Using the ['4C]bicarbonate fixation assay, 3-methylcrotonyl-CoA carboxylase has been detected in the following mammalian tissuesm: bovine kidney, liver, adrenals, lung, skeletal muscle; rat kidney and liver; and human placenta. Antiserum produced in rabbits after injection of the purified bovine kidney enzyme cross-reacts with the enzyme from bovine liver, rat liver, and human placenta3,'°; it has not been tested with other enzyme preparations. Acknowledgments The studies described here were supported by research grants from the National Institutes of Health (HL-16628) and the National Science Foundation(PCM-16251). to B. C. Cochran, M. L. Hector, D. M. Tasset, and R. R. Fall, unpublished observations.
 E n z y m e s o f W a x E s t e r C a t a b o l i s m in J o j o b a 1
By ROBERT A. MOREAU and ANTHONY H. C. HUANG Wax Ester Catabolism in Jojoba The jojoba plant is native to arid regions of southwestern North America. It is the only known plant species whose seeds contain a large amount (50-60% of the fresh weight) of intracellular wax esters in the cotyledons. 2-5 During germination, this reserve wax ester is mobilized to support the growth of the embryonic axis.6 The carbon skeleton of the wax ester is converted efficiently to carbohydrate. The wax ester in the storage wax bodies is hydrolyzed to a fatty acid and a fatty alcohol. The latter is oxidized to produce another fatty acid. The fatty acids are metabolized first to acetate by the fl-oxidation reaction sequence and then to succinate by the glyoxylate cycle in the glyoxysomes. Succinate is metabolized to malate by the tricarboxylic acid cycle in the mitochondria. Finally, malate is converted to sucrose by gluconeogenic and other enzymes in the cytosol. Except for the conversion of 1 mol of wax ester to 2 mol of fatty t Supported by the National Science Foundation. z R. S. McKinney and G. S. Jamieson, Oil Soap, 13, 289 (1936). a T. K. Miwa, J. Am. Oil Chem. Soc. 48, 259 (1971). 4 L. L. Muller, T. P. Hensarling, and T. J. Jacks, J. Am. Oil Chem. Soc. 52, 164 (1975). 5 D. M. Yermonos, J. Am. Oil Chem. Soc. 52, 115 (1975). R. A. Moreau and A. H. C. Huang, Plant Physiol. 60, 329 (1977).
METHODS IN ENZYMOLOGY.VOL. 71
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181971-X
E N Z Y M E S O F W A X ESTER CATABOLISM IN JOJOBA
acids, the pathway in jojoba is identical to that in the triacylglycerolstoring fatty seedlings of castor bean and other species, r The conversion of wax ester to fatty acids in jojoba involves three enzymes. 6"s'9 The first is a wax ester hydrolase that catalyzes the hydrolysis of the wax ester. The fatty alcohol is oxidized to a fatty aldehyde by a fatty alcohol oxidase (fatty alcohol : 02 oxidoreductase) that requires molecular oxygen as the electron acceptor. The fatty aldehyde is oxidized to a fatty acid by fatty aldehyde dehydrogenase (fatty aldehyde : NAD + oxidoreductase) with NAD + as the electron acceptor. All three enzymes are associated with the membrane of the storage wax bodies, and they share many similar enzymatic properties. The activities of the three enzymes are absent in the dry seeds and increase at parallel rates during germination. Other Organisms that Metabolize Wax Esters or Fatty Alcohols Intracellular wax esters are very important in lipid metabolism of marine organisms, lO,ll They are the major type of lipid, often comprising more than 20% of the total dry weight, in at least 120 species of marine invertebrates and vertebrates distributed among 17 orders and 9 phyla. 12 It has been estimated that at least half of all organic substances synthesized initially by phytoplankton is converted into wax at some points in the marine food chain. ~a The activities of wax ester hydrolase in the digestive juices of surf clams and several species of teleost fishes have been partially characterized. ~4-~6However, these hydrolases are probably involved in the digestion of dietary wax esters only, not in the hydrolysis of the intracellular wax reserves in these animals. Although wax esters are very important in the overall metabolism of marine organisms, the biochemical steps involved remain to be elucidated. The techniques described herein should be useful as a guide in the future study of wax ester hydrolysis and fatty alcohol oxidation in marine organisms. Microorganisms are indispensable in the catabolism of large quantities 7 H. Beevers, Ann. N. Y. Acad. Sci. 169, 313 (1969). 8 A. H. C. Huang, R. A. Moreau, and K. D. F. Liu, Plant Physiol. 61, 339 (1978). 9 R. A. Moreau and A. H. C. Huang, Arch. Biochem Biophys. 194, 422 (1979). s0 j. C. Nevenzel, Lipids 5, 308 (1970). 1~ A. A. Benson, R. F'. Lee, and J. C. Nevenzel, Biochern. Soc. Syrup. 35, 175 (1972). 12 j. R. Sargent, R. F. Lee, and J. C. Nevenzel, in "Chemistry and Biochemistry of Natural Waxes" (P. E. Kolattukudy, ed.), pp. 49-91. Elsevier, Amsterdam, 1976. 13 A. A. Benson and R. F. Lee, Sci. Am. 232, 77 (1975). 14 j. S. Patton and J. G. Quinn, Mar. Biol. 21, 59 0973). 1~ j. S. Patton and A. A. Benson, Comp. Biochem. Physiol. 52B, III (1975). ~6 j. S. Patton, J. C. Nevenzel, and A. A. Benson, Lipids 10, 575 (1975).
of wax esters and alkanes of dead plant materials. Again, the biochemical steps involved have been studied inadequately. Some microbes grown in alkanes produce fatty alcohols as intermediates during the to-oxidation of alkanes. The fatty alcohols are then oxidized by a NAD specific fatty alcohol dehydrogense.17-19 The resulting fatty aldehydes are oxidized to fatty acids by a NAD specific fatty aldehyde dehydrogenase.19,2o Discovery of the fatty alcohol oxidase (fatty alcohol : Oz oxidoreductase) raises the possibility that the reported microbial NAD-fatty alcohol dehydrogenase (fatty alcohol: NAD ÷ oxidoreductase) might actually be an oxidase. In the jojoba enzyme preparation containing both fatty alcohol oxidase and fatty aldehyde dehydrogenase, substrate-dependent NAD ÷ reduction can be demonstrated using either fatty alcohol or fatty aldehyde as a substrate. This apparent "NAD÷-linked dehydrogenase" activity with fatty alcohol is due to the activities of both the oxidase and the dehydrogenase and is inhibited by eliminating oxygen from the system. The microbial fatty alcohol dehydrogenase has not been purified to homogeneity or separated from the fatty aldehyde dehydrogenase, and the effect of anaerobiosis on its activity has not been tested. Although this microbial fatty alcohol oxidoreductase may indeed utilize NAD ÷ as the electron acceptor, the possibility exists that it actually utilizes oxygen as the electron acceptor, since its activity was assayed together with the NAD+-fatty aldehyde dehydrogenase. Isolation of Wax Bodies and Their Membranes Seeds ofjojoba (Simmondsia chinensis) are dusted with Phaltan (Chevron Chem. Co., Richmond, California) to retard fungal growth and allowed to germinate in moist vermiculite in darkness at 28°. A small quantity of seeds can be obtained from the Office of Arid Lands Studies, University of Arizona, Tucson, Arizona, and large quantities can be purchased from American Jojoba Industries Inc., Bakersfield, California. All chemical reagents are purchased from Sigma Corp. St Louis, Missouri, except as otherwise noted. Wax bodies are isolated from the cotyledons of seedlings 15-20 days old. All steps are performed at 0-4 ° . Ten grams of cotyledons are chopped with a razor blade in a petri dish containing 20 ml of grinding medium consisting of 0.6 M sucrose, 1 mM EDTA, l0 mM KC1, 1 mM MgC12, 2 lr E. Azoulayand M. T. Heydman,Biochim. Biophys. Acta 73, 1 (1963). is B. Roche and E. Azoulay,Eur. J. Biochem. 8, 426 (1969). 19j. M. LeBeault,B. Roche, Z. Duvnjak,and E. Azoulay,Biochim. Biophys. Acta 220, 373 (1979). 20M. T. Heydman,and E. Azoulay,Biochim. Biophys. Acta 77, 545 (1963).
ENZYMES OF WAX ESTER CATABOLISM IN JOJOBA
mM dithiothreitol, 0.15 M Tricine buffer, adjusted with KOH to a pH of 7.5, until pieces 1-2 mm z in size are obtained. The pieces are further homogenized with a mortar and pestle. The homogenate is filtered through a piece of Nitex cloth with a pore size of 44/~m 2 (Tetko Inc., Elmsford, New York). The filtrate is centrifuged at 10,000 g for 30 min, and the resulting lipid pad is removed with a spatula. The lipid pad is resuspended in 10 ml of grinding medium and shaken with a Vortex mixer; the resuspended material is centrifuged at 10,000g for 30 min. The lipid pad is then removed and resuspended with grinding medium to make a total volume of 5 ml. Examination under the electron microscope reveals that the resuiting suspension contains essentially only wax bodies. 8 Wax bodies are spherical organelles approximately 1 /zm in diameter. Each organelle is packed with wax ester and is surrounded by a membrane. The membranes of isolated wax bodies are obtained by removing the wax with diethyl ether. The isolated wax bodies, in a suspension of 5 ml prepared as described in the preceding paragraph, is mixed with 10 ml of cold diethyl ether. After shaking with a Vortex mixer, the mixture is allowed to settle for 1 hr at 0-4 °. The upper diethyl ether layer is removed by aspiration. The ether extraction procedure is repeated twice. After extraction, the trace amount of diethyl ether remaining is evaporated under a stream of nitrogen. The resulting aqueous resuspension of wax body membranes contains approximately 5 mg of proteins in 5 ml. The wax body membranes (or in some cases intact wax bodies) are used as a source of enzymes for the following enzymatic studies. Wax Ester Hydrolase O
R--C--O--R2 + H20 ---> R - - C - - O H + R2--OH Wax ester Fatty acid Fatty alcohol
Fluorometric Assay Wax ester hydrolase activity is routinely assayed by a fluorometric lipase assay. 21'~2The activity is measured at room temperature in a reaction mixture of 4 ml, containing 0.1 M Tris-HCl buffer, pH 9.0, 2 mM 21 G. G. Guilbault and J. Hieserman, Anal. Chem. 41, 2006 (1969). zz S. Muto and H. Beevers, Plant Physiol. 54, 23 (1974).
dithiothreitol, and 10-50/zl (10-50/xg of protein) of enzyme. The reaction is initiated by the addition of 0.83 mM N-methylindoxylmyristate (from I.C.N. Pharmaceuticals) dissolved in 0.1 ml of ethylene glycol monoethyl ether. Fluorescence measurements are made with a Turner Model 111 fluorometer with excitation filter No. 405 (405 nm maxima) and emission filter No. 2A-12 (>510 nm), attached to a X-Y recorder (Model 7034A, Hewlett-Packard Co.). The reaction rate is linear for the first 10 min.
Spectrophotometric Assay A modified colorimetric assay z2 is used to measure hydrolase activity when various wax esters and glycerides are tested as potential substrates. In this method, the fatty acids produced are converted to copper soaps and measured using sodium dithiocarbamate. The reaction is performed at room temperature in a 15-ml tube. The reaction mixture contains in a final volume of 1 ml: 0.1 M Tris-HCl, pH 9.0, 5 mM dithiothreitol, 10 mM substrate, and 100/zl (100/zg of protein) of enzyme. Substrates (100 mM) are first emulsified in 2 ml of 5% gum acacia for 1 rain at low speed with a Bronwill Biosonic IV ultrasonic generator fitted with a microprobe. For each substrate, two enzyme concentrations (50 and 100/xg of protein) are used, and the reaction is stopped at time intervals of 1-2 hr each to ensure that proper kinetics are observed. The reaction is stopped by the addition of 1 ml of copper reagent (0.9 M triethanolamine, 0.1 M acetic acid, 5% cupric nitrate). Copper soaps are extracted into 4 ml of chloroform by shaking the tubes closed with Teflon screw-caps horizontally for 30 rain. Two milliliters of the chloroform layer are removed and added to 0.2 ml of 0.1% sodium dithiocarbamate in l-butanol. The absorbance is measured immediately at 440 nm. Palmitic acid is used to produce a standard curve that is linear up to a concentration of 0.2 t~mol per 2 ml of chloroform. Stearic acid and arachidic acid exhibit the same molar extinction coefficient.
Assessment of the Two Methods The fluorometric assay is' by far the more convenient one. The activity can be continuously monitored on a recorder. Each assay requires about 5 min. The major drawback of this assay is that the substrate, N-methylindoxylmyristate, is an artificial one, and caution must be used in interpreting the physiological significance of results obtained with this substrate. The colorimetric assay is relatively time-consuming and less accurate, since the activity cannot be measured continuously. Furthermore, the
E N Z Y M E S OF W A X ESTER CATABOLISM IN JOJOBA
amount of enzyme required for this assay is 10 times that required for the fluorometric assay. However, the colorimetric assay is essential in testing the activity of the hydrolase with all of the natural, nonfluorescent substrates. Properties Wax ester hydrolase activity is absent in the dry seeds, but increases at a nearly linear rate during the first 20 days of germination. Most of the cellular activity is associated with the membrane of the wax bodies. The enzyme has an optimal activity at pH 9. The apparent K m value for N-methylindoxylmyristate is 9.3 × 10-5 M. It is stable at 40° for 30 min but is inactivated at higher temperatures. Various divalent and monovalent cations at a concentration of 1 mM, such as CaCI2, MgCIs, NaC1, and KCI, and 1 mM EDTA have little effect on the activity. p-Chloromercuribenzoate at 0.1 mM inhibits 81% of the activity, and its effect is reversed by subsequent addition of 5 mM dithiothreitol. The enzyme exhibits a broad substrate specificity with natural lipids, having high activities on monoglycerides, wax esters, and the native substrate (jojoba wax), and low activities on diacylglycerols and triacylglycerols. It hydrolyzes the artificial substrate, N-methylindoxymyristate, about 50 times faster than any of the natural substances. /'
Fatty Alcohol Oxidase (Fatty Alcohol : Oz Oxidofeductase) RCH2OH + ½02 ~ RCHO + H20 Fatty alcohol Fatty aldehyde
Oxygen Uptake Assay Fatty alcohol oxidase activity is routinely assayed with an oxygen electrode (Yellow Springs Instrument Co., Model 53 Biological Oxygen Monitor, attached to a Hewlett-Packard X-Y recorder, Model 7034 A). The reaction mixture contains in a volume of 4 ml: 13 mM fatty alcohol, 0.1 M Tris-HC1 buffer, pH 9.0, 5 mM dithiothreitol, 1 mM KCN, and 0.5 ml (0.5 mg of protein) of enzyme. The fatty alcohol (130 mM) is first emulsified in 0.5% Tween 80 (2 ml) for 1 min at low speed with a Bronwill Biosonic IV ultrasonic generator fitted with a microprobe. The temperature of the reaction mixture is allowed to equilibrate for 5 min at 30° in the
water bath before the reaction is initiated by addition of substrate. Oxygen uptake is recorded continuously for 10 min.
Radioisotope Assay Fatty alcohol oxidase activity is assayed using [1-~4C]lauryl alcohol as a substrate, and the fatty aldehyde formed is identified by thin-layer chromatography. Substrate emulsion is prepared by sonicating 0.33/xmol of [ 1-14C]lauryl alcohol (12 ~Ci, from Amersham/Searle or I.C.N. Chemical and Radioisotope Division) and 230/zmol of unlabeled lauryl alcohol in 1 ml of 0.5% Tween 80 for 1 rain at low speed with a Bronwill Biosonic IV ultrasonic generator fitted with a microprobe. The reaction mixture contains in a final volume of 4 ml: 12 mM lauryl alcohol (200 /zl), 0.1 M glycine-NaOH buffer, pH 9.0, 5 mM dithiothreitol, and 0.8 ml (0.8 mg of protein) of enzyme. The reaction mixture is shaken at room temperature. After 0, 3, 6, and 9 hr the reaction is stopped by extraction of the fatty components according to the method of Bligh and Dyer. z3 To each 0.8-ml aliquot of the reaction mixture in a tube are added 2 ml nf methanol and 1 ml of chloroform. The tube is capped, shaken, and incubated at room temperature for 30 min; then 1 ml of chloroform and 1 ml of water are added. The tube is shaken and set aside until the liquid phases separate clearly. The chloroform layer is removed, dried, and resuspended in 500 /zl of chloroform; '\100/zl of this chloroform suspension is spotted onto a thin-layer chromatography (TLC) plate coated with silica gel G (200/xm). Then 0.2 mg each of dodecyl alcohol and dodecyl aldehyde dissolved in 50 /xl of chloroform are added to each spot as carriers. The plate is developed in 80:20:1.5 (v/v/v) hexane/diethyl ether/acetic acid. The lipids are detected by spraying with 0.2% 2,7-dichlorofluorescein. The R s values for fatty alcohol and fatty aldehyde are 0.50 and 0.95, respectively. The identified spots, are scraped into a scintillation vial of 10 ml of 0.5% PPO and 0.01% POPOP in toluene and counted.
Assessment of the Two Methods The oxygen uptake measurement is more convenient than the radioactive assay. A larger quantity of enzyme is required for the oxygen uptake assay because of the low sensitivity of the apparatus. The radioactive assay, aside from its higher sensitivity, is also useful for measuring the combined activities of fatty alcohol oxidase and fatty aldehyde dehydrogenase. The substrate (fatty alcohol), the first product (fatty aldehyde), and the second product (fatty acid), can all be separated by TLC. This z3 E. G. Bligh and W. J. Dyer, Can. J. Biochem. 37, 911 (1959).
E N Z Y M E S OF W A X ESTER CATABOLISM IN JOJOBA
system can be used to identify the electron acceptors involved in the two oxidative reactions catalyzed by the oxidase and the dehydrogenase (see next section). In the presence of oxygen alone and the absence of NAD ÷, fatty alcohol is converted to fatty aldehyde only. When oxygen and NAD ÷ are both present, fatty alcohol is converted to fatty acid with little buildup of fatty aldehyde. Under anaerobic condition, fatty alcohol is not oxidized, even when NAD ÷ is supplied. Properties Fatty alcohol oxidase activity is absent in the dry seeds, but increases at a nearly linear rate during the first 20 days of germination. As for the hydrolase, most of the cellular activity is associated with the wax body membrane. The enzyme utilizes molecular oxygen as the electron acceptot, and has an optimal pH of 9.0 with dodecyl alcohol as substrate. It has an apparent K m value of 4 mM for dodecyl alcohol. Dodecyl alcohol gives the highest activity. Other alcohols give the following activities relative to dodecyl alcohol (100%): myristyl (74%), decyl (51%), palmityl (26%), and stearyl (16%). Arachidyl and behenyl alcohols give rates of oxygen consumption that are barely detectable. The physiological substrates eicosenol (19%) and docosenol (13%) are oxidized at rates higher than those of their saturated analogs. Ethanol and palmitic acid are not substrates. The oxidase is more labile than the hydrolase; freezing overnight results in 50% reduction of its activity. Oxidase activity is cyanide insensitive and is not stimulated by NADH or NADPH, suggesting that the enzyme is not a mixed function oxidase. Measurement of the stoichiometry of the oxidase reaction with and without KCN or commercially purified catalase, suggest that the electrons and Os combine to form H~O, but not HsO2.
Fatty Aldehyde Dehydrogenase (Fatty Aldehyde :N A D + Oxidoreductase) R-CHO
+ NAD + +H20--~
Assay Methods Fluorometric Assay Fatty aldehyde dehydrogenase is assayed by measuring the reduction of NAD + fluorometrically. The reaction mixture contains, in a total vol-
ume of 4 ml: 3.25 mM fatty aldehyde, 0.1 M Tris-HCl buffer, pH 9.0, 5 mM dithiothreitol, 1 mM NAD ÷, and 1-50 /zl (1-50 /zg of protein) of enzyme. The fatty aldehyde (32.5 mM decyl aldehyde) is first emulsified in 2 ml of 0.5% Tween 80 by sonicating for 1 min at low speed with a Bronwill Biosonic 1V ultrasonic generator fitted with a microprobe. The reaction is initiated by addition of substrate. Fluorescence measurements are made on a Turner Model 111 fluorometer with excitation filter No. 7-60 (360 nm maximum) and emission filter No. 2A-12 (> 510 nm) attached to a Hewlett-Packard X-Y recorder Model 7034A. The reaction rate is linear for at least 5 min.
Spectrophotometric Assay The enzyme activity is assayed by measuring the production of fatty acid with the colorimetric method exactly as described for the wax ester hydrolase. The substrate used in routine assays is 3.25 mM decyl aldehyde.
Assessment of the Two Methods The fluorometric assay is sensitive and convenient. It is at least 10 times more sensitive than the conventional spectrophotometric assay of NAD + reduction at 340 nm. More important, unlike absorbance, relative fluorescence measurements are not seriously affected by the high turbidity of the reaction mixture. The colorimetric assay can be used to establish the stoichiometry of the reaction; one fatty acid being formed for each NAD ÷ reduced. It is less convenient than the fluorimetric assay, since it necessitates extracting the fatty acid soaps prior to photometric assay. Properties Like the hydrolase and oxidase previously described, fatty aldehyde dehydrogenase activity is absent from dry seeds and increases during germination to a maximum after about 20 days of germination. The enzyme is also associated with the membrane of the wax bodies. It is stable for several months when frozen. Eighty percent of the activity is destroyed by heating the enzyme at 55° for 30 rain. The optimum pH for activity is 9.0. The enzyme has an apparent Km of 4 × 10-6 M for decyl aldehyde, and an apparent Km of 2.5 x 10-4 M for NAD ÷. Of the various substrate analogs, dodecyl aldehyde exerts the highest activity. Other aldehydes give the following activities relative to dodecyl aldehyde (100%): decyl (68%), myristyl (59%), palmityl (32%), and stearyl (14%).
ENZYMES OF WAX ESTER CATABOLISM IN JOJOBA
Acetaldehyde is not a substrate. N A D + is a much better electron acceptor than N A D P ÷, FAD, or ftavin mononucleotide. Divalent and monovalent cations at a concentration of 1 m M , such as CaCI~, MgCI~, NaC1, and KC1, and l m M E D T A exert little effect on the activity. On the other hand, 1 mM CuSO4 inhibits 60% activity, and 1 m M MnC12 causes 24% increase in activity, p-Chloromercuribenzoate at 0.1 m M causes a complete inhibition of the activity, but its effect is o v e r c o m e by the subsequent addition of 5 m M dithiothreitol.