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Fat metabolism in higher plants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
162, 147-157 (1974)
Fat Metabolism
in Higher
Recent Studies on Plant a-Oxidation w. E. SHINE Department
of Biochemistry
AND
and Biophysics, Received
P.
University November
I<.
Plants Systems’* 2
STUMPI; of California,
Davis,
Calijornia
95616
14, 1973
Both the seed and the leaf a-oxidation systems of higher plants require for activity a reductant and molecular oxygen. The reductant may be NADH or a coupled glucose + glucose oxidase system. D-2-Hydroxypalmitate is the only 2-hydroxy acid which is formed from palmitic acid under a variety of conditions; L-2-Hydroxypalmitate is not formed, but when added does inhibit the initial attack of the a-oxidative enzyme(s) on palmitate. Since the addition of glutathione + gluthathione peroxidase greatly increases the formation of DZhydroxypalmitate from palmitate, with a COW comitant decrease in COZ, it is strongly suggested that the intermediate ina-oxidation is n%hydroperoxypalmitate, which may thenundergo decarboxylation to a pentadecanal and CO2 or be reduced to D-2-hydroxypalmitate. Evidence in support of this hypothesis is presented.
Previous papers in this series described an a-oxidation system in germinating seed tissue that formed CO2 and a C,-l fatty acid in the presence of a hydrogen peroxide generating syst’em and KAD+. Later work revealed that a C,-1 long chain aldehyde is an intermediate (1). More recently, Mead and Levis, with brain tissue (a), and Hitchcock and James, with pea leaf extract.s (3), report’ed that ol-hydroxy fatty acids were intermediat’es in the a-oxidation of fatty acids. The latter \I-orkers made the important observation that the accumulating a-hydroxy fatty acid has the n-configuration (4). Based on isotopic competition experiments, Hitchcock, Morris, and James (5) suggested, however, that L-2-hydroxy fatty acid was the transitory intermediate in the a-degradation of a fabty acid to COz and a C,-1 fatty acid. They further reported that the pea leaf system required only molecular 1 This is paper LVIII in a series. Paper LVIl appeared in Plant Physiol. (1973) 62, 1X-161. Paper LTX is the following paper (Arch. Biochem. Bio. phys. 162,158-165,1974). 2 This research was supported in pa,rt by NSF grant 37880X. @ 1974 by .4cademic Press, of rearoduction in any form
Inc. reserved.
MATERIALS
AND
METHOD::
Materials. [l-%]16:01 (55 Ci,/mole) was purchased from Amersham/Searle. The Hitchcock and James method (6) was used for the preparat,ion of DL-2-hydroxy[l-W]16:0. After preparation of the methyl ester, separation of the D- and I,-isomers was accomplished by preparative thin-layer chromatography of the I,-aretylmandelates, similar to the method of Hitchcock and Rose (71, losing 1 solvent passes followed by elution and rerhromatography of individual spots. PreparaGon of A*-[l‘*C]16:1 utilized a method similar to that rlsed for 2-hydroxy-16:O synthesis, exrept, that (-btlt.anol 3 Abbreviations: 16:0, palmit,ate; ~~-16: 1, 2. hexaderenoic acid; 20H-lS:O, %hydroxypalmitat)e; ROOH, organic peroxide; glc, gas liquid chromatography.
147 Copyright All rights
oxygen, in contrast’ to the peanut system which required a HzOz generating system. In this paper, we will present data which demonstrate that both the pea leaf and germinating peanut seed a-oxidation systems have identical properties, that n-2-hydroperoxypalmitate, rather than L-2-hydrox\-palmitate, is the probable intermediate in cY-oxidation of palmitate, and t’hat L-Zhydroxypalmitatr is never formed.
148
SHINE
AND
saturated with KOH instead of 1 N NaOH was utilized for hydrolysis, with final purification by preparative gas-liquid chromatography (glc). Methyl esters were prepared with diazomethane. Glucose oxidase and purified glucose oxidase were purchased from Worthington Biochemical Corp. Catalase was from Schwarz/Mann, and horseradish peroxidase (type I) was from Sigma Chemical Company. Bovine erythrocyte superoxide dismutase was from Truett Laboratories, Dallas, Texas and had an activity of about 13,000 units/mg protein. Bio-Gel P-10 (100-200 mesh) was from Bio-Rad Laboratories, Richmond, CA. Enzymes. Peanut acetone powders, prepared as described previously (8), were extracted with 25 mM potassium phosphate buffer, pH 7.5, containing 0.170 lubrol (9), for 30 min at 8°C with stirring. Washed Polyclar AT (10) was added to the suspension which was stirred briefly, squeezed through bolting silk, and the extract was centrifuged at 30,OOOgfor 20 min. The supernatant was brought to 25yo saturation with solid ammonium sulfate, centrifuged as above, and the supernatant brought to 65yo ammonium sulfate saturation. After centrifuging, the pellet was redissolved in 25 mM potassium phosphate buffer, pH 7.5, and desalted by passing through a Bio-Gel P-10 column equilibrated with the same buffer. In some preparations, the eluate was centrifuged at 105,OOOgfor 120 min and the supernatant used for assays. Pea leaf acetone powders and extracts were prepared by the method of Hitchcock and James (6). Extracts were prepared with 200 mM potassium phosphate buffer, pH 7.0, immediately before use in assays (5). For some assays these extracts also were (a) dialyzed for 23 hr against the same buffer at 4°C or (b) centrifuged at 30,OOOgfor 20 min, the pellet resuspended in the same buffer, brought to 657, saturation with solid ammonium sulfate, centrifuged, and the pellet redissolved, and finally desalted on a Bio-Gel P-10 column. Glycolic oxidase was isolated from spinach leaves by the methods of Zelich (11) through the ethanol precipitation step. The precipitate was dissolved in 25 mM potassium phosphate buffer, pH 8.0. The preparation was assayed by the 2,6-dichlorophenolindophenol reduction procedure (12). Glutathione peroxidase was prepared from beef liver by a combination of the methods of Mills (13) and Little and O’Brien (14). The procedure was carried out at 4°C. Beef liver (270 g) was homogenized in 0.15 N NaCl (500 ml). This homogenate was then treated with ethanol (1,200 ml) and centrifuged, the resulting pellet extracted with Tris-EDTA-pmercaptoethanol (100 mM Tris, 5 mM NaaEDTA, 2 mM p-mercaptoethanol, adjusted to pH 7.3) solution and centrifuged. The supernatant was then
STUMPF treated with an equal volume of ethano~chlorof,orm (400:70) and centrifuged. The resulting supernatant was treated with 34 vol of acetone, centrifuged, and the pellet dried under a stream of Nz. The pellet was suspended in 100 mM potassium phosphate buffer, pH 7.5, centrifuged, and t,he supernatant brought to 65$& saturation with solid ammonium sulfate and centrifuged. The pellet was dissolved in 25 mM potassium phosphate buffer, pH 7.5, and desalted on a Bio-Gel P-10 column. In some assays, rat liver glutathione peroxidase, partially purified by DEAE-cellulose chromatography, was utilized. Both the beef liver and the rat liver glutathione peroxidase preparations were assayed by the cumene peroxide assay (15). Differential and sucrose density gradiejzt ce,ltrifugation. Fourteen-day-old pea shoots (100 g) or lo-day-old peanut cotyledons (190 g) were gently chopped in a blender with 150 ml of isolation medium containing 600 Z~.M sorbitol, 1 mM MgC12, 1% sodium ascorbate, end 100 mM potassium phosphate buffer, pH 7.5. After removing the larger pieces of cellular material by filtering through 2 layers of Miracloth, the suspension was successively centrifuged at 120g for 20 min, 9,OOOgfor 20 min, 34,000g for 25 min, and 105,OOOgfor 180 min. The pellets were resuspended in isolation medium. Then 2.5 ml of the resuspended 1209, 9,000g and 34,000g pellets were layered on top of sucrose density gradients (16) consisting of 5 ml of 6097, sucrose cushion, 20 ml of 6&32yo sucrose gradient, and topped with 5 ml of 20% sucrose (w/w). After centrifuging at 20,000 rpm for 3% hr in a SW 27 rotor, tubes were punctured, and fractions were collected. Assays. Assays for a-oxidation activity were carried out in 13 X 45 mm glass test tubes. Assays carried out in No. 1 hollow polyethylene stoppers gave spurious results because of nonenzymatic dehydration of 2-hydroxy-16:O to 16:1 and therefore were not used. A benzene solution of the substrate was added to each glass tube and the benzene evaporated. To each tube was added the standard reaction mixture which contained 12 ~1 of 100 mM glucose, peanut enzyme, glucose oxidase, and 25 mM potassium phosphate buffer, pH 7.5, in a total volume of 0.1 ml; tubes were maintained at 4°C during these additions. After the final addition, tubes were stoppered with rubber caps into which a Kontes plastic center well containing 0.1 ml of Packard Hyamine hydroxide (1 M) was inserted. The reaction mixture was shaken at 26°C for 40 min (under normal laboratory lighting). The reaction was then terminated by injecting 40 ~1 of 0.1 N NC1 and followed by an additional 15.min incubation. The contents of the center well were analyzed for 14C02 using a nonaqueous fluor (0.42 g 1,4-bis12-
I-l!1
PLANT a-OXIUATIO;“; (5.phenyloxaeolyl)I-benzene, 5.1 g 2,5-diphenyloxazole per liter of toluene) and a Beckman LS-230 liquid scintillation spectrometer. The reaction mixtuc was ext,racted with 2 X 1 ml of diethyl ether with added 2-hydroxy-16:O rarrier, evaporated to dryness under it stream of Ns, and methylated with diazomethane. Met,hyl esters, in benzene sollltion, were analyzed on a 4.7 mm X 0.6 m stainless steel c*olllmn packed with 15’:; HI-EFF-2BP on Chromosorb W (80,900 mesh) in a Varian (A!IOP) glc emplovin$ :L t,hermal conductivity detect or coupled to a xuclear-Chicago Biospan (G)!M) r:hdioactivity detector. Methyl esters were tre:tted with I,-(-)-menthylchloroformate reagent (17 I when analysis ol’ w and l~-2-h?-tlroXp[l-14C]16:U was desired. The reaction mixture for t,he pea leaf system cnorltailled sllbstratr, Tween 20 (O.O1yO),50 ~1 of enzyme extract,, and 200 m&t potassium phosphate buf’fer, pH 5.0, in a total volume of 0.1 ml. After inc\lb:tting al 26°C for 30 min under aerobic ronditions, JObI of 1 N IICl was injected and the reaction prodl~(*ts were analyzed as described above. Superoxide dismrltase was assayed by the nit ro bllt~ tetrazolillm spectrophotometric method, in whic*h the srlperoxide anion reduces nitro blue tetr:tzoli\ml (18). RESULTS In 195’3, Martin and Stumpf (8) proposed a mechanism to c>xplain the ol-oxidat’ive degradation of free fatty acids. The mechanism involvod a direct, clectrophillic attack on the woarbon of a fatty acid by a perferryl cation with a subscqwnt8 release of CO2 and the format,ion of a long chain aldehyde containing ow 1~~scarbon. The long chain aldehyde was thchn oxidized by KAD+ and a long chain aldrhydc dehydrogenasr. In 1964, Hitchcock and James (3) made the important,
observation
actiw
that, in pea leaf extracts
cr-oxidation system degraded fatty acids to CO, and a C,-1 fat’ty acid, but that w2-hydroxy fatty acid (4) also accumulated. Bawd on compct,ition c>xperimcnt)s (5) with both II- and L-2-hydroxy-16:O and [l-‘“Cl16:O in which that D-%-hydroxy-1B:O caused slight inhibition of 14COsformation, whereas that L-%-hydroxj.-16:O resulted in a considwablr inhibition of WOS formation, Hitchcock and Morris (19) proposed that th(J substrate, I(i:O, was hydroxylated to both th(l D- and th(l L-isomers of 2-hydroxylG:O, the wisomor was slowly dcgradcd to ~1
CC& arid pmtadecaxd, whilv thv I,-isonwr was so rapidly degraded to thcl pc~ntad(~c*:1noic acid that it ncwr accumulat~c~d.Klegani, exprrim&s by Morris and Hitchcock (I!), ZO), thmploying
L-[~H]~G:O
and
~-I”H]l(i:0
as substrates and tracing the labc.1 thro11gh the D-‘&hydroxy-l6:O and finally 10 pc’Jlt:ldwanal, supportc~d thcbir nwchan1s111t’nll~,.
Abseuce of r,-~-h!~rl/,o.c~!/paln~itate ,ffwu/otiorl. In contrast, t(J the data of Hit,cahcaoc*katd Row (i) \vho sho\vcid ‘-3 5; of L-2-lluhY)s~1B:O awmiulating, bawd on their I:-:u*cWlmandelyl hydroq. fatty acid prowdurc~ ior the> separat,ion of’ D- and I,-isonl~%, :\I:\&()vetz et al. (9) was not abl(b to show th(j appearanw of L-Zhydrox!, fatty :t&l VIIIploying the L-n1c~nthylchloroforn1nt (h ~)roeedure of Hammarstrijm (21). In rj~~lc~ tc examine thcscb rwults mow closc~ly, irlwcsusing amounts of DL-2.hydrosy-I (i:O \v(w’ added to a wac.tion 1nixturc with (1-iV’l 1(;:(I as subst)rate. As sho\vn in lcig. I, ait bough the pool size of nonradioactivc~ DI,-L’-hycirc~sy1G:O was incwawd from 0 to 0.1 rimc~lc~\\ it11 2.4 Iimolc+
although
of (I-‘V]lti:O
thrw
as substrate*,
was markcld inhibition
14COs forniation, the formation droxy-I ti: 0 \V:LS simultaneously
arid
01
of ~)-L’-liygr’cbatI!- I’(%-
duced \vith 110 appcwancc of r,-~-h~~tlrox~~[1-L4CJ1B:0. If that c-ornpc+it.ion c~xpc%mc~nt: of Hit,chrock and .Jamw invoked tl1ct cliluI,-l)-ll~.droxytion of 110~1~ synthcsizcd [l-14C]1A:0 with rlc)1vadioact,ivc, L-2hydrosyl(i:O with a c*oncomitant dc,crclascL in 11(‘( ):! thrn Id-Zhydroxy-lfi: 0 should havcb li)c~~ dctrrtcd in this wp(~rinwnt. Indwd, ~Iarko vetz et al. shohvchd in l!R? (9) that inc*rcGrt~ the col~c~ntration of L-?-hydroxy-l(i:O ill :
reaction rnixtuw containing [I -14C’\lO:O, w duwd that synthesis of n-2.hydrox\.-1 ri:O a: ~~11 as the cwjlution of “CN2, \\-hcww n-Zhydroxy-16: 0 had only a slight c#t’wt OI t>hc reaction. Th(w rwults strongly ~uggw that r,-‘2.hydroxy-16: 0 bchavw as a11i1rhil)i
150
SHINE
RADIO-GLC
Substrate: +ZDL-OHl6:O: Products, ID-OHII-%]U:O:
2.4 nmoles[l~‘CJl&0 0 nmoles O.C+nmoles
To?: O.IPnmoks 1
MASSwith
AND
PATTERNS
: O.OSnmoles
:O.l nmoles
: hOlrmoles
: o.o2mloks
: O.?7lnmoles
: 008 m&s
carrier2DL-OHNO
FIG. 1. Effect of unlabeled 2-hydroxy-16:O on pea or-oxidation. Reaction mixtures contained the indicated substrates, 50 ~1 of pea extract, and a total of 20 pmoles of potassium phosphate buffer, pH 7.0, in a total volume of 0.1 ml. Samples were incubated at 26°C for 30 min. “C peaks match the derivative D-2-OH-16:0 menthylchloroformate mass peaks.
tor at the initial step in the reaction of 16:0 with the a-oxidation system, whereas D-2hydroxy-16:O has little if any effect on the initial attack. In summary, these experiments suggest that L-2-hydroxy-16:0 is an inhibitor in the initial reaction of substrate with the cyoxidation enzymes and not a product of a-oxidation of 16:0 where D-2-hydroxy16:0 normally accumulates. However, since addition of D-2-hydroxy-16:0 had little effect on 14C02 release, clearly the n-2-hydroxy16:0 could be excluded as a required intermediate for a-oxidation. This of course assumes that added n-2-hydroxypalmitate equilibrates with any enzyme-bound D-2hydroxypalmitate which might be produced. Rate of oxidation of proposed intermediates. If L-2-hydroxy-16:0 were an intermediate in the conversion of 16:0 to COz and pentadecanoic acid, an important requirement would be that its rate of oxidation would be at least as large as that of 16:0, the initial
STUMPF
substrate. As summarized in Table I, and repeated a number of times, [1-J4C]16:0 is the most effective substrate, with L-2-hydroxy-16:O at the same concentration being about 75% as effective and D-2-hydroxy16:0 about 25%. On the other hand, L-2hydroxy-16:O is the most effective substrate for spinach glycolate oxidase (Table I), with n-2-hydroxy-16:0 being about 35% and 16:0 about 15 % as effective. These results would suggest that L-2-hydroxy-16:0 is not an intermediate in the a-oxidation of palmitate and that other enzymes such as endogeneous glycolate oxidase could contribute to 2-hydroxy-16: 0 decarboxylation. Role of glucose oxidase with both the peanut and the pea leaf a-oxidation system. The peanut or-oxidation system required the glucose oxidase “peroxide” generating system for CO2 formation from 16:0 as well as from 2-hydroxy-16:O. In contrast, the pea system appeared to require only molecular OZ. Figure 2 shows that with both the pea and the from peanut systems, 14C02 formation [1J4C]16:0 peaked at a level of 2.6 X 10e3 units of glucose oxidase activity; glc analysis indicated a similar pattern for D-2-hydroxy[l-14C]16:0 formation, although the amount TABLE ENZYMATIC
I
DECARBOXYLATION SUBSTRATES~
OF VARIOUS
Peanut a-oxidation system
Spinach glycolate oxidase
Substrate*
nmoles Substrate _ Palmitate n-2-hydroxy-16:O L-2-hydroxy-16:0 nL-2-hydroxy -16:( 3
COz
nmoles Substrate
COz
0.50 0.50 0.50 -
0.004 0.009 0.024 -
__~ 0.25 0.25 0.25 0.50
0.104 0.027 0.071 0.095
0 The reaction mixture contained substrate, a total of 2.5 Mmoles potassium phosphate buffer, pH 7.5, 1.2 rmoles glucose, and (a) 20 ~1 of peanut enzyme and 0.07 IU glucose oxidase, or (b) 40 ~1 spinach enzyme and 0.02 IU glucose oxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min (peanut,) or 80 min (spinach). * All substrates were carboxyl-14C labeled.
I Al
PLANT (r-0XII)ATIOS
dophenol + &colic acid systm ~~wmd
~10. 2. Effect of glucose oxidase additions on a-oxidation. Reaction mixtures contained 1.2 @moles of glucose and (a) 0.67 nmoles of substrate, 10 ~1 of (NH&SO4-fractionated peanut enzyme, and a t,otal of 2.5 pmoles of potassium phosphate buffer, pH 7.5, or (b) 1.0 nmole substrate, 50 ~1 of (NH&SOr-fractionated pea enzyme, and a total of 20 rmoles of potassium phosphate buffer, pH 7.0, as well as the indicated amounts of glucose oxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 30 min.
VW always much less than the amount of 14CO2 formed. li’ormat’ion of 14C02 from m-%-hydroxy-16:O was increased by glucose oxidase addit’ions in both plant systems. ,4t lower glucose oxidase levels, much more 14C02was formed from [l-14C]16:0 than from m-2-hydroxy[l-*4C]16:0. These experiments clearly indicated that’, alt’hough earlier reports suggest#ed a major difference between the seed and the leaf system in that the former required a Hz02 generating system while the latt’er required molecular oxygen, both systems responded in a very similar manner when the concent’ration of glucose oxidase + glucose was carefully monitored. These results were further substantiated when it was observed t,hat the pea leaf extracts of Hitchcock and James contained a heat-stable component which rapidly reduced 2,6-dichlorophenolindophenol. Moreovw, glycolic acid oxidase which was normally assayed by the 2, B-dichlorophenolin-
in their preparations. Finally, lvhcn lwa leaf preparat#ious, obtained according t,o Hitchcock (5, Ci), \vcr(’ first dialyzed t(J rcmc~ve the heat-stable compon&, only :I ~~11 amount of 14C02 was now form(~tl from [I-W]l(i:O; however, this activit!. \\-a.< inweawl six- and tenfold in the prt~senw of glucose + glucose oxidase or O..i ml1 glycolate, rcspwtively. Glucose osidnw plus glucose also increased activity in :L pea leaf preparation which was partially purified by ammonium sulfatc~ fractionation (E’ig. 2). It is now clear that cndogeneous gl~c~olic oxidase which has been repeatedly c1t.tIIIJIlstrated in leaf extracts by a munlw (Jf workers (22) is responsible for the convwsion of L-a-hydroxy-16:O to CO, and a C,,.~, fut’ty acid. The addition of a H&g~wrnting system such as glucose oxidaae + glucose increased thr rate of YQ wleastld i’n~nl L-2-hydroxy-16:O a2xhas been r’c~portc~tlIJ~ a number of workrrs for short chain t’:rtty acidsL (““) -- . l@‘ecl oJ’ phtathione
peroxidase
OH pro/l l&s
of reactiom. The possibility that :I h!droperoxy function \\‘as involved in a-oxidation was investigated. Neither c:at:dasc nor horseradish peroxidase increased the r:ltio of 2-hydroxy[l-14C]16:0 to ‘“CO2 fornwd from [ 1-14C]16: 0 (Table II) ; nssayx rcpea t tad in the prewnce c~f catalase indicated no In contrast, glutathione 1~111s inhibition. glutat8hione peroxidase caused a lurgc’ increase in u-2-hydroxy[l-W]lG:O ant1 a marked decwase in W02 (Tablrl I I ). This striking effe& of glut,athiow p(wJsidase, \vhich caatalyzes the reduction of pc’roxides, as s~o-c\-I~ in l:ig. 3, \vas invrstigated in more detail. l;igure 3 shows th:lt I)-(,)hydroxy[l-W]lG: 0 increased and l’C’( j2 dccreased with increasing glutathionc pm~sidase lcvcls. Glutathionc pcroxidaw in tlw a,bsencc of glutat8hiont: as w~>ll as glutat hionr~ (2 mhr) in the abscncc of glutathionr, l)fwG dase had no effect. Since moat, of thr! CC), produwd (luring woxidstion has hw11 prwiously postulated (4) to lw dcriwd from L-2-hydroxy-l(i:O, use of glutathiotw plus glutathiono pwoxidasr should haw rwulted in a large incwasr in the L-isomer. Xt a lewl of glut;~thiom~ peroxidaw which caused 50 9; inhibit iI JII (it’
152
SHINE TABLE
EFFECT OF PALMITATE
AND
STUMPF
II
PEROXIDMES ON 2-HYDROXY AND CO? FORMATION FROM PALMITATV
Peroxidase added
nmoles Product 2.hy droxy 16:0
Ratio 2-OH16:O/CO2
co2 ‘_
-_
None Catalase Horseradish peroxidase Glutathione peroxidase + glutathione
0.04 0.02 0.04
0.36 0.29 0.35
0.1 0.1 0.1
0.13
0.13
1.0
I
a Reaction mixtures contained 1 nmole [l-W]16:0, a total of 2.5 pmoles potassium phosphate buffer, pH 7.5, 1.2 @moles glucose, 0.01 IU glucose oxidase, 20 ~1 peanut enzyme, and, as indicated, catalase (150 units), horseradish peroxidase (0.3 purpurogallin units), and glutathione peroxidase plus 0.2 Imoles glutathione in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min.
I 10
I 5
0
Glutathione
I 15
I 20
peroxidase
(h)
FIG. 3. Effect of glutathione tions on a-oxidation. ROOH
+ 2 GSH
peroxidase
addi-
GSH peroxidase ROH
14C02 formation as well as a 250% increase in 2-hydroxy[l-‘4C]16:0 only D-2-hydroxy16:0 accumulated. We suggest therefore that n-2-hydroperoxy-16:O is being formed and the glutathione peroxidase is reducing this product to n-2-hydroxy-16:O. Role of reductants in the wosidation process. No inhibition of a-oxidation was observed by carbon monoxide nor did p-hydroxymercuribenzoate treatment increase the 2-hydroxypalmitate/COZ ratio, thus suggesting the absence of cytochrome P-450 hydroxylation and of cytochrome P-450 (P-420) type peroxidase (23) activity. Also, neither methyl palmitate nor A2-16:l were active substrates. In addition to the glutathione peroxidase-glucose oxidase system described above, other generating systems were utilized in an attempt to increase the ratio of 2-hydroxy-16:O to COZ formed from 16:0. When ascorbate plus FAD was incubated with the peanut extract (conditions were not optimized), SO2 was formed from [lJ4C]16:0 (Table III) but little if any 2-hydroxy[l-14C]16:0 was detected. An equal amount of H20z was only one-third as effective as ascorbate, although when added together their effect was synergistic. These
, 25
+ GSSG + Hz0
Reaction mixtures contained 1.1 nmoles of [l-W]16:0, 0.2 pmoles of glutathione, 1.2 pmoles of glucose, 10 ~1 of glucose oxidase, 20 ~1 of peanut enzyme, a total of 2.5 pmoles of potassium phosphate buffer, pH 7.5, and the indicated amounts of glutathione peroxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min. TABLE
III
DECARBOXYLATION OF P~LMIT~TE BY PEANUT ENZYME AND ASCORB~TE PLUS FAD5 Additions
CO2 (nmoles)
Ascorbate + FAD Ascorbate + FAD + boiled enzyme Ascorbate + FAD + enzyme Ascorbate + enzyme FAD + enzyme Ascorbate + FAD + enzyme + HzOz FAD + enzyme + H202
0.003 0.002 0.035 0.013 0.002 0.074 0.013
D Reaction mixtures contained 1 nmole [l-‘“Cl16:0, a total of 2.5 rmoles potassium phosphate buffer, pH 7.5, and, as indicated, 0.1 pmole ascorbate, 0.012 pmoles FAD, 0.1 pmole HsOz, and 20 ~1 of peanut enzyme in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min.
PLANT
a-OXIl)ATIOX
results are similar to the riboflavin-ascorbatfb-H202 system described by Vorhaben and Steele (fL4) in which a flavin-oxygen udduct was postulated to occur. These results again suggested that 2-hydroxy-1B:O is not an intermrdiatc in a-oxidation. Presumably, 2-hydroxy-16:O was not formed in detwtablc amounts because ascorbate is not a strong rcductant. The results also suggwtc>d that a flavoprotrin could be involved in oc-oxidation. In contrast to the ascorbate results, both Y02 and 2-hvdroxv[l-‘4C]1Ci:0 were formed ., . \vh(an SADH was added to the peanut enZJTl”’ system (Table IV) ; the ammonium sulfate fractionatc>d pea system gave similar rcsultx. Again CO, formation greatly rxccc>dcd 2-hydroxy-16: 0 formation. Of intcwst, however, was the observat’ion that in some aways 14C02 formation exceeded th(l amouny of SADH added (Table IV). At the opt,imum concentration (7 FM) KLIDl’H was as c#fective as NADH. Because previous results had suggested involvcm& of a Aavoprotcin in a-oxidation, the, cbffwt of flavin, with NADH as the added reductant, was tested. Ut.ilizing the optimum conwntration of iYADH (7 PM), addition of FAD (25 PM) resulted in a threefold stimulation of CO2 format’ion, and halfmaxinlal stimulation was observed at 6 ~1~1 I:;\D. 1’AD appclawd to be more effective
SADH added (nmoles)
0.0 0.035 0.070 0.28 0.70 2.x x.4 11.0
Product (nmoles) ~~____~ 2-OH-16:O CO2 0.013 0.010 0.017 0.037 0.035 0.033 0.023 0.027
0.10 0.17 0.22 0.56 0.80 0.70 0.52 0.50
Ratio products/ NADH -__.-~ 2.2 1.8 1.7 1.0 0.22 0.05 0.03
” Ilrat*tion mixtures rontained 2.8 nmoles 11J4C]16:0, a total of 2.5 rmoles potassium phosphate buffer, pH 7.5, and 10 ~1 of peanut enzyme in :a tot’al volume of 0.1 ml. Samples were inellbated al 26°C for 40 min.
I ,-I:;
t,han F51K. Utilizing [U-‘4C]16:0 as sul)strate, there was no evidence that addcxd flavin increased activity by wgwrat iny NADH via thr aldchyde dc~lydrogc,n:tsc,. Thew results suggest,, thcrrfow, that ;t flavoprotein may bc dircct,ly involvc~ti ill woxidation. Dcfinitivc involvtmrnt of :I flavoprotcin must await the purifiwtic)n of the oc-oxidation enzymw. I?/fmrellzdat~ locnfiorr CJ’ a-o.l~itlaficJr/ Wfi/~ity. Both diffcrcntial and swrow dfwit~ gradknt wntrifugat,ion indivatcbd that ( ~v(‘r 80% of th(L pea leaf a-oxidation activity was &her soluhl~~ or nssociatrd lzith :I light microsomal f’rac+ion which could tiot t)(b ptlllrted by wntrifugation at 105,OOO!qFor :< hr. On the other hand, owr X0’; 01’ I 11,. peanut w)tyl(hdotl a-oxidation activity \\.:I:: associatc>d with a heavy microsonlal i’r:rc,tion, 7.i’Z of which could tw p(~llt~t(ql t)! ccwtrifugation at 105,OOOqfor :3 hr. Su(ww density gradicwt wntrifugation itltiiwtc~tl that a srxtll amount of a-oxidation ac.tivit\ leas also associated with a hwviw frac~tiou (dwsity 1.17 g, ml). No oc-oxidat,ion :tchtivit;\. was asswiatctd with chloroplasts, and 1’01bcrt (25) has found I~OIW assoc+atc~d with leaf peroxisr~ttws. :$s has bccrl rvportcyl with th(l lignowric acid a-h;vdrox~l:rtin~ cyst (‘111 (‘X), IY(’ :tlso obswvtld sonw swrow i trhit )ition of woxidation. Ammonium sulfatcb frantic wLt ic)II (Z-37 5, :37-45 “i, and 45-O;i ‘,; ) (pi’ thn high-spwd p(la supcrnatant, followd I)y (1~ salting OII Bio-Ckl I’-10, resultcad in wzynw preparations with somrwhat diffcwtlt pry)I)crtirs; thaw fractions sho\vt>d good tw)xidativc activity in th,, abscww of added r~~tluc:tartt’. Thea Y-37 “; t’ra&on was stinlulattid about’ 23 “; bv. glucost~ plus ghCOSC ()sitl:i,~;c~, Jvhilo the, (Jthcr t\\-o fractions \vw’ irihihitcd in thch prcwnco 01’ this rclduckg sJ,stcattl. .\ diminishw~ depcndwlw on c~xtc~rnal rc~l~~ taut Ivitli increasing purificat~ion hatt pt’(‘viously hwn obswvcd for th(L pcwlut ~,IY~~‘III. Clarification of thaw obwrvatiolls nlust ))(b dofwwd until purificatir)n of th(s tr-osi(l:i.ti,,tr f~rizvmcs is ohtaincd. ~U’up~~widr rlismutase qfect. I)cdttul :~rl> moniutii sulfate, fractions from pw I1savc5 which contained cY-oxidat8ion wc+ivitJ, :LISO containc~tl .+upwosid(~ dismutaw :ic*tivit\. (about 100 units, ring protc~iri). ‘I%~ hi&
154
SHINE
AND STUMPF
speed peanut cotyledon pellet, which contained most of the a-oxidation activity, had only a small amount of superoxide dismutase activity (2 units/mg protein). No correlation was observed between a-oxidation and superoxide dismutase activities. Furthermore, additions of 700 units of bovine erythrocyte superoxide dismutase to assays never inhibited a-oxidation more than 30 %.
The observation that CO, formation occurred more readily from 16:0 than from suggested the either D- or n-2-hydroxy-16:0 possibility that 2-hydroxy-16:O was not an intermediate in COZ formation from 16:O. The rapid decarboxylation of L-2-hydroxy16:0 which was catalyzed by the spinach glycolate oxidase preparation furt’her suggested that glycolic oxidase present in many plant extracts might be responsible for this DISCUSSION decarboxylative activity. Of interest, rat Our results clearly show that in both the liver glycolate oxidase has been observed to peanut and pea leaf systems a-oxidation decarboxylate n-lactate as well as L-lactate required an electron source and molecular (29), as was also observed with D- and L-2oxygen. The a-oxidation activity in the hydroxy-16:O and spinach glycolate oxidase peanut cotyledon, as in the brain (27), is (Table I). associated with a microsomal fraction which In order to determine what intermediate might actually be involved in a-oxidation, can be partially solubilized by employing detergents. Activity from the pea leaf is various enzymes which react with hydroperoxides were tested. Neither catalase nor apparently loosely associated with a light microsomal fraction and readily yields a horseradish peroxidase had any effect. Glutasoluble a-oxidation system. thione peroxidase, however, caused a strikWhen the endogenous cofactors necessary ing decrease in CO:! formation and an infor the pea leaf system were removed by crease in 2-hydroxy-16: 0. These latter results dialysis or ammonium sulfate fractionation, were similar to observations for the reduction the requirement for glycolic acid, glucose of linoleic acid hydroperoxide: namely, (a) plus glucose oxidase, or NADH could be linoleic hydroperoxide was not decomposed demonstrated. Moreover, both the peanut by catalase or horseradish peroxidase (30), and pea leaf extracts showed the same re- and (b) it was reduced to the corresponding sponse to increasing levels of glucose oxidase, hydroxy fatty acid by glutathione peroxii.e., an increase in the glucose oxidase level dase (14, 31). Therefore, the results strongly resulted in an activity peak when 16:0 was suggest that 2-hydroperoxy-16:O is an interthe substrate. The peanut extract showed a mediate in a-oxidation of 16:0 to CO* and similar activity peak with increasing levels pentadecanal. This would also explain why little if any 2-hydroxy-16:O was detected of NADH. At high reductant levels significant in- when a-oxidation was carried out in the hibition occurred suggesting that excess presence of the ascorbate plus flavin system; reductant may cause inhibition of 14C02 presumably, the hydroperoxide was not rerelease from palmitate. Similar effects have duced but was undergoing decarboxylation. been observed in other a-oxidation systems. The NADH results further suggest that less For example, formation of cerebronic acid than two electrons are required for the forfrom lignoceric acid by rat brain enzymes mation of each CO2 molecule, since the ratio of nmoles COZ formed to NADH added showed a distinct plateau with increasing NADH levels (26) ; on the other hand, in a approached 2. Because we detected only n-2-hydroxyrat liver mitochondrial system, CO2 formation from ol-hydroxyphytanic acid was in- 16:0 in numerous assays, the report by creased 50 % by NADPH (28). Glucose plus Hitchcock and Rose (7) of the probable glucose oxidase was strongly inhibitory in biosynthesis of the n-isomer in the pea leaf system is difficult to reconcile. Furthermore, the rat brain system. The latter inhibition when may be explained either by the presence of we detected only n-2-hydroxy-16:0 excess reductant or by the generation of assays were carried out in the presence of glutathione peroxidase. Moreover, we were excess H202.
PLANT
or-OSI1).4TIOS
unablts to dt$wt racemization of either isomer in the prcsencr of the peanut cnzymc txtract. In addition, \w d~trctcd no L-Zhydroxy-16:O in tbit’hcr t,he peanut or the pt~r. leaf systenl when various amounts of unlabeled DL-g-hydroxy-16 : 0 wrc added to thts reaction mixture. These results do not thtwfow support thtl assumption of HitchfYJt’k :LIK~blorris (19) that added unlabeled rJ-L’-hytlrosy-1ci:O causes a dccrrasc in latwlt~d C’02 IJ~ diluting the label. Markovet’z rt al. (!I) have px&JUSly reported that added unhtbelt~d L-I’-hydrosy-16:O caused a marktld dtuww ill labeled D-2-hydroxy-16:O as wll :LS in L4COe product~ion, \\hercas D-Z hgdrosy-16:O had little> if any efftct on product formation. Wc, therefore, suggest that thth L-istJln(T is probably an inhibitor of the initial attack of t’he mzynw on the n-t~arbc~n of thcl fatt,!: acid, although it could :dso bo inhibitmg t’he subsequent format ion of oc-hJ,droperoxy fatt,\- acid. ln prtsvious work, \\-e reported t,hat for full activatic 111of tht: swtl system a hydrogenI)c’rositlt~-g~~nt,ratiiig system was an essential compouttnt of thtt a-oxidation process (1). Thus, tlw Fystems, glycolic acid plus glycolic acid cbxidaw, gluwsc~ oxidase plus glucose, L-:unintb wit1 oxidase plus leucine, all of which 1i:iw in c~onimon the formation of H202, wre cffwtive in the cY-oxidation prtwss. Howrvt~r, ivtl had earlier obscrvcd that thtb direct addition of H&z to the 01. oxidation sgstcw in the absenw of these gcnwating syst clns \vas wscntially ineffw tivt:. It is now clt>ar that the important t’OI11n1011 fc~atuw of all these systems is the flavin ct ,mponont of the Aavoprotoin oxidastrs. Thwt~ oxidastw arc’ probably rcsponsik~lv for thtl gcntwtion of an active species of molecular oxygtw required to initiate the ol-oxidativt~ procwx. WC thewforcb propose in l;ig. 4 an a-oxidation schtmle which involves only t’hc Distmcrs, namely D-Z-hydroperoxy-16: 0 and D-L’-h~droxp-1li:O. WC suggest that, a flavoprotein(s) rathw than cytochromr P-450 may btl involved, since we did not observe C’O inhibition of oc-oxidat,ion and added flavin incrrhascd product’ formation significant81y. Mhcrs have also reported no CO inhibition of cu-oxidation in other systems
1.Y.i
(26). The scheme show a rcquircnwnt ~OI electrons in order to form XH. (e.g., flavin semiquinone) which abstracts Ii,. from thch cu-carbon of the substrattl to form the full! reduced X (XH2) and a free radical, \vhich then re:tcts with the subsequent,ly osygt~natcd XH, to form D-2-hydroprroxy-16:O and thereby wgtwerate XH. . By a t~onwrtt~tl intramolecular ni&2mism, n-%-hydroptt1*oxy-16:0 can decarboxylatc to i’ornl (YJ? and aldchydc. Alternately, tho hydroptw)side cm lw rt~duwd to form r)-Z-hytlrosy16:O.
Tht, schcmc is consistent \vith data rt’ported by others and results prtwntt~tl htw. For cxamplcl, only, the D-isomtxr \v:ts formcld during t ht. Lu-hydroxylation Of lignowric acid (26). In addition, Morris and Hittalrcock (20) reported the loss of slIMrate D-Y-“H and ret&ion of substrate L-‘_‘-~H in w%hydrosy-16:0 which accurnulatrs during a-oxidation. These results are predictctl in our proposed mechanism. They observed a similar patt’ern in the pent’adccanal t’c)rmod by decarboxylation (19). Inhibition ()I CYoxidation by wcess rcductant (glucow osidasc or NADH) could br caused bv rtlduction of XH., thus prewnting tht: iliitial E-I. abstraction from thcb oc-carbon of tlw s\lhstratt>. Involvcmtwt 01’ a flavin in tlicl r+ action is suggested Kay a significant (thrt:cxfold) 1~hD rtlharwtnent of N-I ,sidation n-h1 Gthcr ascorbic acid (Tablt~ ITT i or
FIG. 4. Proposed fatty acids.
mechanism
for ~-oxidation
of
156
SHINE
AND
NADH was the added reductant. Furthermore, the decreased dependence on an external reductant with increasing purification is consistent with involvement of a flavoprotein whose active state, the semiquinone, shows a high degree of acceptor specificity (32). Although we have previously shown that EDTA inhibits a-oxidation (9), as have others (26), in the present study we observed that more highly purified enzyme preparations were either unaffected or only slightly inhibited by 3 mM EDTA. We have also observed that in contrast to a stimulation reported by others (26), Mg2+ appeared to reverse inhibition caused by high concentrations of peanut protein, but was inhibitory at lower protein concentrations. Further studies will be necessary, however, in order to demonstrate conclusively whether a Aavoprotein is involved in a-oxidation. An oxygenated flavin has also been postulated to be an intermediate in the hydroxylation of aromatic substrates by flavoproteins such as p-hydroxybenzoate hydroxylase (33). Our results obtained with ascorbate as a reductant (Table III) bear some resemblance to the nonenzymatic riboflavinH202-Cu2+-ascorbate system described by Vorhaben and Steele (24), which hydroxylates aromatic substrates. Our scheme also has some similarities with the scheme proposed for aryl-hydroxylations by Howes and Steele (34) ; however, we found no evidence that a-oxidation is inhibited by superoxide dismutase. This would suggest that superoxide anions (or perhydroxyl radicals) involved in a-oxidation are formed at or near the site of n-2-hydroperoxy-16:0 formation. We believe that our proposal presents a unifying picture of a-oxidation which may elucidate not only the a-oxidation of fatty acid in plant systems but also similar reactions in animal systems. Further work is now under way to define precisely the presence and role of a flavin in the plant LYoxidative systems. ACKNOWLEDGMENTS We thank Dr. A. L. Tappel for the sample of rat liver glutathione peroxidase. We also acknowledge
STUMPF the helpful evaluation
criticism of .Dr. Lloyd Ingraham of proposed mechanisms.
in the
REFERENCES 1. STUMPF, P. K. (1956) J. Biol. Chem. 223,643. 2. MEND, J. F., AND LEVIS, G. M. (1963) J. Biol. Chem. 238, 1634. 3. HITCHCOCK, C., AND JAMES, A. T. (1964) Biothem. J. 93, 22~. 4. HITCHCOCK, C., MORRIS, L. J., AND JAMES, A. T. (1968) Eur. J. Biochem. 3,473. 5. HITCHCOCK, C., MORRIS, L. J., AND JAMES, A. T. (1968) Eur. J. Biochem. 3,419 (1966) Bio6. HITCHCOCK, C.,~ND JAMES, A.T. chim. Biophys. Acta 116, 413. 7. HITCHCOCK, C., AND ROSE, A. (1971) Biochem. J. 126, 1155. 8. MARTIN, R. O., AND STUMPF, P. K. (1959) J. Biol. Chem. 234, 2548. AND H.~M9. MARKOVETZ, A.J., STUMPF, P.K., MARSTReM, S. (1972) Lipids 7, 159. 10. LOOMIS,W. D., ANDBATThILE, J. (1966) Phytochemistry 6, 423. 11. ZELITCH, I. (1955) i?~ Methods in Enzymology (Colowick, S. P., and Kaplan, N. 0. eds.), Vol. 1, p. 528, Academic Press, New York. 12. LATZKO, E., AND GIBBS, M. (1972) in Methods in Enzymology (San Pietro, A., ed.), Vol. 24, Part B, p. 261, Academic Press, New York. 13. MILLS, G. C. (1960) Arch. Biochem. Biophys. 86,1. 14. LITTLE,C.,ANDO'BRIEN, P.J. (1968) Biochem. Biophys. Res. Commun. 31, 145. 15. LITTLE, C., OLINESCU, R., REID, K. G., AND O’BRIEN, P. J. (1970) J. BioZ. Chem. 246, 3632. 16. LORD, J. M., KAGAWA, T., AND BEEVERS, H. (1972) Proc. Nat. Acad. Sci. USA 69, 2429. 17. ANNETT, R. G., .~ND STUMPF, P. K. (1972) Anal. Biochem. 4’7, 638. 18. BE~UCHAMP, C., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276. 19. HITCHCOCK,C., AND MORRIS, L.J. (1970) Eur. J. Biochem. 17, 39. 20. MORRIS, L. J., AND HITCHCOCK,C. (1968) Eur. J. Biochem. 4, 146. 21. HAMMARSTR~M, S. (1969) Fed. Eur. Biochem. sot. L&t. 6, 192. 22. ZELITC!H, I., AND OCHOA, S. (1953) J. Biol. Chem. 201, 707. 23. HRYCAY, E. G., AND O’BRIEN, P. J. (1971) Arch. B&hem. Biophys. 147, 14. 24. VORH.~BEN, J. E., END STEELE, R. H. (1967) Biochemistry 6, 1404.
PLANT
cr-OSII)4’I’lO?;
25. TOLI~~HT, N. E., personal communication. 26. H~sHI,M., G-D KISHIMOTO, ‘I'. (1973) J. Bid. C’hw. 248, 4123. 27. I,EVIS, (:. M., AND MEAD, J. F. (196-k) J. f&l. C’hcm. 239, ii. 28. Ts.11, S.-<‘., STEINIIWG, I)., AVIG:IX, J., ML) F.iI.k.s, II. M. (1973) J. Biol. Chenz. 243,109l.
47,185. 30. O’Rmm, I’. J. (1969) CCW. J. Biochem. Iii+ 31. CHRISTOPHEKSEN, B. 0. (1968) Hiochim. phi/s. Acta 164, 35. 32. I)rsos, M. (1971) Wzwhim. Biophys. ilctu 226,
2!).
31. Howb:s,
liaHl.riM.t,
165, 361.
Y.
(1973)
Arch.
Riochem.
Biophys.
269.
33. ~I’E:CTOl~, T., .\ND bl\ssw, Chem. t’orrrm.
247,
\‘. (1972) ./. /{;
5632.
II. M., .IND STEELE, It. H. (1972) li’tas. C’henc. Pathol.
Pharmacol.
3, S-19.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
162, 147-157 (1974)
Fat Metabolism
in Higher
Recent Studies on Plant a-Oxidation w. E. SHINE Department
of Biochemistry
AND
and Biophysics, Received
P.
University November
I<.
Plants Systems’* 2
STUMPI; of California,
Davis,
Calijornia
95616
14, 1973
Both the seed and the leaf a-oxidation systems of higher plants require for activity a reductant and molecular oxygen. The reductant may be NADH or a coupled glucose + glucose oxidase system. D-2-Hydroxypalmitate is the only 2-hydroxy acid which is formed from palmitic acid under a variety of conditions; L-2-Hydroxypalmitate is not formed, but when added does inhibit the initial attack of the a-oxidative enzyme(s) on palmitate. Since the addition of glutathione + gluthathione peroxidase greatly increases the formation of DZhydroxypalmitate from palmitate, with a COW comitant decrease in COZ, it is strongly suggested that the intermediate ina-oxidation is n%hydroperoxypalmitate, which may thenundergo decarboxylation to a pentadecanal and CO2 or be reduced to D-2-hydroxypalmitate. Evidence in support of this hypothesis is presented.
Previous papers in this series described an a-oxidation system in germinating seed tissue that formed CO2 and a C,-l fatty acid in the presence of a hydrogen peroxide generating syst’em and KAD+. Later work revealed that a C,-1 long chain aldehyde is an intermediate (1). More recently, Mead and Levis, with brain tissue (a), and Hitchcock and James, with pea leaf extract.s (3), report’ed that ol-hydroxy fatty acids were intermediat’es in the a-oxidation of fatty acids. The latter \I-orkers made the important observation that the accumulating a-hydroxy fatty acid has the n-configuration (4). Based on isotopic competition experiments, Hitchcock, Morris, and James (5) suggested, however, that L-2-hydroxy fatty acid was the transitory intermediate in the a-degradation of a fabty acid to COz and a C,-1 fatty acid. They further reported that the pea leaf system required only molecular 1 This is paper LVIII in a series. Paper LVIl appeared in Plant Physiol. (1973) 62, 1X-161. Paper LTX is the following paper (Arch. Biochem. Bio. phys. 162,158-165,1974). 2 This research was supported in pa,rt by NSF grant 37880X. @ 1974 by .4cademic Press, of rearoduction in any form
Inc. reserved.
MATERIALS
AND
METHOD::
Materials. [l-%]16:01 (55 Ci,/mole) was purchased from Amersham/Searle. The Hitchcock and James method (6) was used for the preparat,ion of DL-2-hydroxy[l-W]16:0. After preparation of the methyl ester, separation of the D- and I,-isomers was accomplished by preparative thin-layer chromatography of the I,-aretylmandelates, similar to the method of Hitchcock and Rose (71, losing 1 solvent passes followed by elution and rerhromatography of individual spots. PreparaGon of A*-[l‘*C]16:1 utilized a method similar to that rlsed for 2-hydroxy-16:O synthesis, exrept, that (-btlt.anol 3 Abbreviations: 16:0, palmit,ate; ~~-16: 1, 2. hexaderenoic acid; 20H-lS:O, %hydroxypalmitat)e; ROOH, organic peroxide; glc, gas liquid chromatography.
147 Copyright All rights
oxygen, in contrast’ to the peanut system which required a HzOz generating system. In this paper, we will present data which demonstrate that both the pea leaf and germinating peanut seed a-oxidation systems have identical properties, that n-2-hydroperoxypalmitate, rather than L-2-hydrox\-palmitate, is the probable intermediate in cY-oxidation of palmitate, and t’hat L-Zhydroxypalmitatr is never formed.
148
SHINE
AND
saturated with KOH instead of 1 N NaOH was utilized for hydrolysis, with final purification by preparative gas-liquid chromatography (glc). Methyl esters were prepared with diazomethane. Glucose oxidase and purified glucose oxidase were purchased from Worthington Biochemical Corp. Catalase was from Schwarz/Mann, and horseradish peroxidase (type I) was from Sigma Chemical Company. Bovine erythrocyte superoxide dismutase was from Truett Laboratories, Dallas, Texas and had an activity of about 13,000 units/mg protein. Bio-Gel P-10 (100-200 mesh) was from Bio-Rad Laboratories, Richmond, CA. Enzymes. Peanut acetone powders, prepared as described previously (8), were extracted with 25 mM potassium phosphate buffer, pH 7.5, containing 0.170 lubrol (9), for 30 min at 8°C with stirring. Washed Polyclar AT (10) was added to the suspension which was stirred briefly, squeezed through bolting silk, and the extract was centrifuged at 30,OOOgfor 20 min. The supernatant was brought to 25yo saturation with solid ammonium sulfate, centrifuged as above, and the supernatant brought to 65yo ammonium sulfate saturation. After centrifuging, the pellet was redissolved in 25 mM potassium phosphate buffer, pH 7.5, and desalted by passing through a Bio-Gel P-10 column equilibrated with the same buffer. In some preparations, the eluate was centrifuged at 105,OOOgfor 120 min and the supernatant used for assays. Pea leaf acetone powders and extracts were prepared by the method of Hitchcock and James (6). Extracts were prepared with 200 mM potassium phosphate buffer, pH 7.0, immediately before use in assays (5). For some assays these extracts also were (a) dialyzed for 23 hr against the same buffer at 4°C or (b) centrifuged at 30,OOOgfor 20 min, the pellet resuspended in the same buffer, brought to 657, saturation with solid ammonium sulfate, centrifuged, and the pellet redissolved, and finally desalted on a Bio-Gel P-10 column. Glycolic oxidase was isolated from spinach leaves by the methods of Zelich (11) through the ethanol precipitation step. The precipitate was dissolved in 25 mM potassium phosphate buffer, pH 8.0. The preparation was assayed by the 2,6-dichlorophenolindophenol reduction procedure (12). Glutathione peroxidase was prepared from beef liver by a combination of the methods of Mills (13) and Little and O’Brien (14). The procedure was carried out at 4°C. Beef liver (270 g) was homogenized in 0.15 N NaCl (500 ml). This homogenate was then treated with ethanol (1,200 ml) and centrifuged, the resulting pellet extracted with Tris-EDTA-pmercaptoethanol (100 mM Tris, 5 mM NaaEDTA, 2 mM p-mercaptoethanol, adjusted to pH 7.3) solution and centrifuged. The supernatant was then
STUMPF treated with an equal volume of ethano~chlorof,orm (400:70) and centrifuged. The resulting supernatant was treated with 34 vol of acetone, centrifuged, and the pellet dried under a stream of Nz. The pellet was suspended in 100 mM potassium phosphate buffer, pH 7.5, centrifuged, and t,he supernatant brought to 65$& saturation with solid ammonium sulfate and centrifuged. The pellet was dissolved in 25 mM potassium phosphate buffer, pH 7.5, and desalted on a Bio-Gel P-10 column. In some assays, rat liver glutathione peroxidase, partially purified by DEAE-cellulose chromatography, was utilized. Both the beef liver and the rat liver glutathione peroxidase preparations were assayed by the cumene peroxide assay (15). Differential and sucrose density gradiejzt ce,ltrifugation. Fourteen-day-old pea shoots (100 g) or lo-day-old peanut cotyledons (190 g) were gently chopped in a blender with 150 ml of isolation medium containing 600 Z~.M sorbitol, 1 mM MgC12, 1% sodium ascorbate, end 100 mM potassium phosphate buffer, pH 7.5. After removing the larger pieces of cellular material by filtering through 2 layers of Miracloth, the suspension was successively centrifuged at 120g for 20 min, 9,OOOgfor 20 min, 34,000g for 25 min, and 105,OOOgfor 180 min. The pellets were resuspended in isolation medium. Then 2.5 ml of the resuspended 1209, 9,000g and 34,000g pellets were layered on top of sucrose density gradients (16) consisting of 5 ml of 6097, sucrose cushion, 20 ml of 6&32yo sucrose gradient, and topped with 5 ml of 20% sucrose (w/w). After centrifuging at 20,000 rpm for 3% hr in a SW 27 rotor, tubes were punctured, and fractions were collected. Assays. Assays for a-oxidation activity were carried out in 13 X 45 mm glass test tubes. Assays carried out in No. 1 hollow polyethylene stoppers gave spurious results because of nonenzymatic dehydration of 2-hydroxy-16:O to 16:1 and therefore were not used. A benzene solution of the substrate was added to each glass tube and the benzene evaporated. To each tube was added the standard reaction mixture which contained 12 ~1 of 100 mM glucose, peanut enzyme, glucose oxidase, and 25 mM potassium phosphate buffer, pH 7.5, in a total volume of 0.1 ml; tubes were maintained at 4°C during these additions. After the final addition, tubes were stoppered with rubber caps into which a Kontes plastic center well containing 0.1 ml of Packard Hyamine hydroxide (1 M) was inserted. The reaction mixture was shaken at 26°C for 40 min (under normal laboratory lighting). The reaction was then terminated by injecting 40 ~1 of 0.1 N NC1 and followed by an additional 15.min incubation. The contents of the center well were analyzed for 14C02 using a nonaqueous fluor (0.42 g 1,4-bis12-
I-l!1
PLANT a-OXIUATIO;“; (5.phenyloxaeolyl)I-benzene, 5.1 g 2,5-diphenyloxazole per liter of toluene) and a Beckman LS-230 liquid scintillation spectrometer. The reaction mixtuc was ext,racted with 2 X 1 ml of diethyl ether with added 2-hydroxy-16:O rarrier, evaporated to dryness under it stream of Ns, and methylated with diazomethane. Met,hyl esters, in benzene sollltion, were analyzed on a 4.7 mm X 0.6 m stainless steel c*olllmn packed with 15’:; HI-EFF-2BP on Chromosorb W (80,900 mesh) in a Varian (A!IOP) glc emplovin$ :L t,hermal conductivity detect or coupled to a xuclear-Chicago Biospan (G)!M) r:hdioactivity detector. Methyl esters were tre:tted with I,-(-)-menthylchloroformate reagent (17 I when analysis ol’ w and l~-2-h?-tlroXp[l-14C]16:U was desired. The reaction mixture for t,he pea leaf system cnorltailled sllbstratr, Tween 20 (O.O1yO),50 ~1 of enzyme extract,, and 200 m&t potassium phosphate buf’fer, pH 5.0, in a total volume of 0.1 ml. After inc\lb:tting al 26°C for 30 min under aerobic ronditions, JObI of 1 N IICl was injected and the reaction prodl~(*ts were analyzed as described above. Superoxide dismrltase was assayed by the nit ro bllt~ tetrazolillm spectrophotometric method, in whic*h the srlperoxide anion reduces nitro blue tetr:tzoli\ml (18). RESULTS In 195’3, Martin and Stumpf (8) proposed a mechanism to c>xplain the ol-oxidat’ive degradation of free fatty acids. The mechanism involvod a direct, clectrophillic attack on the woarbon of a fatty acid by a perferryl cation with a subscqwnt8 release of CO2 and the format,ion of a long chain aldehyde containing ow 1~~scarbon. The long chain aldehyde was thchn oxidized by KAD+ and a long chain aldrhydc dehydrogenasr. In 1964, Hitchcock and James (3) made the important,
observation
actiw
that, in pea leaf extracts
cr-oxidation system degraded fatty acids to CO, and a C,-1 fat’ty acid, but that w2-hydroxy fatty acid (4) also accumulated. Bawd on compct,ition c>xperimcnt)s (5) with both II- and L-2-hydroxy-16:O and [l-‘“Cl16:O in which that D-%-hydroxy-1B:O caused slight inhibition of 14COsformation, whereas that L-%-hydroxj.-16:O resulted in a considwablr inhibition of WOS formation, Hitchcock and Morris (19) proposed that th(J substrate, I(i:O, was hydroxylated to both th(l D- and th(l L-isomers of 2-hydroxylG:O, the wisomor was slowly dcgradcd to ~1
CC& arid pmtadecaxd, whilv thv I,-isonwr was so rapidly degraded to thcl pc~ntad(~c*:1noic acid that it ncwr accumulat~c~d.Klegani, exprrim&s by Morris and Hitchcock (I!), ZO), thmploying
L-[~H]~G:O
and
~-I”H]l(i:0
as substrates and tracing the labc.1 thro11gh the D-‘&hydroxy-l6:O and finally 10 pc’Jlt:ldwanal, supportc~d thcbir nwchan1s111t’nll~,.
Abseuce of r,-~-h!~rl/,o.c~!/paln~itate ,ffwu/otiorl. In contrast, t(J the data of Hit,cahcaoc*katd Row (i) \vho sho\vcid ‘-3 5; of L-2-lluhY)s~1B:O awmiulating, bawd on their I:-:u*cWlmandelyl hydroq. fatty acid prowdurc~ ior the> separat,ion of’ D- and I,-isonl~%, :\I:\&()vetz et al. (9) was not abl(b to show th(j appearanw of L-Zhydrox!, fatty :t&l VIIIploying the L-n1c~nthylchloroforn1nt (h ~)roeedure of Hammarstrijm (21). In rj~~lc~ tc examine thcscb rwults mow closc~ly, irlwcsusing amounts of DL-2.hydrosy-I (i:O \v(w’ added to a wac.tion 1nixturc with (1-iV’l 1(;:(I as subst)rate. As sho\vn in lcig. I, ait bough the pool size of nonradioactivc~ DI,-L’-hycirc~sy1G:O was incwawd from 0 to 0.1 rimc~lc~\\ it11 2.4 Iimolc+
although
of (I-‘V]lti:O
thrw
as substrate*,
was markcld inhibition
14COs forniation, the formation droxy-I ti: 0 \V:LS simultaneously
arid
01
of ~)-L’-liygr’cbatI!- I’(%-
duced \vith 110 appcwancc of r,-~-h~~tlrox~~[1-L4CJ1B:0. If that c-ornpc+it.ion c~xpc%mc~nt: of Hit,chrock and .Jamw invoked tl1ct cliluI,-l)-ll~.droxytion of 110~1~ synthcsizcd [l-14C]1A:0 with rlc)1vadioact,ivc, L-2hydrosyl(i:O with a c*oncomitant dc,crclascL in 11(‘( ):! thrn Id-Zhydroxy-lfi: 0 should havcb li)c~~ dctrrtcd in this wp(~rinwnt. Indwd, ~Iarko vetz et al. shohvchd in l!R? (9) that inc*rcGrt~ the col~c~ntration of L-?-hydroxy-l(i:O ill :
reaction rnixtuw containing [I -14C’\lO:O, w duwd that synthesis of n-2.hydrox\.-1 ri:O a: ~~11 as the cwjlution of “CN2, \\-hcww n-Zhydroxy-16: 0 had only a slight c#t’wt OI t>hc reaction. Th(w rwults strongly ~uggw that r,-‘2.hydroxy-16: 0 bchavw as a11i1rhil)i
150
SHINE
RADIO-GLC
Substrate: +ZDL-OHl6:O: Products, ID-OHII-%]U:O:
2.4 nmoles[l~‘CJl&0 0 nmoles O.C+nmoles
To?: O.IPnmoks 1
MASSwith
AND
PATTERNS
: O.OSnmoles
:O.l nmoles
: hOlrmoles
: o.o2mloks
: O.?7lnmoles
: 008 m&s
carrier2DL-OHNO
FIG. 1. Effect of unlabeled 2-hydroxy-16:O on pea or-oxidation. Reaction mixtures contained the indicated substrates, 50 ~1 of pea extract, and a total of 20 pmoles of potassium phosphate buffer, pH 7.0, in a total volume of 0.1 ml. Samples were incubated at 26°C for 30 min. “C peaks match the derivative D-2-OH-16:0 menthylchloroformate mass peaks.
tor at the initial step in the reaction of 16:0 with the a-oxidation system, whereas D-2hydroxy-16:O has little if any effect on the initial attack. In summary, these experiments suggest that L-2-hydroxy-16:0 is an inhibitor in the initial reaction of substrate with the cyoxidation enzymes and not a product of a-oxidation of 16:0 where D-2-hydroxy16:0 normally accumulates. However, since addition of D-2-hydroxy-16:0 had little effect on 14C02 release, clearly the n-2-hydroxy16:0 could be excluded as a required intermediate for a-oxidation. This of course assumes that added n-2-hydroxypalmitate equilibrates with any enzyme-bound D-2hydroxypalmitate which might be produced. Rate of oxidation of proposed intermediates. If L-2-hydroxy-16:0 were an intermediate in the conversion of 16:0 to COz and pentadecanoic acid, an important requirement would be that its rate of oxidation would be at least as large as that of 16:0, the initial
STUMPF
substrate. As summarized in Table I, and repeated a number of times, [1-J4C]16:0 is the most effective substrate, with L-2-hydroxy-16:O at the same concentration being about 75% as effective and D-2-hydroxy16:0 about 25%. On the other hand, L-2hydroxy-16:O is the most effective substrate for spinach glycolate oxidase (Table I), with n-2-hydroxy-16:0 being about 35% and 16:0 about 15 % as effective. These results would suggest that L-2-hydroxy-16:0 is not an intermediate in the a-oxidation of palmitate and that other enzymes such as endogeneous glycolate oxidase could contribute to 2-hydroxy-16: 0 decarboxylation. Role of glucose oxidase with both the peanut and the pea leaf a-oxidation system. The peanut or-oxidation system required the glucose oxidase “peroxide” generating system for CO2 formation from 16:0 as well as from 2-hydroxy-16:O. In contrast, the pea system appeared to require only molecular OZ. Figure 2 shows that with both the pea and the from peanut systems, 14C02 formation [1J4C]16:0 peaked at a level of 2.6 X 10e3 units of glucose oxidase activity; glc analysis indicated a similar pattern for D-2-hydroxy[l-14C]16:0 formation, although the amount TABLE ENZYMATIC
I
DECARBOXYLATION SUBSTRATES~
OF VARIOUS
Peanut a-oxidation system
Spinach glycolate oxidase
Substrate*
nmoles Substrate _ Palmitate n-2-hydroxy-16:O L-2-hydroxy-16:0 nL-2-hydroxy -16:( 3
COz
nmoles Substrate
COz
0.50 0.50 0.50 -
0.004 0.009 0.024 -
__~ 0.25 0.25 0.25 0.50
0.104 0.027 0.071 0.095
0 The reaction mixture contained substrate, a total of 2.5 Mmoles potassium phosphate buffer, pH 7.5, 1.2 rmoles glucose, and (a) 20 ~1 of peanut enzyme and 0.07 IU glucose oxidase, or (b) 40 ~1 spinach enzyme and 0.02 IU glucose oxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min (peanut,) or 80 min (spinach). * All substrates were carboxyl-14C labeled.
I Al
PLANT (r-0XII)ATIOS
dophenol + &colic acid systm ~~wmd
~10. 2. Effect of glucose oxidase additions on a-oxidation. Reaction mixtures contained 1.2 @moles of glucose and (a) 0.67 nmoles of substrate, 10 ~1 of (NH&SO4-fractionated peanut enzyme, and a t,otal of 2.5 pmoles of potassium phosphate buffer, pH 7.5, or (b) 1.0 nmole substrate, 50 ~1 of (NH&SOr-fractionated pea enzyme, and a total of 20 rmoles of potassium phosphate buffer, pH 7.0, as well as the indicated amounts of glucose oxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 30 min.
VW always much less than the amount of 14CO2 formed. li’ormat’ion of 14C02 from m-%-hydroxy-16:O was increased by glucose oxidase addit’ions in both plant systems. ,4t lower glucose oxidase levels, much more 14C02was formed from [l-14C]16:0 than from m-2-hydroxy[l-*4C]16:0. These experiments clearly indicated that’, alt’hough earlier reports suggest#ed a major difference between the seed and the leaf system in that the former required a Hz02 generating system while the latt’er required molecular oxygen, both systems responded in a very similar manner when the concent’ration of glucose oxidase + glucose was carefully monitored. These results were further substantiated when it was observed t,hat the pea leaf extracts of Hitchcock and James contained a heat-stable component which rapidly reduced 2,6-dichlorophenolindophenol. Moreovw, glycolic acid oxidase which was normally assayed by the 2, B-dichlorophenolin-
in their preparations. Finally, lvhcn lwa leaf preparat#ious, obtained according t,o Hitchcock (5, Ci), \vcr(’ first dialyzed t(J rcmc~ve the heat-stable compon&, only :I ~~11 amount of 14C02 was now form(~tl from [I-W]l(i:O; however, this activit!. \\-a.< inweawl six- and tenfold in the prt~senw of glucose + glucose oxidase or O..i ml1 glycolate, rcspwtively. Glucose osidnw plus glucose also increased activity in :L pea leaf preparation which was partially purified by ammonium sulfatc~ fractionation (E’ig. 2). It is now clear that cndogeneous gl~c~olic oxidase which has been repeatedly c1t.tIIIJIlstrated in leaf extracts by a munlw (Jf workers (22) is responsible for the convwsion of L-a-hydroxy-16:O to CO, and a C,,.~, fut’ty acid. The addition of a H&g~wrnting system such as glucose oxidaae + glucose increased thr rate of YQ wleastld i’n~nl L-2-hydroxy-16:O a2xhas been r’c~portc~tlIJ~ a number of workrrs for short chain t’:rtty acidsL (““) -- . l@‘ecl oJ’ phtathione
peroxidase
OH pro/l l&s
of reactiom. The possibility that :I h!droperoxy function \\‘as involved in a-oxidation was investigated. Neither c:at:dasc nor horseradish peroxidase increased the r:ltio of 2-hydroxy[l-14C]16:0 to ‘“CO2 fornwd from [ 1-14C]16: 0 (Table II) ; nssayx rcpea t tad in the prewnce c~f catalase indicated no In contrast, glutathione 1~111s inhibition. glutat8hione peroxidase caused a lurgc’ increase in u-2-hydroxy[l-W]lG:O ant1 a marked decwase in W02 (Tablrl I I ). This striking effe& of glut,athiow p(wJsidase, \vhich caatalyzes the reduction of pc’roxides, as s~o-c\-I~ in l:ig. 3, \vas invrstigated in more detail. l;igure 3 shows th:lt I)-(,)hydroxy[l-W]lG: 0 increased and l’C’( j2 dccreased with increasing glutathionc pm~sidase lcvcls. Glutathionc pcroxidaw in tlw a,bsencc of glutat8hiont: as w~>ll as glutat hionr~ (2 mhr) in the abscncc of glutathionr, l)fwG dase had no effect. Since moat, of thr! CC), produwd (luring woxidstion has hw11 prwiously postulated (4) to lw dcriwd from L-2-hydroxy-l(i:O, use of glutathiotw plus glutathiono pwoxidasr should haw rwulted in a large incwasr in the L-isomer. Xt a lewl of glut;~thiom~ peroxidaw which caused 50 9; inhibit iI JII (it’
152
SHINE TABLE
EFFECT OF PALMITATE
AND
STUMPF
II
PEROXIDMES ON 2-HYDROXY AND CO? FORMATION FROM PALMITATV
Peroxidase added
nmoles Product 2.hy droxy 16:0
Ratio 2-OH16:O/CO2
co2 ‘_
-_
None Catalase Horseradish peroxidase Glutathione peroxidase + glutathione
0.04 0.02 0.04
0.36 0.29 0.35
0.1 0.1 0.1
0.13
0.13
1.0
I
a Reaction mixtures contained 1 nmole [l-W]16:0, a total of 2.5 pmoles potassium phosphate buffer, pH 7.5, 1.2 @moles glucose, 0.01 IU glucose oxidase, 20 ~1 peanut enzyme, and, as indicated, catalase (150 units), horseradish peroxidase (0.3 purpurogallin units), and glutathione peroxidase plus 0.2 Imoles glutathione in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min.
I 10
I 5
0
Glutathione
I 15
I 20
peroxidase
(h)
FIG. 3. Effect of glutathione tions on a-oxidation. ROOH
+ 2 GSH
peroxidase
addi-
GSH peroxidase ROH
14C02 formation as well as a 250% increase in 2-hydroxy[l-‘4C]16:0 only D-2-hydroxy16:0 accumulated. We suggest therefore that n-2-hydroperoxy-16:O is being formed and the glutathione peroxidase is reducing this product to n-2-hydroxy-16:O. Role of reductants in the wosidation process. No inhibition of a-oxidation was observed by carbon monoxide nor did p-hydroxymercuribenzoate treatment increase the 2-hydroxypalmitate/COZ ratio, thus suggesting the absence of cytochrome P-450 hydroxylation and of cytochrome P-450 (P-420) type peroxidase (23) activity. Also, neither methyl palmitate nor A2-16:l were active substrates. In addition to the glutathione peroxidase-glucose oxidase system described above, other generating systems were utilized in an attempt to increase the ratio of 2-hydroxy-16:O to COZ formed from 16:0. When ascorbate plus FAD was incubated with the peanut extract (conditions were not optimized), SO2 was formed from [lJ4C]16:0 (Table III) but little if any 2-hydroxy[l-14C]16:0 was detected. An equal amount of H20z was only one-third as effective as ascorbate, although when added together their effect was synergistic. These
, 25
+ GSSG + Hz0
Reaction mixtures contained 1.1 nmoles of [l-W]16:0, 0.2 pmoles of glutathione, 1.2 pmoles of glucose, 10 ~1 of glucose oxidase, 20 ~1 of peanut enzyme, a total of 2.5 pmoles of potassium phosphate buffer, pH 7.5, and the indicated amounts of glutathione peroxidase in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min. TABLE
III
DECARBOXYLATION OF P~LMIT~TE BY PEANUT ENZYME AND ASCORB~TE PLUS FAD5 Additions
CO2 (nmoles)
Ascorbate + FAD Ascorbate + FAD + boiled enzyme Ascorbate + FAD + enzyme Ascorbate + enzyme FAD + enzyme Ascorbate + FAD + enzyme + HzOz FAD + enzyme + H202
0.003 0.002 0.035 0.013 0.002 0.074 0.013
D Reaction mixtures contained 1 nmole [l-‘“Cl16:0, a total of 2.5 rmoles potassium phosphate buffer, pH 7.5, and, as indicated, 0.1 pmole ascorbate, 0.012 pmoles FAD, 0.1 pmole HsOz, and 20 ~1 of peanut enzyme in a total volume of 0.1 ml. Samples were incubated at 26°C for 40 min.
PLANT
a-OXIl)ATIOX
results are similar to the riboflavin-ascorbatfb-H202 system described by Vorhaben and Steele (fL4) in which a flavin-oxygen udduct was postulated to occur. These results again suggested that 2-hydroxy-1B:O is not an intermrdiatc in a-oxidation. Presumably, 2-hydroxy-16:O was not formed in detwtablc amounts because ascorbate is not a strong rcductant. The results also suggwtc>d that a flavoprotrin could be involved in oc-oxidation. In contrast to the ascorbate results, both Y02 and 2-hvdroxv[l-‘4C]1Ci:0 were formed ., . \vh(an SADH was added to the peanut enZJTl”’ system (Table IV) ; the ammonium sulfate fractionatc>d pea system gave similar rcsultx. Again CO, formation greatly rxccc>dcd 2-hydroxy-16: 0 formation. Of intcwst, however, was the observat’ion that in some aways 14C02 formation exceeded th(l amouny of SADH added (Table IV). At the opt,imum concentration (7 FM) KLIDl’H was as c#fective as NADH. Because previous results had suggested involvcm& of a Aavoprotcin in a-oxidation, the, cbffwt of flavin, with NADH as the added reductant, was tested. Ut.ilizing the optimum conwntration of iYADH (7 PM), addition of FAD (25 PM) resulted in a threefold stimulation of CO2 format’ion, and halfmaxinlal stimulation was observed at 6 ~1~1 I:;\D. 1’AD appclawd to be more effective
SADH added (nmoles)
0.0 0.035 0.070 0.28 0.70 2.x x.4 11.0
Product (nmoles) ~~____~ 2-OH-16:O CO2 0.013 0.010 0.017 0.037 0.035 0.033 0.023 0.027
0.10 0.17 0.22 0.56 0.80 0.70 0.52 0.50
Ratio products/ NADH -__.-~ 2.2 1.8 1.7 1.0 0.22 0.05 0.03
” Ilrat*tion mixtures rontained 2.8 nmoles 11J4C]16:0, a total of 2.5 rmoles potassium phosphate buffer, pH 7.5, and 10 ~1 of peanut enzyme in :a tot’al volume of 0.1 ml. Samples were inellbated al 26°C for 40 min.
I ,-I:;
t,han F51K. Utilizing [U-‘4C]16:0 as sul)strate, there was no evidence that addcxd flavin increased activity by wgwrat iny NADH via thr aldchyde dc~lydrogc,n:tsc,. Thew results suggest,, thcrrfow, that ;t flavoprotein may bc dircct,ly involvc~ti ill woxidation. Dcfinitivc involvtmrnt of :I flavoprotcin must await the purifiwtic)n of the oc-oxidation enzymw. I?/fmrellzdat~ locnfiorr CJ’ a-o.l~itlaficJr/ Wfi/~ity. Both diffcrcntial and swrow dfwit~ gradknt wntrifugat,ion indivatcbd that ( ~v(‘r 80% of th(L pea leaf a-oxidation activity was &her soluhl~~ or nssociatrd lzith :I light microsomal f’rac+ion which could tiot t)(b ptlllrted by wntrifugation at 105,OOO!qFor :< hr. On the other hand, owr X0’; 01’ I 11,. peanut w)tyl(hdotl a-oxidation activity \\.:I:: associatc>d with a heavy microsonlal i’r:rc,tion, 7.i’Z of which could tw p(~llt~t(ql t)! ccwtrifugation at 105,OOOqfor :3 hr. Su(ww density gradicwt wntrifugation itltiiwtc~tl that a srxtll amount of a-oxidation ac.tivit\ leas also associated with a hwviw frac~tiou (dwsity 1.17 g, ml). No oc-oxidat,ion :tchtivit;\. was asswiatctd with chloroplasts, and 1’01bcrt (25) has found I~OIW assoc+atc~d with leaf peroxisr~ttws. :$s has bccrl rvportcyl with th(l lignowric acid a-h;vdrox~l:rtin~ cyst (‘111 (‘X), IY(’ :tlso obswvtld sonw swrow i trhit )ition of woxidation. Ammonium sulfatcb frantic wLt ic)II (Z-37 5, :37-45 “i, and 45-O;i ‘,; ) (pi’ thn high-spwd p(la supcrnatant, followd I)y (1~ salting OII Bio-Ckl I’-10, resultcad in wzynw preparations with somrwhat diffcwtlt pry)I)crtirs; thaw fractions sho\vt>d good tw)xidativc activity in th,, abscww of added r~~tluc:tartt’. Thea Y-37 “; t’ra&on was stinlulattid about’ 23 “; bv. glucost~ plus ghCOSC ()sitl:i,~;c~, Jvhilo the, (Jthcr t\\-o fractions \vw’ irihihitcd in thch prcwnco 01’ this rclduckg sJ,stcattl. .\ diminishw~ depcndwlw on c~xtc~rnal rc~l~~ taut Ivitli increasing purificat~ion hatt pt’(‘viously hwn obswvcd for th(L pcwlut ~,IY~~‘III. Clarification of thaw obwrvatiolls nlust ))(b dofwwd until purificatir)n of th(s tr-osi(l:i.ti,,tr f~rizvmcs is ohtaincd. ~U’up~~widr rlismutase qfect. I)cdttul :~rl> moniutii sulfate, fractions from pw I1savc5 which contained cY-oxidat8ion wc+ivitJ, :LISO containc~tl .+upwosid(~ dismutaw :ic*tivit\. (about 100 units, ring protc~iri). ‘I%~ hi&
154
SHINE
AND STUMPF
speed peanut cotyledon pellet, which contained most of the a-oxidation activity, had only a small amount of superoxide dismutase activity (2 units/mg protein). No correlation was observed between a-oxidation and superoxide dismutase activities. Furthermore, additions of 700 units of bovine erythrocyte superoxide dismutase to assays never inhibited a-oxidation more than 30 %.
The observation that CO, formation occurred more readily from 16:0 than from suggested the either D- or n-2-hydroxy-16:0 possibility that 2-hydroxy-16:O was not an intermediate in COZ formation from 16:O. The rapid decarboxylation of L-2-hydroxy16:0 which was catalyzed by the spinach glycolate oxidase preparation furt’her suggested that glycolic oxidase present in many plant extracts might be responsible for this DISCUSSION decarboxylative activity. Of interest, rat Our results clearly show that in both the liver glycolate oxidase has been observed to peanut and pea leaf systems a-oxidation decarboxylate n-lactate as well as L-lactate required an electron source and molecular (29), as was also observed with D- and L-2oxygen. The a-oxidation activity in the hydroxy-16:O and spinach glycolate oxidase peanut cotyledon, as in the brain (27), is (Table I). associated with a microsomal fraction which In order to determine what intermediate might actually be involved in a-oxidation, can be partially solubilized by employing detergents. Activity from the pea leaf is various enzymes which react with hydroperoxides were tested. Neither catalase nor apparently loosely associated with a light microsomal fraction and readily yields a horseradish peroxidase had any effect. Glutasoluble a-oxidation system. thione peroxidase, however, caused a strikWhen the endogenous cofactors necessary ing decrease in CO:! formation and an infor the pea leaf system were removed by crease in 2-hydroxy-16: 0. These latter results dialysis or ammonium sulfate fractionation, were similar to observations for the reduction the requirement for glycolic acid, glucose of linoleic acid hydroperoxide: namely, (a) plus glucose oxidase, or NADH could be linoleic hydroperoxide was not decomposed demonstrated. Moreover, both the peanut by catalase or horseradish peroxidase (30), and pea leaf extracts showed the same re- and (b) it was reduced to the corresponding sponse to increasing levels of glucose oxidase, hydroxy fatty acid by glutathione peroxii.e., an increase in the glucose oxidase level dase (14, 31). Therefore, the results strongly resulted in an activity peak when 16:0 was suggest that 2-hydroperoxy-16:O is an interthe substrate. The peanut extract showed a mediate in a-oxidation of 16:0 to CO* and similar activity peak with increasing levels pentadecanal. This would also explain why little if any 2-hydroxy-16:O was detected of NADH. At high reductant levels significant in- when a-oxidation was carried out in the hibition occurred suggesting that excess presence of the ascorbate plus flavin system; reductant may cause inhibition of 14C02 presumably, the hydroperoxide was not rerelease from palmitate. Similar effects have duced but was undergoing decarboxylation. been observed in other a-oxidation systems. The NADH results further suggest that less For example, formation of cerebronic acid than two electrons are required for the forfrom lignoceric acid by rat brain enzymes mation of each CO2 molecule, since the ratio of nmoles COZ formed to NADH added showed a distinct plateau with increasing NADH levels (26) ; on the other hand, in a approached 2. Because we detected only n-2-hydroxyrat liver mitochondrial system, CO2 formation from ol-hydroxyphytanic acid was in- 16:0 in numerous assays, the report by creased 50 % by NADPH (28). Glucose plus Hitchcock and Rose (7) of the probable glucose oxidase was strongly inhibitory in biosynthesis of the n-isomer in the pea leaf system is difficult to reconcile. Furthermore, the rat brain system. The latter inhibition when may be explained either by the presence of we detected only n-2-hydroxy-16:0 excess reductant or by the generation of assays were carried out in the presence of glutathione peroxidase. Moreover, we were excess H202.
PLANT
or-OSI1).4TIOS
unablts to dt$wt racemization of either isomer in the prcsencr of the peanut cnzymc txtract. In addition, \w d~trctcd no L-Zhydroxy-16:O in tbit’hcr t,he peanut or the pt~r. leaf systenl when various amounts of unlabeled DL-g-hydroxy-16 : 0 wrc added to thts reaction mixture. These results do not thtwfow support thtl assumption of HitchfYJt’k :LIK~blorris (19) that added unlabeled rJ-L’-hytlrosy-1ci:O causes a dccrrasc in latwlt~d C’02 IJ~ diluting the label. Markovet’z rt al. (!I) have px&JUSly reported that added unhtbelt~d L-I’-hydrosy-16:O caused a marktld dtuww ill labeled D-2-hydroxy-16:O as wll :LS in L4COe product~ion, \\hercas D-Z hgdrosy-16:O had little> if any efftct on product formation. Wc, therefore, suggest that thth L-istJln(T is probably an inhibitor of the initial attack of t’he mzynw on the n-t~arbc~n of thcl fatt,!: acid, although it could :dso bo inhibitmg t’he subsequent format ion of oc-hJ,droperoxy fatt,\- acid. ln prtsvious work, \\-e reported t,hat for full activatic 111of tht: swtl system a hydrogenI)c’rositlt~-g~~nt,ratiiig system was an essential compouttnt of thtt a-oxidation process (1). Thus, tlw Fystems, glycolic acid plus glycolic acid cbxidaw, gluwsc~ oxidase plus glucose, L-:unintb wit1 oxidase plus leucine, all of which 1i:iw in c~onimon the formation of H202, wre cffwtive in the cY-oxidation prtwss. Howrvt~r, ivtl had earlier obscrvcd that thtb direct addition of H&z to the 01. oxidation sgstcw in the absenw of these gcnwating syst clns \vas wscntially ineffw tivt:. It is now clt>ar that the important t’OI11n1011 fc~atuw of all these systems is the flavin ct ,mponont of the Aavoprotoin oxidastrs. Thwt~ oxidastw arc’ probably rcsponsik~lv for thtl gcntwtion of an active species of molecular oxygtw required to initiate the ol-oxidativt~ procwx. WC thewforcb propose in l;ig. 4 an a-oxidation schtmle which involves only t’hc Distmcrs, namely D-Z-hydroperoxy-16: 0 and D-L’-h~droxp-1li:O. WC suggest that, a flavoprotein(s) rathw than cytochromr P-450 may btl involved, since we did not observe C’O inhibition of oc-oxidat,ion and added flavin incrrhascd product’ formation significant81y. Mhcrs have also reported no CO inhibition of cu-oxidation in other systems
1.Y.i
(26). The scheme show a rcquircnwnt ~OI electrons in order to form XH. (e.g., flavin semiquinone) which abstracts Ii,. from thch cu-carbon of the substrattl to form the full! reduced X (XH2) and a free radical, \vhich then re:tcts with the subsequent,ly osygt~natcd XH, to form D-2-hydroprroxy-16:O and thereby wgtwerate XH. . By a t~onwrtt~tl intramolecular ni&2mism, n-%-hydroptt1*oxy-16:0 can decarboxylatc to i’ornl (YJ? and aldchydc. Alternately, tho hydroptw)side cm lw rt~duwd to form r)-Z-hytlrosy16:O.
Tht, schcmc is consistent \vith data rt’ported by others and results prtwntt~tl htw. For cxamplcl, only, the D-isomtxr \v:ts formcld during t ht. Lu-hydroxylation Of lignowric acid (26). In addition, Morris and Hittalrcock (20) reported the loss of slIMrate D-Y-“H and ret&ion of substrate L-‘_‘-~H in w%hydrosy-16:0 which accurnulatrs during a-oxidation. These results are predictctl in our proposed mechanism. They observed a similar patt’ern in the pent’adccanal t’c)rmod by decarboxylation (19). Inhibition ()I CYoxidation by wcess rcductant (glucow osidasc or NADH) could br caused bv rtlduction of XH., thus prewnting tht: iliitial E-I. abstraction from thcb oc-carbon of tlw s\lhstratt>. Involvcmtwt 01’ a flavin in tlicl r+ action is suggested Kay a significant (thrt:cxfold) 1~hD rtlharwtnent of N-I ,sidation n-h1 Gthcr ascorbic acid (Tablt~ ITT i or
FIG. 4. Proposed fatty acids.
mechanism
for ~-oxidation
of
156
SHINE
AND
NADH was the added reductant. Furthermore, the decreased dependence on an external reductant with increasing purification is consistent with involvement of a flavoprotein whose active state, the semiquinone, shows a high degree of acceptor specificity (32). Although we have previously shown that EDTA inhibits a-oxidation (9), as have others (26), in the present study we observed that more highly purified enzyme preparations were either unaffected or only slightly inhibited by 3 mM EDTA. We have also observed that in contrast to a stimulation reported by others (26), Mg2+ appeared to reverse inhibition caused by high concentrations of peanut protein, but was inhibitory at lower protein concentrations. Further studies will be necessary, however, in order to demonstrate conclusively whether a Aavoprotein is involved in a-oxidation. An oxygenated flavin has also been postulated to be an intermediate in the hydroxylation of aromatic substrates by flavoproteins such as p-hydroxybenzoate hydroxylase (33). Our results obtained with ascorbate as a reductant (Table III) bear some resemblance to the nonenzymatic riboflavinH202-Cu2+-ascorbate system described by Vorhaben and Steele (24), which hydroxylates aromatic substrates. Our scheme also has some similarities with the scheme proposed for aryl-hydroxylations by Howes and Steele (34) ; however, we found no evidence that a-oxidation is inhibited by superoxide dismutase. This would suggest that superoxide anions (or perhydroxyl radicals) involved in a-oxidation are formed at or near the site of n-2-hydroperoxy-16:0 formation. We believe that our proposal presents a unifying picture of a-oxidation which may elucidate not only the a-oxidation of fatty acid in plant systems but also similar reactions in animal systems. Further work is now under way to define precisely the presence and role of a flavin in the plant LYoxidative systems. ACKNOWLEDGMENTS We thank Dr. A. L. Tappel for the sample of rat liver glutathione peroxidase. We also acknowledge
STUMPF the helpful evaluation
criticism of .Dr. Lloyd Ingraham of proposed mechanisms.
in the
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