Fat metabolism in higher plants

Fat metabolism in higher plants

ARCHIVES OF BIOCHEMISTRY AND 117, 604-614 (1966) BIOPHYSICS Fat Metabolism XXXII. Control of Plant DAVID Department of Biochemistry in Highe...

999KB Sizes 13 Downloads 158 Views

ARCHIVES

OF

BIOCHEMISTRY

AND

117, 604-614 (1966)

BIOPHYSICS

Fat Metabolism XXXII. Control

of Plant DAVID

Department

of Biochemistry

in Higher

Acetyl-CoA

BURTON and Biophysics,

Plants

Carboxylase

Activity

AND P. K. STUMPF’ University

of California,

Davis,

California

Received September 23, 1966 Intact lettuce chloroplasts readily utilize acetate-l% for incorporation into long chain fatty acids in the presence of HCOa-, ATP, Mg++, and light. However, disrupted chloroplasts no longer utilize either acetate-i4C or acetyl-CoA-r4C as a substrate. Present investigations show an almost complete absence of acetyl-CoA carboxylase activity in preparations of disrupted chloroplasts. Attempts to stimulate this activity by employing the allosteric effecters of mammalian and yeast acetyl-CoA carboxylase gave negative results. Further study showed the presence of an inhibitor in preparations of disrupted chloroplasts which markedly depressed the activity of wheat germ acetyl-CoA carboxylase. Its general properties are described. In addition, wheat germ acetyl-CoA carboxylase, unlike its counterparts in other tissues, is not stimulated by phosphorylated sugars, dicarboxylic and tricarboxylic acids.

Acetyl-CoA carboxylase is a key enzyme of fatty acid biosynthesis in animals, microorganisms, and plants. Earlier studies in this laboratory on fatty acid biosynthesis in avocado mesocarp preparations indicated that acetyl-CoA carboxylase was absent from a soluble system, which was nevertheless capable of synthesizing long chain fatty acids from malonyl-CoA (1). Similarly, the soluble enzymes of disrupted butter lettuce chloroplasts, although capable of fatty acid synthesis, could not utilise acetate or acetylCoA nearly as efficiently as malonyl-CoA, whereas intact chloroplasts could readily convert acetate to fatty acids (2, 3). These findings suggested that acetyl-CoA carboxylase or acetyl thiokinase, or both, are absent or inactive in such preparations. In animal systems it is well established that fatty acid synthesis is profoundly affected by the levels of certain intermediates of carbohydrate metabolism, notably tricarboxylic acids (4-6). In extracts of rat adipose tissue, fatty acid synthesis is greatly

stimulated by tricarboxylic acid cycle intermediates. Martin and Vagelos have demonstrated that acetyl-CoA carboxylase (7, 8) is activated by such intermediates. Citrate, the most effective compound, causes an accompanying change in the sedimentation coefficient of the enzyme (9). Similar conclusions were reached by Waite and Wakil, who used chicken liver acetyl-CoA carboxylase (lo), and by Lynen et al. (II), who used the rat liver enzyme. More recently, White and Klein (12) have shown that citrate, fructose 1,6-diphosphate, and L-(Yglycerophosphate stimulate fatty acid biosynthesis from acetate in yeast by activating acetyl-CoA carboxylase, while Wakil et al. (13) have shown that the fatty acid-synthesizing systems of pigeon liver and Escherichia coli are stimulated by various phosphorylated sugars and inorganic phosphate, fructose 1,6-diphosphate being the most effective. In the latter case it appears that the site of stimulation is the acetyl-CoA transacylase or condensing enzyme This paper reports the results of investigations into the reasons for the apparent ab-

1 Supported in part by NSF grant GB 2352, and USDA contract 12.14.lOO-7990(74). 604

PLANT

ACETYL-COA

CARBOXYLASE:

of acetyl-CoA carboxylase in preparat ions of broken butter lettuce chloroplasts. l
liSI’EHI~Il~NTrlL

METHODS

Zateriuls. Adenosine triphosphate, GHS, CoA, and 9-palmityl-CoA were purchased from the Sigma Chemical Company. KH”COI was prepared from Ba13C03 whirh was obtained from Oak Ilidge National Laboratories. Malonyl-2-1%~CoA was prepared by the method of Eggerer and Lynen (l-1) starting from malonic acid-2-l%, which was prlrrhased from the New England Nuclear Comp:u>p. Acetyl-1-*GCoA and acet,yl-CoA were prepared by the method of Simon and Shemin (15). All reagents used were of analytical qualitsy. l’rotein was determined by the Biuret method (l(i). Preparution

of

butter

lettuce

chloroplasts

and

chloroplust frucfions. Butter lettuce (Latuca sati~a L.) chloroplasts were prepared by homogenizil~g ZOO-gm portions of washed deveined trlltter lettuce leaves in 250 ml of cold buffer bq for 45 seconds in a chilled Waring blendor. The homogetlute was forced through two layers of cheesecloth alltl then gravity filtered through 4 layers. 1 debris was centrifrlged down at 5009 for 1 minute, and chloroplasts were sedimented at 1OOOgfor 10 minlltes. rlfter washing with 1~4buffer, the chloroplasts were resrrspended iu a suitable volume of buffer b4 so as to obtain a final chlorophyll concentrat,ion of l-3 mg per milliliter. Chloroplast 1)reparations were placed in a chilled AmincoFreltch pressllre cell and extruded at 20,000 pounds I)si. The disrllpted rhloroplast suspension was centrifllged at 2000g for 10 minutes to remove ~IIljroken chloroplasts. The resulting supernatant frac t iorl was termed b/r&r lettuce chloroplast 2 Buffer bq contains the following components: sucrose, 0.5 &I; NaCl, 0.01 -“I; Na-ascorbate, 0.04 hl; K-phosphate, 0.01 >r; EDTA (Na salt), 0.001 M.

ACTIVITY

tioi

To separate the graIla from solrltdf~ enzymes (stroma), the pressate was cclltrifugeti at 105,OOOg for 30 miuut,es. The sol\lble stroma proteins were carefully removed and I hc graua were washed with br#l”er bq, receut rifrlged, :~tltl finally resuspended iu the original volumr of I)ltf’l’rt b4. Iu some of the studies on the inhibition of wlirlat germ acetyl-CoA cnrl)oxylase by cliloroplast :11id chloroplast fractiorrs, preparations were made I)y slrbstitlrting 0.01 1, Tris-TICl, pT1 8.6. colrlninitlg 0.001 br $;I>TA for I)rlft’er b.,, and omit ling t hc washilly of “whole chloroplasts” (see text 1. AftfTl, disrllption and cell trifrlgation at 105,OOOg, a clear brownish-green supernntant fraction was obt:tillrti which was highly inhibitory. pressate.

~repurcction

of rchrut

germ

clcet!/l-(‘0-i

c~rrbo.r!~/-

use. W%eat germ aret y-GA carbosylase was prepared from a11 acetone powder of wheat germ by the method of Hatch and Stumpf (li). The purification was taken as far as the stagy of iscb electric precipitation at pH G.G-6.25. Enzyme assa!y. Scctyl-COB carboxylase was assayed by measuring the acetyl-CoA dependent. inrorporat,ion of KIII”CO, into acid-soluble, 11o11volatile compounds. Reactiou rnixtllres contnilrctl: MgCl,, 5 pmoles; ATP, 2.5 pmoles; acetyl-Co.4. 0.55 @moles; KIIl%O,, 10 pmoles; El)TA, 1 *mole; Tris-HCl buffer, pH 8.6, 5 X 1OF >I final COIIcentration. Enzyme was added in the amolln ts specified in the text, and the tot,al volume of ttrr reaction mixt,ure was 1 ml. Incubatiolis were carried oilt for either 15 or 30 minules at 30”. Reactions were terminated by adding 0.2 1111of glacial acetic acid, :uld aliqrmts of 0.1 ml from cacll reaction were dried 011 2.5 X 7.5-cm strips of filter paper. The dried strips were placed ill scintillation counting vials with 10 ml of s~itltillatiotl fluid containing 0.6’;; 2,5-di-phenylox:tzole and 0.057~ p-bis-2’(5’-phenyloxazolyl) brtlzrue ill toluene, and couut.ed in a Pacnknrtl Tri-Cart) liquid scintillation spectrometer. I
606

BURTON

AND STUMPF

phosphate buffer, pH 8.2, 166 pmoles; ATP, 4 pmoles; MnClZ, 1 hmole; MgC12, 1 &mole; KHi4C03, 30 pmoles (8.1 X lo6 dpm); GSH, 8 pmoles; and acetyl-CoA. 1 Bmole. Reactions were carried out for 90 minutes at 30” in a shaking water bath, and were stopped by adding Dowex 5OW-X8. Counting was performed as described above. The identification of the products of KHi4C03 fixation in chloroplast pressates by paper chromatography is described in the text. RESULTS

AND DISCUSSION

The studies of Brooks and Stumpf (2,3) on fatty acid biosynthesis in intact and broken chloroplast preparations reveal a paradox. Since intact chloroplasts effectively catalyze the incorporation of acetate into long chain fatty acids, acetyl-CoA carboxylase must be fully active. However, in broken chloroplast preparations either acetate or acetyl-CoA was ineffective as precursors of fatty acids. Since, in the course of obtaining broken chloroplast preparations, nothing is removed from the system, experiments were designed to clarify the loss in the capacity of the chloroplast system to employ either acetate or acetyl-CoA as a substrate. Pr&iminary experiments showed that butter lettuce chloroplast pressates were capable of incorporating KH14C03 into acidsoluble nonvolatile compounds, and that this incorporation was increased by the addition of various compounds which have been implicated as allosteric activators of acetylCoA carboxylase in animal and microbial systems (4-13). Table I shows the results of such an experiment. The observation that the KH14C03 incorporation was independent of acetyl-CoA suggested that the fixation product was not the result of acetyl-CoA carboxylase activity, namely, malonyl-CoA, and this was confirmed by paper chromatography. Reaction mixtures in which significant carboxylation above the basal level had occurred, i.e., those containing glucose 6-phosphate, fructose 1,6-diphosphate, fructose 6-phosphate, ribulose 5phosphate, and phosphoenol pyruvate, were chromatographed on Whatman No. 3mm paper in a developing solvent of ethanol- 0.1 M potassium phosphate buffer, pH 4.5. When the chromatograms were examined for radioactivity on a Vanguard scanner, no

radioactivity was found associated with the area occupied by authentic malonyl-CoA. As a further confirmation, portions of the reaction mixtures were treated with a neutral solution of hydroxylamine in ethanol. After centrifugation to remove insoluble salts, the concentrated solutions were chromatographed on Whatman No. 3mm paper in a solvent system of iso-pentanol pyridine water (1: 1: 1). No radioactivity was found in the area of the chromatogram corresponding with authentic malonyl monohydroxamate. Instead, the radioactivity was associated with more polar material. The carboxylation products observed with fructose 1,6-diphosphate and phosphoenol pyruvate as substrates were examined more closely. Both carboxylations were independent of the presence of acetyl-CoA and were avidin-insensitive. With fructose 1,6diphosphate as substrate, carboxylation was dependent on the presence of ATP, but that of phosphoenol pyruvate was not. The products of these carboxylations were identified by paper chromatography. The supernatant solutions from the Dowex-treated reaction mixtures (see Experimental Methods) were lyophilized and the residues were taken up in a small volume of 50 % ethanol. Two-dimensional chromatography was performed on Whatman No. 3mm paper in 80 % phenol-water in the first dimension TABLE

I

INCORPORATION OF KHi4C03 INTO ACID-SOLUBLE, NONVOLATILE COMPOUNDS BY BUTTER LETTUCE CHOROPLAST PRESS~TE” Additions

No acetyl-CoA Acetyl-CoA Citrate + acetyl-CoA Glucose-6-P + acetyl-CoA Fructose-l ,6-di P + acetyl-CoA Fructose-6-P + acetyl-CoA 3-PGA + acetyl-CoA Ribulose-5-P + acetyl-CoA n-or-Glycerophosphate + acetyl-CoA PEP + acetyl-CoA

% of “C /I nm ’ Incorporated ___-

30 4 2 3.2 3 1 3 3

0.12 0.14 0.04 0.33 1.22 0.97 0.20 2.98 0.12 1.28

0 Reaction conditions and assay were as described in Experimental Methods.

PLANT

ACETYL-COA

CARBOXYLASE

and n-butanol-propionic acid-water (1245: 620:574) in the second dimension. After thorough drying, radioautograms were prepared by leaving the chromatograms in contact with x-ray film for one week. On development of the radioautograms only one radioactive spot was det#ected in each case. The area of the paper chromatogram corresponding to the radioactive spot was cut out, and the l”C-labeled compound &ted wit’h water. The product from the fructose 1 ,Gdiphosphate-containing reaction mixture was identified as 3-phosphoglyceric acid. The compound was treated with acid phosphatase for 1 hour at 37” and pH S.2; subsequent, co-chromatography of the acidified reaction mixture with authentic glyceric acid and 3-phosphoglyceric acid showed that, the major portion of the 14Cwas associated wit.h glyceric acid, and that the remaining small presumably unhydrolyzed amount was associated with 3-phosphoglyceric acid. The product from the phosphoenol pyruvate-containing reaction mixture was identified as malic acid by co-chromatography of the material eluted from the t’wo-dimensional chromatogram, with authent#ic malic and fumaric acids, using I-pentanol saturated with 5 M formic acid as t’he solvent system. Radioactivity was associated exclusively with malic acid. Undoubtedly malic acid was formed from oxalncetic acid which was synthesized by carboxylation of phosphoenol pyruvate. This conclusion is strengthened by the observation t,hat treat8ment8 of the reaction mixture with aniline original citrate at acid pH (18) caused the release of 60 ‘X of the total 14C incorporated as 14C02, indicating the presence of a P-keto acid, presumably oxalacetic acid. Since oxalacetic acid is unstable on paper chromatography, it would be lost, and thus not detected with malic acid. These results show that’ t,he chloroplast press&es exhibit negligible acetyl-CoA carboxylase activit,y, but. appear to be capable of performing the normal carboxylation reactions of the reductive carbon cycle. The addition of the phosphorylated compounds listed above provides substrates for the reductive carbon cycle which was depleted

ACTIVITY

during preparation of pressates (20). The pressates also possess a phosphoenol pyruvate carboxylating system, which is independent of ATP and is therefore probably phosphoenol pyruvate carboxylase ratsher than the carboxykinasr. Since all attempt’s to demonstrat’e directly acetyl-CoA carboxylase activit,y in 1he chloroplast pressate mere of a negative nature, experiments were designed to elucidate the lack of carboxylase activity. Table II shows the result of an experiment. in which nucleotides other t’han ATP were used in the reaction mixtures. It is clear from t,hese results that a possible different nucleobide specificity is not. t.he reason for t,hc lack of acetyl CoA caarboxylase activity. Table III summarizes t.he results of MI experiment designed t,o test t,hc possibility that the pressate contained an active malonyl-CoA decarboxylase which would decarboxylate newly synthesized malonylCoA with the subsequent regeneration of xcetyl-CoA. The resnlt)s showed that no malonyl-CoA decarboxylase was present, since no labeled :tcetyl hydroxnmat(1 was detected. However, fret malonic acid was detected in the reaction mixture>. Control experiments indicatccl that at’ zero time, appreciable amounts of fire malonic acid were present in the react,ion mixture. Malonyl-CoA it&f showed no free maIonic* acid. Presumably a hydrolysis of free thioester occurred nonenzymicnlly during tlllc workup of the react ion mixture. :jlthough TABLE

II

EFFECT 0~ DIFFERENT NU~LEOTIDES ON ~ARBOXYLATION IN BUTTER L~rrucr: CHLOROPIAST PRESL\TE~ Nucleotide

ATI’ ATP; no acetyl-CoA No Acet,yl-CoA; no nucleotidc CTP GTP UTP ITP

InP

:'; '"C Incorporated

-I 4 1 4 -I 4

0.22 0.23 0.006 0.0”3 0.017 0.057 0.008

Q Reaction conditions were as described in E’xperimental Methods, except for the modifications shown above.

608

BURTON

AND STUMPF

TABLE III INCUBATION OF MALONYL-~-~~C-COA WITH BUTTER LETTUCE PRESSATE' Acetyi bydroxamate

% of ‘4C resent a4 maPmy1 monohydroxamate

0

0

80

30 60

0 0

67 46

Time of incubation (min)

Ms?c

20 33 54

a Malonyl-2-i4C-CoA was incubated for the periods indicated with pressate in the standard system (using unlabeled KHCOI). Reactions were terminated by the immersion of the tubes in boiling water for 20 seconds. The precipitated protein was removed by centrifugation, and the supernatant fraction was treated with 0.5 ml of 2 M neutral hydroxylamine solution. After reaction the solution was lyophilized, and the residue was taken up in ethanol. After removing insoluble material, the solution was chromatographed together with samples of authentic acetyl hydroxamate, malonyl monohydroxamate, and malonic acid on Whatman No 3mm paper using 1-pentanol saturated with 5 M formic acid as solvent. The chromatograms were scanned for radioactivity which was located in two areas corresponding to malonyl monohydroxamate and malonic acid. The relative amounts of r4C in these areas was estimated by cutting out and weighing the areas under the peaks on the scanner trace.

hydrolysis of malonyl-CoA some enzymic appears to occur, after 60 minutes approximately

50 % of the thioester

sults

also indicate

that

by a component

TABLE IV INCUBATION OF ACETYL-IJ4C-C~A WITH BUTTER LETTUCE PRESSATE dpm recovered under:

still remains.

Table IV lists the results of an experiment to test the presence of an acetyl-CoA deacylase in the pressate. If present, such an enzyme could deacylate the acetyl-CoA added to the system as substrate, thus considerably depressing acetyl-CoA carboxylase activity. As indicated in Table IV, no acetyl-CoA deacylase was detected. The rebound

CoA would proceed better at the lower pH, provided that the enzyme was not inactivated. However, when the chloroplast pressate was incubated with KH14C03 at pH 6.5 the incorporation was considerably lower than at pH 8.2, and was still independent of added acetyl-CoA. The results summarized above indicated that acetyl-CoA carboxylase activity was not detectable in extracts of disrupted chloroplasts. Since a large body of information clearly implicated this enzyme as participating in the incorporation of acetate into long chain fatty acids in intact chloroplasts, another approach to the solution of this puzzle suggested that perhaps an inhibitor was released during the disruption of the chloroplasts which specifically blocked acetyl-CoA carboxylase activity. To investigate this possibility, acetyl-CoA carboxylase prepared from wheat germ was used as a model plant enzyme system. Table V summarizes the results of an experiment in which wheat germ acetyl-Coil carboxylase was assayed in the presence and absence of lettuce chloroplast pressate. The experiment demonstrated that the pressate

acetyl-CoA

is not

of the pressate

making it unavailable to the enzyme. At pH 8.2 at which all the incubations

were run, the 1% in the added KH14C0, could be present predominantly as the HCOSion. At a pH of 6.5 a much larger proportion of the W would be present as 14C02. If CO2 rather than HCO,- was the substrate for lettuce chloroplast acetyl-CoA carboxylase, it is possible that the carboxylation of acetyl-

Acid conditions

Alkaline conditions

of assay

Initial acetyl-CoAJ4C Incubated with pressate Acetyl-1-r*C-CoA was incubated with butter lettuce chloroplast pressate in the standard system (using unlabeled KHC03). At the end of the incubation a 0.2-ml aliquot was taken, and after dilution to 5 ml was centrifuged to remove particulate material. One-ml portions of the supernatant solution were treated with 0.2 ml of either 1 N HCl or 1 N KOH, and O.l-ml aliquots of this were assayed for radioactivity as described in Experimental Methods. Any free acetic acid formed by hydrolysis of acetyl-CoA would have been lost because of its volatility in the isolation procedure. Under alkaline conditions, no loss would occur. Thus the difference in counts is a measure of deacylase activity or binding to protein.

PLANT

ACETYL-COA

CARBOXYLASE

does indeed inhibit the acetyl-CoA carboxylasc, and this observation suggests an explanation for t,he apparent absence of the enzyme in disrupted chloroplast prepara-

ACTIVITY

160 I

t,ions.

Table

VI

shows tbe effects of intact TABLE

1’

EFFECT OF LETTUCE CHLOROPLMT PRESSBTE WHE.~T GERM ACETYL CoA C~RBOXI~LUE mumoles

OS

KH’4C03 fixed

Condition

Pressate alone Carboxylase alone Carboxylase + pressate

34 131 50.5

35 0.3 40

-1 130.7 10.5

were as described in Experimental Methods, and contained 0.8 mg enzyme and pressat,e equivalent, to 0.5 mg chlorophyll. Assay was 30 minutes at 30”. Equivalent amounts of buffer, used to suspend the pressate, were added to those reaction mixtures not containing pressat.e. Reaction

TABLE EVFECTS OF FRACTIONS

VI

CHLOKOPL:\STS AND CHLOROPL.~ST ON WHE.~T GERM ACETYL-COA CARBOXYL~SE mpmoles KHWOa fixed

Carboxylase alone Chloroplasts alone Chloroplasts + carboxylase Pressate alone Pressate + carboxylase Grana alone Grana + carboxylase Stroma alone Stroma + carboxylase

146 21.7 47.7 53.8 91.5 0.6 78.4 70.0 157

0.5 15.5 20.1 52.5 61.7 0.4 0.4 71.3 83

145.5 6.2 27.G 1.3 129.8 0.2 ~ 78.0 l-l.3 74

a Reaction mixtures and incubation for 30 minutes were as described in Experimental Methods and contained 800 pg wheat germ carboxylase. Chloroplasts, pressate, and grana were added in amounts equivalent to 0.5 mg chlorophyll, and stroma was added at the level which would be equivalent to 0.5 mg chlorophyll before separation from grana. Amounts of buffer bd equivalent to that added with chloroplasts, grana, pressate, and stroma were added to the control tubes.

0:1

0’

mixtures

Grano

Added

0:2 (mg

0:3

chlorophyll)

1. Relationship of acetyl-CoA carboxylase and increasing concentration of grana. Reaction conditions as in Experimental Methods except for amount, of enzyme which was 210 pg. One mg of chlorophyll is approximately equivalent to 5 mg protein. Assay time, 30 minut.es. FIG. activity

chloroplasts, chloroplast pressate, washed grana, and pressate supernatant (“stroma”) on wheat germ acetyl-CoA carboxylase. Each of these preparations caused inhibition of the enzyme. Since preparations of grana showed negligible endogenous carboxylating abilit,y, further experiments mere performed in which washed grana was used as a wuw of the inhibitor. Figure 1 relates t#he cffecat of ir,care,sirlg amounts of grana on the carboxylation rea(:tion by a fixed amount of enzyme. A dr:lmatic inhibition is caused by tho grana. Among possible explanations, these cffc& may be relat,ed to a binding of t,ht enzyme, or of one of it,s substrat8es lo the: grana, thus reducing the arnounk ava,ilable for rewt,ion. To test these interpretations, experiments described in Table VII and YITI :IW submitted. Table VII summarizes the effect, of pwincubation of the enzyme with grana as related to the extent of inhibition. The data show that the inhibitory reaction is rapid, as evidenced by the smomlts of T
610

BURTON

AND

tion in the presence and absence of grana. Preincubation caused only a small increase in the extent of inhibition, suggesting that the primary inhibitory act is probably nonenzymic. Table VIII describes the effect on the inhibition by addition of biotin to the reacTABLE

VII

EFFECT OF PREINCUBATION OF GRANA ACETYL-COA CARBOXYLASE GERM EXTENT OF INHIBITION Time of preincubation

AND WHEAT ON THE

(min)

0 5 10 20 30

Enzyme preinc. withoklt grana for 30

34.8 31.6 27.2 19.5 19.5 278.

min

Enzyme not preinc. (no grana)

290.

Reaction mixtures were as described in Erperimental Methods, and cont.ained 2-4 mg enzyme. Enzyme was preincubated at 30” for the times indicated with complete reaction mixtures (except for acetyl-CoA and KH1*COB) and grana (d.6 mg chlorophyll). Acetyl-CoA and KH’4C03 were added to start the reactions. Assay time was 15 minutes at 30”. TABLE

STUMPF

tion mixtures. Clearly biotin does not release the inhibition either when preincubated with the enzyme or when added at the start of the assay, indicating that the inhibitory action is not related to an avidin-like effect on the biotinyl group of the acetyl-CoA carboxylase. The possibilities that the inhibitor acts either by deacylating or binding acetyl-CoA, or by decarboxylating malonyl-CoA as soon as it is formed, are discounted by the data presented in Tables III and IV. Since chloroplast pressates, containing grana, do not show these effects, it is reasonable to conclude that isolated grana would also not exhibit them. The inhibitory activities of both grana and pressate supernatant (“stroma”) are relatively heat stable (Fig. 2). On a protein basis stroma showed a higher specific activity of inhibition; however, if both stroma and grana were added at their original concentrations as in the pressate, their in-

0-O

Boiled

d--d

Unbolled

O--Q Boiled H

Unboilcd

,,rono. grono. ~tromo. stroma.

VIII

EFFECT OF BIOTIN ON THE INHIBITION OF WHEAT C;ERM ACETYL-COA CARBOXYLASE BY BUTTER LETTUCE CHLOROPLAST GRANA~ mrmoles KHlaCOa fixed

Conditions

+Biotin

Control (no cubated Control (no bated + grana; + grana;

-B&in

grana);

not prein-

197

199

grana);

preincu-

170

175

not preincubated preincubat,ed

19 10.5

22 10.5

a Reaction mixtures were as described in Experimental Methods. Where applicable enzyme (1.2 mg) was preincubated with biotin (100 rg) in the presence or absence of grana (a.6 mg chlorophyll) with the complete reaction mixture except for acetyl-CoA and KH’“COa, which were added to start the reactions. Incubations were carried out for 30 minutes at 30”.

FIG. 2. Heat stability of inhibitor in grana and stroma. Reaction mixtures were as described in Experimental Methods and contained 260 fig of enzyme. Protein in grana and stroma was determined by weighing ether insoluble trichloroacetic acid-precipitable material in the preparations. “Boiled” connotes immersion in boiling water for 5 minutes. Incubations were carried out at 30” for 30 minutes.

PLANT

ACETYL-COA

CARBOXYLASE

611

ACTIVITY

VCGCUO, a pale green friable powder was obtained which was used in the experiments (see legend to Table IX). Table VI and Fig. 2 indicate that both stroma and grant are inhibitory to the wheat germ acetyl-CoA carboxylase. Because of the ease of handling the soluble preparation, further studies on the nature and characteristics of the inhibit,or were carried out using sbroma. When 0.01 31Tris-HCl, pH S.6, containing 0.001 RI EDTA, was employed as the homogenizing buffer in the preparation of TABLE IX butter lettuce chloroplasts, the isolat.ed EFFIXT~FRUTTERLETTUCECHLOROPLBSTGRANA particles were mainly naked lamellar sysACETONE POWDER ON WHEAT (;ERM ACETYLtems (electron micrographic observations CoA C~RBOXYLASE by Dr. D. Von Wettstcin). These arc chloroAcetone powder added (mg) mpmolesKHWOa fixed plasts which have lost their outer membrane, but which may st’ill he scdiment,ccl at] 212 0 1000~ along with intact chloroplasts. On 1 154 2 disruption in the Aminco-E’rench Pressure 101 60 3 Cell and centrifugation at’ lOFi,OOOg,a grecn5 35 ish brown supernatant solution was obtained which was highly inhibitory. Assuming that, Twenty rng acetone powder was homogenized the inhibitor was the same as t,hat, present in a Potter Elvejhem homogenizer with 2 ml buffer in cxtract,s made in buffer bs, studies were bq, and aliquots were added to each tube as indiperformed on this material. cated. Reaction mixtures were as described in ExAs shown in Fig. 3 the inhibitory preparaperiwlental Methods. Enzyme added = 315 pg. tion was fract,ioned on Rrlphadex G-25. Two Incubat,ion was for 30 minutes at, 30”.

hibitory activities were equal on a per milliliter basis. Since an acetone powder preparation of butt’er lettuce chloroplast grana was capable of inhibiting the carboxylase, the inhibitory activity is probably not associated with lipoidal material (Table IX). The acetone powder was prepared by extracting the grana from 25 butter lettuces with acetone at -20” four times, and then washing with dry diethyl ether. Aft,er thorough drying in

,.-\ ,/ -!20-

.,

SH prodient

(Oualitotivs)

i

inhtbitor -0ctiuity

-50

------E,,,

EZI, -40

-30

-20

0

2

4

6

6

IO

12 14 16 16 20 22 24 FRACTION NUMBER --

26 26

30

0

FIG. 3. Sephadex G-25 fractionation profile of inhibitor. A 20-ml soluble fraction (6 mg dry wt/ml) was passed through a Sephadex G-25 column (37 X 2.2 cm) elut.ing with 0.01 M Tris, pH 8.6. The sample was mixed with mercaptoethanol to give a concentration of 0.01 M. The RSH acts as a marker for small molecular weight material. Its elution was checked with nitroprusside reagent, and a qualitative plot of this elution is shown. Five-ml fractions were collected and the Ezls was determined (19). The absorption in the region of fractions 12-20 is due to %H, not protein. The inhibitory activity was assayed in 0.5-ml aliquots of the fractions. For assay the reaction mixtures were as described in Experimental Methods and contained 167 rg enzyme. Incubation was at 30” for 30 minutes.

612

BURTON

AND STUMPF

fractions with inhibitory activity were obtained. One was associated with material of high molecular weight. The smaller molecular weight material was eluted mainly prior to the elution of mercaptoethanol, suggesting that it had a molecular weight near that of the exclusion limit of Sephadex G-25, and was thus partially excluded and partially included. Dialysis of the inhibitory fractions supported these findings. The smaller molecular weight material was completely dialysable, whereas the inhibitory activity associated with the high molecular weight fraction was nondialysable. As might be expected, the inhibitory activity of the unfractionated preparation was partially dialysable. The inhibition was not related to a direct removal of ATP or Mg++, components of the carboxylation reaction. Experiments carried out in the presence of either 2.5 pmoles of ATP or 20 pmoles in varying amounts of Mg++ did not release the inhibitory effects associated with the soluble inhibition preparation. The effects of papain and of 1 N HCl and 1 N KOH were also investigated. Incubation with papain at 37” and pH 7 did not lead to a loss of inhibitory activity, and heating with 1 N KOH at 70” for 45 minutes also failed to show any marked effect. Heating with 1 N HCl under the same conditions appeared to cause some loss of inhibitory activity. The activity of all inhibitor preparations was lost on ashing, thereby eliminating possible heavy metal ion effects. Treatment with ribonuclease led to no loss, thereby excluding RNA or its derivatives as possible inhibitors. Treatment with Norit A did not cause loss of inhibitory activity. On batchwise treatment with DEAE-cellulose, inhibitory activity, associated with the high molecular weight fraction from Sephadex fractionation, was completely lost, but the inhibitory activity of the smaller molecular weight fraction was unaffected. Acyl carrier protein from E. coli (a generous gift from Dr. R. D. Simoni) showed neither stimulatory nor inhibitory action on wheat. germ acetyl-CoA carboxylase. These studies indicated that butter lettuce chloroplast preparations contain a potent

inhibitor of wheat germ acetyl-CoA carboxylase activity. Efforts to clarify its nature have indicated in preliminary experiments that it exerts its effect on the enzyme itself rather than on its substrates or cofactors. The inhibitor(s) appears to be heat-stable and exists in at least two molecular species. Its chemical characterization is now under investigation. As was mentioned in the introduction to this paper, acetyl-CoA carboxylase from animal and microbial systems is subject to allosteric regulation by a variety of compounds (4-13). Having a partially purified preparation of wheat germ acetyl-CoA carboxylase made available the opportunity to test these allosteric effecters on a plant system. Table X summarizes the effects of phosphorylated compounds on the wheat germ carboxylase, and Table XI shows the effects of polycarboxylic acids. These data make it clear that none of the compounds tested exhibit the dramatic activating effects (demonstrated in other systems) on the wheat germ carboxylase. Although citrate caused inhibition at high concentration (30 mM), further experiments showed that this effect was less marked at 10 ells and was not exhibited at all at 5 IIWI. The inhibition was probably due to chelation of Mg++, since EDTA at 30 nnvr caused complete loss of enzyme activity. TABLE

X

EFFECT OF SOME PHOSPHORYLATED COMPOUNDS ON WHEAT GERM ACETYL-COA CARBOXYLASE

Additive

-

Fructose 1,6-diphosphate Fructose 1,6-diphosphate n-a-Glycerophosphate Ribose 5-phosphate Phosphoenol pyruvate Phosphoenol pyruvate + avidin (100 rg) Avidin (100 pg)

rnM

-

5 30 2.5 5 5 5 -

m@loles KH’eCOa fixed +A;&yl-

100 89 85 110 105 680 670 12

-A&tyl-

0.3 0.9 0.3 1.0 504 -

Reaction mixtures were as described in Experimental Methods. Amount of enzyme was 1.37 mg and incubation time was 30 minutes.

PLANT TABLE EFFECT

Additive

GERM

ACETYL-COA nm -

Citrate Malate Isocitrale Huccinate Fumarate a-Ket,oglutarate Glucose-6-P Fructose-6-P Pyruvate

30 10 10 10 10 10 4 3.3 10

CARBOXTLAW

XI

AND TCA

OF GLYC~LYTIC

ON WHEAT

ACETYL-COA

INTERMEDIATES

CARBOXYLASE

mFmoles KH’*COa fixed +Acetyl-CoA

125 14 120 132 129 129 112 110 124 124

-Acetyl-CoA

0.44 0.50 0.70 0.20 0.10 0.60 3.0 3.0 4.0 2.1

Reaction mixtures were as described in Experimental Methods. Amount of enzyme was 0.8 mg and incubation was for 30 minutes at 30”.

Long chain fatty acyl-CoA compounds have been shown to inhibit a variety of enzymes, among these being glucose 6phosphate dehydrogenase (21, 22), fatty acid synthetase (12, 23), citrate synthetase (24), and acetyl-CoA carboxylase (23, 25). It has been suggested that these CoA derivatives might be involved in the regulation of TPNH formation, and hence of various metabolic systems. However, Taketa and Pogell (21) have suggested that since many enzymes of diverse metabolic function are inhibited by palmityl-CoA, rather than being specific, these effects are due t’o the detergent properties of the acylGA’s, leading to conformational changes in enzyme structure, and hence in many cases to loss of enzymic activity. The wheat germ acetyl-CoA carboxylase was found to be unaffected by palmityl-CoA up to 0.5 mnl, but at 1 rnnf a marked inhibition was apparent. However, since a white, flocculent precipitate was formed when palmityl-CoA was added to the reaction mixture, it is probable that the observed inhibition is due to llIg++ binding (21). It would therefore appear that acetyl-CoA carboxylnse in disrupted chloroplasts has its activity curtailed markedly because of the presence of an inhibit’or. Since acetate is an effective precursor with intact chloroplasts, the inhibition is presumably masked in these chloroplasts. The precise role of this in-

61:;

ACTIVITY

hibitor in controlling fatty acid synthesis is as yet not, known. However, since the corn plete carbon dioxide reduction cycle is fully ac$ive in chloroplast, preparations and since malonyl-CoA serves as an cffertive substrate of fatt)y acid synthesis in t,hcse same preparations, it, is obvious that, thch cnzymcs participating in these systems :W not effected by the inhibitor, and suggests I hat the inhibition has considerable spccificit,.v for the acetyl-Co-4 carboxylase. Chemical characterization of t,his inhibitor must first be carried out before its regulatory role can be determined. These results would explain the earlier results of Brooks and St.umpf (a), who showed that malonyl-Cob is :I far more effect,ive precursor of long &in filt t,y acids &an acctyl-GA + Co,. Of considerable &c:rcst, from t hc comparative point, of view, is the hk of nctivit,y of the allosteric cffectors of t’he mammalian and yeast ncctyl-CoA carhoxylasc wit811botll the chloroplast and wheat germ :tc*ctylXo:1 carhoxylasc. Preliminary cxpclrimr>nts, honever, indicate that as the plant c~arboxylusc undergoes purificlation c~on~itlcrable loas of :lctivity is observed, and thi:: :lc+iv&y ca:mnot be rcstorrd by a number of compounds ef’fcctive with the mnmmalian and yc:~l~ system*. Further work is cont.emplatetl lo elucidat,c~ the considerable instability of t h(> morn purified carboxylase prep:rr:rtions.

1. YANG, 8. F., .1x1) STIJMPF, I'. Biophys. Acta 98, 19 (1965).

K.,

Biochim.

.\NII STIXW, 1'. K., Biochim. AC/U 98, 213 (1965). 3. BROOKS, J. L., .\XD SITXPF. 1’. K., -lrch. Riothem. Biophys. 116, 108 (1966). S. J.. .J. Riol. 4. BRODY, R. O., AND (~ROIX, C’hem. 199, 4’51 (1952). 5. POR,TER, J. W., WAKIL, S. J.. TIETZ, A., JZCOB, AI. I., .\N) (;IBSOS, I). RI., ICorhinr. Biophysl. Acta 26, 35 (1957). 2. BROOKS,

J. L.,

Biophys.

6. ABR.IHAM,

S., MATTHES,

K. J., ANI) CH~\II(OFF,

I. L., J. Biot. Chem. 236, 2551 (1060). 7. MIRTIN, II. B., ASI) V.\GELOS, P. It., Hiochem. Biophys. lies. C’ommun. 7, 101 (1962). 8. M.IRTIN, I). B.. ANI) V~GEMX. I’. I?.., J. Biol. CIh,ern. 237, 1787 (1962). 9. V.\GEI,OS, P. It., ALBEIZTS, A. W., .ZND N’LHTIN, 1). B., ./. Rio/. Chem. 238, 533 (19fi3).

614 10. WAITE, M., AND WAKIL,

11.

12. 13.

14. 15. 16.

17.

BURTON

AND STUMPF

S. J., J. Biol. Chem. 237, 2750 (1962). LYNEN, F., MATSUHASHI, M., NUMA, S., AND SCHWEIZER, E., in “Control of Lipid Metabolism” (J. K. Grant, ed.). Academic Press, New York (1963). WHITE, D., AND KLEIN, H. P., Biochem. Biophys. Res. Commun. 20, 78 (1965). WAKIL, S. J., GOLDMAN, J. K., WILLIAMSON, I. P., AND TAAMEY, R. E., Proc. Natl. Acad. Sci. U.S. 66, 880 (1966). EGGERER, H., AND LYNEN, F., Biochem. 2.336, 540 (1962). SIMON, E. J., AND SHEININ, D., J. Am. Chem. Sot. 76, 2520 (1953). GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). HATCH, M. D., AND STUMPF, P. K., J. Biol. Chem. 236, 2879 (1961).

18. UMBREIT, W. W., BURRIS,R.

19. 20. 21. 22.

H., AND STAUFFER, J. F.,“Manometric Methods,” p. 211. Burgess Publishing Co., Minneapolis (1957), 3rd ed. MURPHY, J. B., AND KIES, M. W., Biochem. Biophys. Acta 46,382 (1960). BAMBERGER, E. S., AND GIBBS, M., Plant Physiol. 40, 919 (1966). TABETA, K., AND POGELL, B. M., J. Biol. C&m. 240, 651 (1965). EGER-NEUFELDT, I., TEINZER, A., WEIBS, L., AND WIELAND,

O., Biochem.

Biophys.

Res.

Commun. 19, 43 (1965). 23. TUBBS, P. K., AND GARLAND, P. B., Biochem. J. 93, 550 (1964). 24. WJELAND. O., AND WEISS, L., Biochem. Biophys. Res. Commun. 13, 26 (1963). 25. BORTZ, W. M., AND LYNEN, F., Biochem. 2.337, 505 (1963).