seedlings. These differences in activity are considered real since (i) precautions were taken to prevent enzyme inactivation during extraction, and (ii) subsequent studies have shown that similar low activity can be induced in etiolated seedlings by a brief light irradiation (manuscript in preparation). The lower activity in green seedlings may reflect the relative flux through the pathway if, as in animal systems (6), the enzyme is rate limiting. In green seedlings the highest activity occurs in the apical bud which correlates with the finding that gibberellins (23) are produced mainly in these tissues. As the tissue matures there is a decrease in reductase activity. The decline in activity may be due to a decline in enzyme level and thus may reflect either a decrease in synthesis or an increase in destruction, or both. However, the causal mechanism is not known. Further studies on the regulation of this enzyme in pea seedlings are in progress and these results will be reported in a separate communication. REFERENCES 1. KIRTLEY, M. E., AND RUDNEY, istry 6, 230-238.
H. (1967)
Biochem-
FROM
PEA
SEEDLINGS
729
2. KAWACHI, T., ANDRUDNEY, H. (1970) Biochemistry 9, 1700-1705. 3t . KNAPPE, J., RINGELMANN, E., AND LYNEN, F. (1959) Biochem. 2. 332, 195-213. 4. SHEFER, S., HAUSER, S., LAPAR, V., AND MOSBACH, E. H. (1972) J. Lipid Res. 13, 402-412. 5. BUCHER, N. L. R., OVERATH, P., AND LYNEN, F. (1960) Biochim. Biophys. Acta 40, 491-501. 6. SHAPIRO, D. J., AND RODWELL, V. W. (1971) J. Biol. Chem. 246, 3210-3216. 7. ANDERSON, D. G., AND PORTER, J. W. (1967) Annu. Reu. Plant Physiol. 18, 197-228. 8. GRAEBE, J. E., DENNIS, D. J., UPPER, C. D., AND WEST, C. A. (1965) J. Biol. Chem. 240, 1847-1854. 9. SEMBDNER, G., AND SCHREIBER, K. (1971) Fed. Eur. Biochem. Sot. Lett. 15, 1-7. 10. SHORT, K. C., AND TORREY, J. G. (1972) Plant Physiol. 49, 155. 11. GOODWIN, T. W. (1958) &o&en. J. 70, 612-617. 12. HEPPER, C. M., AND AUDLEY, B. G. (1969) Bio&em. J. 114, 379-386. 13. GRAEBE, J. E. (1967) Science 157, 73-75. 14. GOLDFARB, S., AND PITOT, H. (1971) J. Lipid Res. 12, 512-515. 15. HILZ, H., KNAPPE, J., RINGELMAN, E., AND LYNEN, F. (1958) Biochem. 2. 329, 476-489. 16. Louw, A. I., BEKERSKY, I., ANDMOSBACH, E. (1969) J. Lipid Res. 10, 683-686. 17. LOOMIS, W. D., AND BA~AILE, J. (1966) Phytochemistry 5, 423-438. 18. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. WOLF, D. E., HOFFMAN, C. H., ALDRICH, P. E., SKEGGS, H. R., WRIGHT, E. D., ANDFOLKERS, K. (1957) J. Amer. Chem. Sot. 79, 1486-1487. 20. BEG, Z. H., AND LUPIEN, P. J. (1972) Biochim. Biophys. Acta 260, 439-448. 21. WHITE, L. W., AND RUDNEY, H. (1970) Biochemistry 9, 2713-2724. 22. JONES, R. L., AND LANG, A. (1968) Plant Physiol. 43, 629-634. 23. PHILLIPS, I. D. J. (1971) Planta 101, 277.
OF BIOCHEMISTRY
AND
Properties Coenzyme
167, 723-729
BIOPHYSICS
of Microsomal A Reductase
JOHN Department
3-Hydroxy-3-Methylglutaryl From Pisum
D. BROOKER2 of
Biochemistry, Received
(1975)
AND
University February
sativum
Seedlings1
DAVID
W. RUSSELL
of
Dunedin,
Otago,
New
Zealand
12, 1974
The properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the microsomal fraction of Pisum sat&m seedlings have been described. The enzyme requires NADPH for activity and NADH does not support the reaction. The presence of a thiol compound such as dithiothreitol, is required for activity and a concentration of 10 mM is optimal. The pH optimum is 6.8 and the K, (apparent) for DL-3-hydroxy-3-methylglutaryl coenzyme A is about 100 pM. Activity of the enzyme is not affected by mevalonic acid at the concentrations tested (up to 1.0 mM). 3-Hydroxy-3-methylglutaric acid and free CoA cause substantial inhibition, whereas gibberellic acid has no effect. The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase is twice as high in etiolated seedlings as in green seedlings. In green seedlings activity is highest in the apical bud, declines sharply in semimature leaves, and there is almost no activity in mature leaves.
The reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA)~ to mevalonic acid (MVA) (reaction 1) is catalysed by HMG-CoA reductase [mevalonate: NADP oxidoreductase (acylating CoA). EC 1:1.1.34] and is an irreversible reaction occurring early in the biosynthetic pathway of isoprenoid compounds. HMG-CoA
+ 2NADPH + MVA
+ 2H+ + 2 NADP+
+ CoASH
(1)
The enzyme has been described and purified from both yeast and mammalian tissue (l-4) and subsequent tracer and feeding experiments with mammalian systems (5, 6) have shown that it is probably a 1 Supported in part by grants from the University of Otago andi the Department of Scientific and Industrial Research to D. W. Russell. * Supported by a Post Graduate Scholarship from the University Grants Committee. 3The abbreviations used are: HMG-CoA, 3hydroxy-3-methylglutaryl coenzyme A; MVA, mevaionic acid; GA,, gibberellic acid; HMG; 3-hydroxy-3methylglutaric acid; DBED, dibenzylethylenediamine. 723 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
major control point in the isoprenoid pathway. In higher plants the products of this pathway comprise a wide range of compounds including carotenoids (7), sterols, isoprenoid quinones, and three major plant growth regulators, gibberellic acid (8), abscisic acid (9), and some naturally occurring cytokinins (10). However there have been very few studies of the enzymes which catalyse the early reactions up to farnesyl pyrophosphate and almost nothing is known about the regulation of the pathway. Previous tracer studies with intact tissues from higher plants, which showed incorporation of acetate (ll), HMG-CoA (12), or MVA (13), provide evidence that the isoprenoid pathway in plants is similar to that described for animals and microorganisms. Further work by Hepper and Audley (12) on the cell free synthesis of rubber from HMG has added support to the in vivo tracer work and has provided some evidence of cofactor requirements for the conversion of acetate to polyisoprenoids. However enzyme studies are re-
724
BROOKER
quired to conclusively establish the precise pathway and are essential for analysis of the regulation of biosynthetic activity. In the present work the occurrence of HMG-CoA reductase in a higher plant (Pisum satiuum) has been demonstrated for the first time and the properties of the enzyme are described. EXPERIMENTAL
PROCEDURES
Materials. Sources of chemicals were as follows: HMG, CoA (free acid), MVA (DBED salt), MVA-lactone, NJ’-dicyclohexylcarbodiimide, NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, dithiothreitol, and 2-mercaptoethanol from Sigma; [3-“C]HMG from International Chemical and Nuclear Corporation; [2-“C]MVA-1actone from Amersham; and insoluble polyvinylpyrrolidone (Polyclar AT) from General Aniline & Film Corporation. Preparation of substrate. [3-“C]HMG anhydride was synthesized from [3-“C]HMG by the dicyclohexylcarbodiimide method of Goldfarb and Pitot (14). HMG-CoA was prepared from the anhydride as described by Hilz et al. (15) and was chromatographitally purified on paper using n-butanol:acetic acid: water (5:2:3) as the developing solvent (16). HMGCoA solutions were stored in 0.01 M sodium phosphate buffer (pH 5.8) at -15°C for several months without significant degradation. Plants. Pea seeds, variety Alaska (Dalgety N.Z. Limited, Timaru, New Zealand), were soaked in water for about 6 h and planted either in trays of moist washed vermiculite for etiolated peas, or in a standard potting mixture for light grown peas. Light-grown pea seedlings were maintained under long-day conditions at 23°C * 2” with a 7 h dark period. The plants were grown under 80 W fluorescent tubes and 60 W incandescent bulbs in the ratio of 2:l. Tissues for assay were harvested from plants 15-25 cm high. Etiolated peas were grown in darkness at 27°C i 1’. Illumination for inspection and watering was provided by a green safelight consisting of a 46 cm green fluorescent tube fitted with a green filter with maximum transmission at 520 nm and a band width at half peak height of 20 nm. Seedlings were harvested at the third internode stage. Enzyme extraction. Apical buds were removed from the seedlings and homogenized in a cold mortar in 5 vol of homogenizing medium and 10% w/w prewashed (17) polyclar AT. The homogenizing medium contained 10 mM Tris-HCl (pH 7.0); 0.35 M sucrose; 0.03 M EDTA; and 10 mM mercaptoethanol (added fresh). The slurry was squeezed through two layers of muslin and the crude homogenate centrifuged at 12,OOOg for 15 min to sediment cell debris, nuclei, chloroplasts, and mitochondria. The superna-
AND
RUSSELL
tant was recentrifuged at 18,OOOg to remove any remaining mitochondria and then centrifuged at 50, OOOg for 1 h to sediment the microsomal fraction which contained the enzyme. The pellet contained 95% of the activity associated with the total microsomal fraction sedimented at 105,OOOg for 1 h. The 50,OOOg pellet was suspended in 0.1 M potassium phosphate buffer (pH 6.8; 0.3 ml/g fresh weight of tissue). An aliquot was removed for estimation of protein and the remainder was made to 10 mM dithiothreitol. Each assay contained 0.05 ml of this preparation. Protein was estimated by the Lowry method (18) using bovine serum albumin as a standard. HMG-CoA reductase assay. Two standard procedures were used: (a) a radiochemical assay employing thin layer chromatography (tic); (b) a spectrophotometric assay. Analyses were carried out in duplicate or triplicate. (a) Radiochemical assay. Each incubation mixture (final volume 0.1 ml) contained the following: 2.0 pmol potassium phosphate buffer (pH 6.81; 1.0 rmol dithiothreitol; 2.0 pmol glucose-6-phosphate; 0.1 unit glucose-6-phosphate dehydrogenase; 0.2 pmol NADP+; 60.0 nmol DL-[3-*“C]HMG-CoA (0.83 &i/ rmol); and the enzyme suspension, not exceeding 0.8 mg protein. Where necessary the NADPH generating system was replaced by at least 0.2 rmol NADPH. The reaction was started by the addition of HMG-CoA, incubated at 30°C for up to 20 min and stopped by the addition of 0.01 ml 6 M HCl and 10 pmol of carrier MVA. The solution was allowed to stand at room temperature for at least 1 h to ensure maximum conversion of MVA to its la&one, then centrifuged at half speed in a bench centrifuge to sediment the precipitated protein. Half of the supernatant was then streaked directly onto a 5 x 20 cm activated silica gel thin layer plate. The chromatogram was developed (16 cm) in chloroform:acetone (2:1, v/v). (The chloroform contained 1% w/w ethanol as a preservative.) The thin layer plates were scanned by a Packard radiochromatogram scanner and the peak corresponding to MVA-lactone scraped directly into a counting vial. A neighbouring background area was also counted. Samples were counted in toluene scintillation fluid (efficiency 80%) in a Packard Tricarb liquid scintillation counter. Recovery of MVA-lactone from the thin layer plates was 98%. The enzyme activity was expressed as nmoles MVA/mg protein/h or enzyme units/g fresh wt of tissue. One enzyme unit is defined as the amount of enzyme required to catalyze the formation of 1 nmol of MVA per min under the assay conditions described. (bl Spectrophotometric assay. The incubation mixture contained in a volume of 1.0 ml: 90 Fmol potassium phosphate buffer (pH 6.8); 10 rmol dithiothreitol; 0.15 wmol DL-HMG-CoA; 0.3 pmol NADPH,
HMG-CoA
REDUCTASE
and the enzyme preparation not exceeding 1.0 mg of protein. The reaction was carried out at 30°C in a Pye Unicam SP18C0 spectrophotometer (double beam) with a chart recorder and the enzyme activity was calculated from the change in absorbance at 340 nm. Using either of these assay procedures, the measured rate of MVA synthesis was linear with enzyme concentration up to at least 0.8 mg protein and linear with time for up to 20 min. Under all assay conditions NADPH concentration was nonlimiting. The radiochemical assay was used more frequently than the spectrophometric assay because of high absorbing material which masked smaller A,,, changes, especially in extracts of green tissue. Effect of storage conditions on enzyme activity. Microsomal HMG-CoA reductase from pea seedlings was unstable in the absence of a low molecular weight thiol compouncl such as 2-mercaptoethanol or dithiothreitol, but could be stored for up to a week at - 15°C in the presence of 10 mM dithiothreitol and 10% (v/v) glycerol with B 28% loss in activity. Storage under these conditions for 1 month resulted in a 52% loss in activity. Continual freezing and thawing of an enzyme preparation over a period of 1 week resulted in a 40% loss of enzyme activity. Storage in the presence or absence of a thiol compound at 0°C resulted in a 60-70% loss of activity in 24 h. Storage for 24 h at -15°C in the presence or absence of 10% glycerol (without dithiothreitol) caused a 70-80% loss in activity, but in the presence of both dithiothreitol (15 mM) and glycerol (10% v/v) only 13% of the activity was lost. RESULTS
Product identification. When DL- [3“C]HMG-CoA was incubated in a reaction mixture containing substrate, cofactors and the enzyme preparation, a radioactive product was formed which had a mobility on tic similar to MVA-lactone. Control assays omitting either enzyme, HMG-CoA, or NADPH gave no product. The radioactive product was positively identified as MVA by thin layer and paper chromatography, derivative formation, and recrystallization without loss of specific radioactivity. Thin layer chromatography. Standards chromatographed on activated silica gel thin layer plates in chloroform:acetone (2:1, v/v) were located and their R,‘s calculated (Table I). MVA-lactone had an R, between 0.65 and 0 .70 . Paper chromatography. The radioactive product isolated by tic was eluted with acetone, reduced to a small volume, and
FROM
PEA
725
SEEDLINGS TABLE tic
Compound
R,
MVA-lactone
0.65
MVA HMG CoA HMG-CoA
0.036 0.68 0.00 0.018
Acetoacetate
0.98
Acetate
0.45
I
STANDARDS~ Means
of detection
Radioactivity ylamine-FeCl, Radioactivity Radioactivity uv quenching Radioactivity quenching Semicarbazide action Radioactivity
and hydroxtest
and
uv
colour
re-
o Standards were spotted on activated silica thin layer plates (5 x 20 cm) and developed chloroform:acetone (2:l) at 20°C.
gel in
rechromatographed on Whatman No. 1 paper in a solvent of tertiary butanol:formic acid:water (20:5:8). The developed strip was scanned by a Packard radiochromatogram scanner. Only one radioactive peak was apparent with an R, identical to standard MVA-lactone. Derivative formation. The product was eluted from a thin layer plate as above, diluted with unlabelled MVA-lactone, and converted to the benzhydrylamide derivative by the method of Wolf et al. (19). The product formed was recrystallized five times from hot benzene/petroleum ether. The specific radioactivity was calculated for each crystallization (Table II) and found to be constant. These results identify the reaction product as MVA and establish the validity of the assay. Properties of HMG-CoA reductase from pea seedlings. The effect of pH on the activity of the microsomal enzyme is shown in Fig. 1. Optimum activity was observed at pH 6.8 with a marked decline above and below this figure. Activity at pH 5.5 and pH 7.5 was 40% of that at pH 6.8. Examination of pyridine nucleotide specificity showed that the enzyme has an absolute and specific requirement for NADPH (Table III). When saturating concentrations of NADPH were either added directly to a reaction mixture or produced by an NADPH generating system, good activity was obtained. By contrast virtu-
726
BROOKER TABLE
IDENTIFICATION OF THE
OF RADIOACTIVE BENZHYDRYLAMIDE
AND
II
step
Specific radioactivity (dpmk)
17500
Initial value Crystallization Crystallization Crystallization Crystallization Crystallization
No. No. No. No. No.
1 2 3 4 5
17980 17858
17604 18410
17848
a Carrier MVA-lactone was added to the radioactive product and the benzhydrylamide was prepared by the method of Wolf et al. (19). The derivative was recrystallized from hot benzene/petroleum ether. Crystals recovered were weighed and a small sample counted in toluene scintillation fluid. 55
7:
had no effect upon reductase activity, suggesting that the enzyme did not require free (or loosely bound) metal cations for activity. Comparative studies on the effectiveness of different thiol compounds showed that dithiothreitol was about 20% more effective than 2-mercaptoethanol or glutathione at the concentration tested (Table IV). The optimal concentration of dithiothreitol was 10 mM (Fig. 2); optimal concentrations of 2-mercaptoethanol and glutathione were not determined. In the absence of any thiol compound enzyme activity was about 50% less than optimum. Preincubation with the thiol compound was not necessary for its effect. Studies on the relationship between velocity and substrate concentration showed that the enzyme is saturated with DLHMG-CoA at a concentration of about 0.2
M
PRODUCT BY SYNTHESIS DERIVATIVE AND
RECRYSTALLIZATION”
Crystallization
RUSSELL
50
TABLE ABILITY
45
OF PYRIDINE
HMG-CoA
III
NUCLEOTIDES REDUCTASE
TO SUPPORT ACTIVITY”
40
Pyridfme3nucl)eotide m
35
30
0.35 42.75
NADH NADPH
25
a NADH or NADPH in an aqueous solution (pH 7.01, were added to the reaction mixture containing a microsomal preparation from etiolated pea seedlings and the enzyme activity determined spectrophotometrically.
20
15
10
" ? z c
HMG-CoA reductase activity (nmol/mg protein/h)
TABLE
5
THE
EFFECT
OF VARIOUS
HMG-CoA Thiol
FIG. 1. Effect
of
pH on enzyme activity. microsomal enzyme was assayed over a pH range 5.5 to 7.5 and the specific activity calculated. following buffers (0.1 M) were used: citrate, pH sodium phosphate, pH 6.0; potassium phosphate,
The from The 5.5; pH
6.5-7.5.
ally no activity was obtained with NADH at a similar concentration (Table III). The effect of EDTA on the activity of the microsomal enzyme was examined in order to determine possible metal ion requirements. Concentrations of EDTA up to 0.1
compound
(4.0
rnM)
2-Mercaptoethanol Glutathione Dithiothreitol
IV THIOL
REDUCTASE
COMPOUNDS
ON
ACTIVITY’
HMG-CoA reductase activity (nmolhg protein/h) 37.9 37.1
48.4
“The reaction mixture contained the microsomal fractjon, prepared in the absence of any thiol compound, from etiolated pea seedlings. Various tbiol compounds were added to the reaction mixture and the reaction started immediately by addition of substrate. Preincubation with thiol compound was not necessary for its stimulatory effect.
HMG-CoA
V
m 0
eg
9
c
16.
REDUCTASE
FROM
PEA
727
SEEDLINGS
.:
2 0
2
4 DTT
6
6 contentr~ltmn
10
12 Cm
14
16
16
20
M)
FIG. 2. Effect of dithiothreitol (DTI’) on HMG-CoA reductase activity. A microsomal enzyme extract was prepared in the absence of any thiol compound and aliquots were added to incubation mixtures containing increasing concentrations of DTT. The specific enzyme activity was calculated for each concentration.
mM and no inhibition occurred at substrate concentrations up to 0.6 mM. From a double reciprocal plot (Fig. 3) the apparent K, for DL-HMG-CoA is about 100 PM. Under the assay conditions described, NADPH was always present in saturating concentrations and the affinity of the enzyme for NADPH was not examined. The possibility of product inhibition was examined by incubating the reaction mixture in the presence of a range of concentrations of MVA (Table V). Concentrations up to 1 mM MVA had no effect on the reductase activity. The presence of either HMG or free CoA in the reaction mixture was examined for any effect on the reductase activity. Both HMG and CoA were inhibitory although CoA had the greatest effect (Table V). At a concentration of 0.5 mM, CoA inhibited enzyme activity by about 50%, whereas a concentration of 3.0 mM HMG was required for the same effect. The effect of gibberellic acid (GA,), on enzyme activity was examined with a view to testing for possible feed-back inhibition by this isoprenoid plant hormone. GAB, up to a concentration of 0.2 mM, was included in the reaction mixture and the results of these assays showed that GA, had no effect on reductase activity. There is considerable variation in the level of reductase activity in different tissues. Microsomal reductase activity in apical buds of etiolated seedlings is more than 2-fold higher than in apical bud tissues of green seedlings (Table VI). In green seedlings, assays of microsomal fractions from apical buds, immature and mature leaves
FIG. 3. Double reciprocal plot of velocity against HMG-CoA concentration. A microsomal enzyme preparation was assayed in the presence of varying amounts of DL-HMG-CoA. The enzyme activity was calculated and results are shown as a typical Lineweaver-Burk plot. The apparent Km obtained from this plot is approx 100 pM.
showed that apical buds contain about 0.6 units/g, whereas semimature leaves contain 66% of this value and fully mature leaves contain only 7% of the apical bud activity (Table VI). Thus there are striking differences in microsomal reductase activity between tissues of different developmental age and the results show that high levels of microsomal HMG-CoA reductase activity are associated only with young and developing tissues. DISCUSSION
A rapid assay technique for HMG-CoA reductase from pea seedlings has been described and the properties of the micro-
728
BROOKER TABLE
EFFECT
OF HMG,
HMG-CoA Compound
V
CoA,
AND SOME PRODUCTS REDUCTASE ACTIVITYO
Concentration
HMG-CoA reductase activity (nmol/mg protein/h)
MVA HMG
O-l.0 0.15
ON
mM rnM
34.0 38.2
% of control 100
96.5
1.0 rnM 10.0 rnM
30.4 5.4
76.8 13.6
CoA
0.025 mM 0.5 rnM
26.8 20.8
67.0 52.0
GA,
o-o.2
42.5
rnM
100
“The reaction mixture contained the microsomal preparation from etiolated pea seedlings. The various compounds were added to the reaction mixture and the reaction started by addition of substrate. TABLE LEVEL
OF HMG-CoA
VI
REDUCTASE
IN DIFFERENT
TISSUES~
Tissue
Etiolated seedlings Apical bud Green seedlings Apical bud 1st leaf (semimature) 2nd leaf (mature)
Relative HMG-CoA reductase levels
225
100 66 7
“The microsomal fraction was prepared from tissues of different ages obtained from the same green pea seedlings. Reductase activity was assayed by the standard procedure described under Methods.
somal enzyme have been determined. The reaction product has been identified as MVA by chromatography and benzhydrylamide derivative formation. Results show that the microsomal reductase has an absolute requirement for NADPH which can be satisfied either by an NADPH generating system or added NADPH. Almost no reaction occurs when saturating levels of NADPH are substituted by similar concentrations of NADH. The presence of a low molecular weight thiol compound is required for optimal activity and experiments with EDTA indicate that the enzyme does not require free metal cations for
AND
RUSSELL
activity. Optimum activity occurs at pH 6.8 and the apparent K, for DL-HMG-CoA is about 100 PM. The microsomal HMG-CoA reductase from pea seedlings is similar to the yeast and rat liver enzymes (1, 4, 6) in its pH optimum, specificity for NADPH, and requirement for a protective thiol compound. The inhibition studies indicate that the microsomal HMG-CoA reductase from pea seedlings is not regulated by product inhibition since concentrations of MVA up to 1.0 mM had no effect on activity. However enzyme activity is affected by both free CoA and HMG. This has also been reported in mammalian systems (4) and Beg and Lupien (20) have suggested that HMG may be a regulator of cholesterogenesis in rat liver. However it is doubtful whether the inhibitory effect of HMG has any significance in vivo since HMG-CoA has been shown to arise from the condensation of acetoacetyl-CoA and acetyl-CoA in animal systems (21), and thus free HMG is not an intermediate. Hence high levels of HMG are likely to arise only from the cleavage of HMG-CoA. But the affinity of the reductase for its substrate suggests that significant cleavage of HMG-CoA would occur only if the reductase is inhibited. Thus HMG effects on reductase activity in vivo are likely to be secondary. The hormone GA,, one of the products of the isoprenoid pathway in higher plants, had no effect on the enzyme activity within the concentrations tested (O-O.2 mM). The normal in uiuo concentration of this hormone is not definitely known but a concentration of 1 PM is generally regarded as close to the physiological optimum. Although the presence of GA, in pea tissue has not been conclusively demonstrated, there is evidence for the existence of GA, (22) which differs from GA, only in the absence of the A1 double bond. In addition GA, is known to be biologically active in pea seedlings. Undoubtedly other gibberellins must be tested before it can be definitely concluded that none of these hormonal end products exert an inhibitory effect upon the reductase. Consideration of the probable pathway of biosynthesis also raises the possibility that branch point
HMG-CoA
REDUCTASE
intermediates such as gibberellin A,z, geranylgeranyl pyrophosphate and farnesyl pyrophosphate may exert a regulatory effect. Tissues at different developmental stages show large differences in enzyme activity. Of particular interest is the high activity found in etiolated seedlings compared with the somewhat lower activity in green plants. Microsomal reductase activity in the apical buds of green seedlings was about 50% less than in apical buds of etiolated seedlings. These differences in activity are considered real since (i) precautions were taken to prevent enzyme inactivation during extraction, and (ii) subsequent studies have shown that similar low activity can be induced in etiolated seedlings by a brief light irradiation (manuscript in preparation). The lower activity in green seedlings may reflect the relative flux through the pathway if, as in animal systems (6), the enzyme is rate limiting. In green seedlings the highest activity occurs in the apical bud which correlates with the finding that gibberellins (23) are produced mainly in these tissues. As the tissue matures there is a decrease in reductase activity. The decline in activity may be due to a decline in enzyme level and thus may reflect either a decrease in synthesis or an increase in destruction, or both. However, the causal mechanism is not known. Further studies on the regulation of this enzyme in pea seedlings are in progress and these results will be reported in a separate communication. REFERENCES 1. KIRTLEY, M. E., AND RUDNEY, istry 6, 230-238.
H. (1967)
Biochem-
FROM
PEA
SEEDLINGS
729
2. KAWACHI, T., ANDRUDNEY, H. (1970) Biochemistry 9, 1700-1705. 3t . KNAPPE, J., RINGELMANN, E., AND LYNEN, F. (1959) Biochem. 2. 332, 195-213. 4. SHEFER, S., HAUSER, S., LAPAR, V., AND MOSBACH, E. H. (1972) J. Lipid Res. 13, 402-412. 5. BUCHER, N. L. R., OVERATH, P., AND LYNEN, F. (1960) Biochim. Biophys. Acta 40, 491-501. 6. SHAPIRO, D. J., AND RODWELL, V. W. (1971) J. Biol. Chem. 246, 3210-3216. 7. ANDERSON, D. G., AND PORTER, J. W. (1967) Annu. Reu. Plant Physiol. 18, 197-228. 8. GRAEBE, J. E., DENNIS, D. J., UPPER, C. D., AND WEST, C. A. (1965) J. Biol. Chem. 240, 1847-1854. 9. SEMBDNER, G., AND SCHREIBER, K. (1971) Fed. Eur. Biochem. Sot. Lett. 15, 1-7. 10. SHORT, K. C., AND TORREY, J. G. (1972) Plant Physiol. 49, 155. 11. GOODWIN, T. W. (1958) &o&en. J. 70, 612-617. 12. HEPPER, C. M., AND AUDLEY, B. G. (1969) Bio&em. J. 114, 379-386. 13. GRAEBE, J. E. (1967) Science 157, 73-75. 14. GOLDFARB, S., AND PITOT, H. (1971) J. Lipid Res. 12, 512-515. 15. HILZ, H., KNAPPE, J., RINGELMAN, E., AND LYNEN, F. (1958) Biochem. 2. 329, 476-489. 16. Louw, A. I., BEKERSKY, I., ANDMOSBACH, E. (1969) J. Lipid Res. 10, 683-686. 17. LOOMIS, W. D., AND BA~AILE, J. (1966) Phytochemistry 5, 423-438. 18. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. WOLF, D. E., HOFFMAN, C. H., ALDRICH, P. E., SKEGGS, H. R., WRIGHT, E. D., ANDFOLKERS, K. (1957) J. Amer. Chem. Sot. 79, 1486-1487. 20. BEG, Z. H., AND LUPIEN, P. J. (1972) Biochim. Biophys. Acta 260, 439-448. 21. WHITE, L. W., AND RUDNEY, H. (1970) Biochemistry 9, 2713-2724. 22. JONES, R. L., AND LANG, A. (1968) Plant Physiol. 43, 629-634. 23. PHILLIPS, I. D. J. (1971) Planta 101, 277.