Nicotinamide cofactors (NAD and NADP) in glyoxysomes, mitochondria, and plastids isolated from castor bean endosperm

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 215, No. 1, April 15, pp. 274-279, 1982

Nicotinamide Mitochondria,

Cofactors and Plastids

(NAD and NADP) in Glyoxysomes, Isolated from Castor Bean Endosperm’

ROBERT P. DONALDSON Department of Biologicd

seienees, Gemge Washington Unkersity,

Washington, D. C, %%A%?

Received June 17, 1981, and in revised form November 3, 1981

Glyoxysomes, mitochondria, and plastids were separated from the cytosol of germinating castor bean endosperm by sucrose gradient centrifugation in a vertical rotor (25 min, 5O,OOOg,,).The amounts of nicotinamide cofactors, NAD(H) and NADP(H), retained in the isolated organelle fractions were measured by enzyme cycling techniques. The NAD(H) was equally distributed between the cytosol and the mitochondria with a small amount in the glyoxysomes. The mitochondria retained 4 pmol of NAD(H)/ pg protein, about seven times as much as the glyoxysomes. Most of the NADP(H) was in the cytosol. However, the glyoxysomes and plastids retained significant amounts, both having 0.3 pmol NADP(H)/pg protein, twice that in the mitochondria. The subcellular distribution of NADP(H) was compared to the location of dehydrogenases capable of using this cofactor. The cytosol and plastids contained 6-phosphogluconate dehydrogenase. NADP isocitrate dehydrogenase was found in the glyoxysomes, in mitochondria, and in an unidentified subcellular fraction obtained at 1.16 g/ml in the density gradients. Knowledge of the quantities of NADP(H) and NAD(H) retained in the isolated organelles should make it possible to investigate their reduction and reoxidation in intact organelles.

The glyoxysome is an organelle occurring in germinating oil seeds, such as castor bean, which convert fatty acid to sucrose. Acetyl CoA released by p-oxidation of fatty acids in the glyoxysome is converted to succinate by the glyoxylate cycle enzymes enclosed in the organelle (2, 3). Acetyl-CoA apparently cannot penetrate the glyoxysome membrane (10) and, therefore, is not catabolized in the mitochondria. Instead the acetyl-CoA is committed to the glyoxylate cycle and, thus, to gluconeogenesis. Once this commitment is established with the formation of succinate further processing takes place else1 The abbreviation, NAD(H), is used here to inditotal NAD+ plus NADH. NADP(H) indicates total NADP+ plus NADPH. cate

0003-9861/82/050274-06$02.00/O Copyright All rights

Q 1982 by Academic Press. Inc. of reproduction in any form resewed.

where, the conversion of succinate to oxalacetate in the mitochondria (2) and the construction of hexose phosphate and sucrose in the cytosol (18). The recent discovery (14) of o-oxidation in rat liver peroxisomes suggests that some acetyl-CoA is being directed into some nonmitochondrial metabolism in this tissue as well. Fatty acids required to produce the membranes enclosing the organelles in castor bean (4, 7) are synthesized in the plastids (21) using acetyl-CoA released from citrate (11). The compartmentation of these specific metabolic activities implies the inclusion of the requisite cofactors along with the enzymes, especially if the cofactors do not traverse the enclosing membrane. NAD+ does not seem to penetrate the glyoxysoma1 membrane (10). NAD+ accepts H in 274

NAD

AND

NADP

IN GLYOXYSOMES,

the glyoxysome during b-oxidation and during the glyoxylate cycle. Thus some NAD+ should be contained in glyoxysomes. A similar line of reasoning would lead to the prediction of NADP+ compartmentation in the plastids. The purpose of this study was to determine the NAD(H) and NADP(H) content of subcellular fractions isolated from germinating castor bean endosperm, the cytosol, the mitochondria, the glyoxysomes, and the plastids. METHODS

Endosperm fractionation. Castor beans (R&&us communis var. Hale) were germinated directly in wet vermiculite at 30°C for 4.5 days in the dark. The excised endosperm (25 g) was homogenized in an equivalent amount of medium (25 ml) consisting of 13% (w/w) sucrose, 150 mru Tricine: pH 7.5, 10 mM KCl, 1 mEd MgClr, and 1 mM EDTA, pH 7.5. The chilled endosperm was chopped 10 min with four razor blades, filtered through four layers of cheesecloth, and centrifuged at 27Og for 10 min. The fat was removed and 10 ml of the 27Og supernatant was slowly pipetted onto a linear gradient (prepared using an Isco Model 570 gradient maker) of 20 ml 20 to 56% (w/w) sucrose with a 4-ml layer of 18% (w/w) sucrose (each sucrose solution had 1 m?d EDTA, pH 7.5). The 270~ pellet was resuspended in 10 ml of 13% (w/w) sucrose, 50 mM Tricine, 1 mM EDTA, pH 7.5, and pipetted onto a sucrose gradient identical to that described above. The sucrose gradients were centrifuged in a Dupont/Sorvall TV850 vertical rotor for 25 min at 25,006 rpm (5O,OOOg~~), preceded by about 9 min automatic slow acceleration to 25,000 rpm and followed by active deceleration to 1060 rpm after which the rotor was allowed to coast to a stop. The gradients were removed from the rotor within 1 h of placing the rotor in the centrifuge. Previously described preparations required at least 2 h centrifugation not including acceleration and deceleration (8). As we were developing this procedure, the use of the vertical rotor for the rapid fractionation of castor bean endosperm was reported by Gregor (12). The fractions were collected using an Isco Model 135. The supernatant fractions were obtained in 6- to 3ml increments, the ER and mitochondria in 1.5-ml increments, the glyoxysomes in 0.75-ml increments. The 270g pellet gradient containing the plastids was fractionated in 6- to 3-ml volumes until 30% sucrose ’ Abbreviations used: Tricine, N-tris(hydroxymethyl)methylglycine; ER, endoplasmic reticulum; Hepes, N-2-hydroxyethylpiperazine-N‘-2-ethanesulfonic acid; Ml’??, thiasolyl blue; Mes, 2-(N-morpho1ino)etbanesulfonic acid.

MITOCHONDRIA,

AND

PLASTIDS

was reached and the remainder All procedures described above

2-5°C. NAD(H) and NADP(H)

275 as 1.5-ml volumes. were performed at

analysis. Immediately after all the fractions were collected, two 50-pl samples of each were placed in 13 X 150-mm Pyrex test tubes which were then placed in a boiling water bath for 1.5 min followed by transfer to an ice water bath. To each sample 0.9 ml of the reaction mixture described below (lacking the enzyme) was added. The samples were kept in the dark at room temperature for at least 30 min before the enzyme was added. During this time any enzyme-independent dye reduction was exhausted. The total NAD+ plus NADH was measured using a procedure adapted from two others (1,19). The reaction mixture consisted of 65 mM Hepes, pH 7.5,340 mM ethanol, 0.2 mM MTT (thiazolyl blue), 0.9 mhb phenazine ethosulfonate, of which 0.9 ml was added to each 50-J sample. After the preincubation period, the samples were centrifuged and analyzed five at a time in a Perkin-Elmer Model 555 spectrophotometer under the following conditions: 3O”C, 560 nm, 2-nm bandwidth slit, 0.1 A full scale. The absorbance of each sample was recorded at 0.7-min intervals for 1.4 min first without enzyme to assure that there was no nonspecific dye reduction and then for 5 min after the addition of 50 ~1 of alcohol dehydrogenase (1.25 mg/ml or 375 units/ml, Sigma Catalog No. 3263). Standards were also included in each experiment, ranging from 5 to 200 ng of NAD+ in 1 mM Mes, pH 6. The concentration of the standard was confirmed by measuring Ass indicating that 1 nmol NAD+ (Sigma Catalog No. N7004) was equivalent to 720 ng. One picomole of NAD’ usually produced a rate of about 3.6 X 1O-4 A/min. Total NADP+ plus NADPH was determined using the same reaction mixture and conditions as for NAD+ with the omission of the ethanol and the addition of 3 mg glucose 6-phosphate. The reaction was initiated by adding 10 ~1 of glucose-6-phosphate dehydrogenase (0.32 mg/ml or 60 units/ml, Sigma Catalog No. G4134) (19). One picomole (0.345 ng) of NADP+ standard produced a rate of about 7 X lo-’ A&min. These procedures measure the total amount of nicotinamide dinucleotide without distinguishing the reduced form from the oxidized. The reduced forms, NADH and NADPH, could be destroyed by treating the boiled samples with 0.2 Y HCl for 30 s, neutralizing with 0.2 M NaOH, and then adding the reaction mixture (15). Enzymes, protth, sucrose mmsurements. The enzymes fumarase (2), isocitrate lyase (2), and 6-phosphogluconate dehydrogenase (18) were measured using the published procedures. NADP isocitrate dehydrogenase was measured as reported (2) except that 100 mM Tricine buffer, pH 7.5, was used. Protein was estimated using the Folin reagent. Sucrose per-

276

ROBERT P. DONALDSON

centage (w/w)

was read directly

using 1466RN).

a hand

re-

a considerable amount sedimented at 270s and upon centrifugation in the sucrose gradient moved to 45.5% (w/w) sucrose, RESULTS 1.21 g/ml. This enzyme has been reported, along with other activities (e.g., ribulose Vertical rotor fractionation. One other report (11) has been made of the use of a diphosphate carboxylase), in plastids obtained at a similar density from developvertical rotor to isolate organelles from ing (20) and germinating (18) castor bean castor bean. The convenience of the verendosperm, and pea roots (17). Thus it is tical rotor is that the time of centrifugalikely that this enzyme is indicative of tion is much shorter, 25 min at 25,000 rpm plastids. Note that very little of this enrather than 2 h or more. Also, the rotor zyme was found in the vicinity of the holds eight tubes so that a large amount giyoxysomes and mitochondria in the 2709 of a particular organelle can be obtained supernatant fractionation. That is these in a short time or a variety of conditions organelles were not contaminated with may be examined at once. Figure 1 demplastids. However, the plastids appeared onstrates that the separation of organelles to be contaminated with glyoxysomes. is identical to that obtained with swinging NAD(H) distribution. Much of the bucket rotors (8) although somewhat better separations may be obtained using a NAD(H) was in the cytosol fraction (Fig. 1). Also, a large amount was associated non-reorienting zonal rotor (9). The leftwith the mitochondria. The glyoxysomes hand panel of Fig. 1 shows the fractionretained a small amount. The NAD(H) in ation of the 270~ supernatant. The four peaks of protein represent, from left to the 270s pellet was mainly associated with the small amount of mitochondria which right, the cytosol, the ER, the mitochonof NAD(H) in dria, and the glyoxysomes. Isocitrate lyase obscured the measurement confirms the location of glyoxysomes in the plastids. Table I confirms the impressions given about 50% (w/w) sucrose, density, 1.23 g/ retained alml. Fumarase indicates the position of the by Fig. 1. The mitochondria mitochondria in 40% (w/w) sucrose, den- most half of the total NAD(H) in germisity, 1.18 g/ml. No marker enzyme for ER nating castor bean endosperm. Relative to had (28% sucrose, 1.12 g/ml) is reported here protein content, the mitochondria much more NAD(H) than any other subsince no NAD or NADP was particularly cellular site. The glyoxysomes had much associated with that fraction. The cytosol less NAD(H) relative to protein. The value contained 6-phosphogluconate dehydroreported here is less than that determined genase (20) and NADP isocitrate dehydrogenase (2) as previously reported. The sep- using fluorescence (5) but greater than aration between the glyoxysomes and that given by Mettler and Beevers (16). in the mitochondria was mitochondria can be increased by using a The NAD(H) completely oxidized. In the isolated gly34 to 56% (w/w) sucrose gradient rather oxysomes the ratio, NADH/NAD+, was than the 20 to 56% shown (Fig. 1). The right-hand panel of Fig. 1 shows the 0.24. Glyoxysomes from less developed endosperm, 4-day rather than 5-day, had a fractionation of the 2709 pellet. The largconsistently higher ratio, about 0.4. This est amount of protein was found in a fracis clearly not a reflection of the in tivo tion at 36% (w/w) sucrose, 1.16 g/ml, condition but indicative of the oxidative associated with NADP isocitrate dehycapabilities of the isolated organelles; midrogenase. The cellular origin of this tochondria are able to oxidize NADH and fraction is unknown. Very little fumarase was found in the 2709 pellet fractions. A glyoxysomes cannot (2). NADP(H) distribution. Although the cysignificant amount of isocitrate lyase was obtained in these fractions. This indicates tosol again accounted for a major portion that some glyoxysomes sediment at 270g of the cofactor, the NADP(H) content of but that few mitochondria do. Although the various organelles was quite different much of the 6-phosphogluconate dehydrothan NAD(H). The mitochondria had some genase was found in the cytosol fraction, NADP(H) but the glyoxysomes had more. fractometer

(Bausch

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NAD

AND

NADP

IN GLYOXYSOMES,

MITOCHONDRIA,

AND

277

PLASTIDS

PELLET

SUPERNATANT P

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0 a *

30-

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FUMARASE

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-1

l-

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ISOCITRATE

DEHYDROGENASE -100

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DEHYOROGENASE

z ‘, s! 0 E

030201-

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10 VOLUME

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(ml)

FIG. 1. Fractionation of castor bean endosperm by gradient centrifugation in a vertical rotor. Left: The separation of subcellular components from a 2’70s supernatant of homogenized endosperm. The cytosol was collected in fractions from 0 to 13 ml which included the original 10 ml of supernatant. The ER was in fractions from 13 to 22 ml. The mitochondria (fumarase) were between 22 and 29 ml. The glyoxysomes (isocitrate lyase) were obtained between 29 and 34 ml. Right: Fractions obtained from 270s pellet, including some residual cytosol from 0 to 15 ml, NADP isocitrate dehydrogenase containing fractions between 15 and 24 ml, and plastids (6-phosphogluconate dehydrogenase) from 24 to 30 ml.

0.2

278

ROBERT TABLE

NAD(H)

Cytosol Mitochondria Glyoxysomes Plastids

I

CONTENT OF FRACTIONS BEAN ENDOSPERM NAD(H) (pmol/ag protein) 0.33 4.05 0.60 0.41

P. DONALDSON

FROM CASTOR

Percentage of total in homogenateb 47.6 46.9 3.5 2.0

n Four different preparations, standard deviations less than 25% of value given. *Calculated from summations of values represented in Fig. 1. All fractions are included.

NADP(H) in the 2709 pellet was clearly correlated with 6-phosphogluconate dehydrogenase, the plastid marker. The second small peak of NADP(H) obtained from the 2’709 pellet was associated with the isocitrate lyase, the glyoxysomes. Table II shows that more than half of the total NADP(H) in the homogenate was in the cytosol. The mitochondria and plastids each had about 10% of the NADP(H); the glyoxysomes had 16%. On a protein basis, the glyoxysomes and plastids were the outstanding repositories of NADP(H). The glyoxysomes had half as much NADP(H) as NAD(H) relative to protein. The mitochondria had less than half as much NADP(H) as the glyoxysomes or plastids. Most of the NADP(H) in the glyoxysomes and cytosol was in the oxidized form, that in the plastids and mitochondria was completely oxidized.

somes may be less than the in wivo amount. Some NAD(H) may be lost during isolation because of damage to the membrane (16), or perhaps the membrane is normally permeable to NAD+ and/or NADH. The NAD(H) content of the isolated organelle could be the consequence of NAD(H) binding to the enclosed enzymes rather than confinement by the membrane. Knowledge of the amounts of cofactors retained in the isolated glyoxysomes will make it possible to investigate mechanisms of reoxidation of endogenous NADH and NADPH reduced, respectively, by /3oxidation and isocitrate dehydrogenase activities in the intact organelle. Once reduced, electrons from NADH must somehow be transmitted to the mitochondrial electron transport system (2). One possibility, which has not been eliminated, is that NADH simply leaves the glyoxysomes and is directly reoxidized by the mitochondria. A second possibility is the reoxidation of NADH by a malate-aspartate shuttle (16). Thirdly, the NADH in the glyoxysomes may donate electrons to flavins and cytochromes (detected as cytochrome c reductase) in the glyoxysomal membrane (10) which may be discharged to external acceptors. This possibility is explored in the following article (23). If the quantities of NAD(H) measured here are any indication of the in vivo distribution then the relatively large amount of NAD(H) in the mitochondria would cycle more slowly than that in the glyoxysomes since the mitochondria are dependent on TABLE

II

DISCUSSION NADP(H)

Glyoxysomes utilize NAD+ to oxidize fatty acid (P-hydroxyl acyl-CoA dehydrogenase) and malate (2, 3). It would seem that glyoxysomes should retain some NAD(H) during isolation since it is thought not to pass through the membrane (10). Since @-oxidation and the glyoxylate cycle are the raison d ‘are of germinating castor bean endosperm and since these activities are confined to the glyoxysome, it is surprising that so little NAD(H) is found in the isolated organelle. The amount of NAD(H) measured in the isolated glyoxy-

IN SUBCELLULAR FRACTIONS HOMOGENATES OF ENDOSPERM NADP(H) (pmol/ag protein)

Cytosol Mitochondria Glyoxysomes Plastids

FROM

Percentage of total in homogenate

0.07 0.13 0.29 0.31

a Three different preparations, standard tions less than 30% of value given, except cytosol which had a range of 0.02 to 0.15.

63.9 9.5 15.8 10.8 deviafor the

NAD

AND

NADP

IN GLYOXYSOMES,

the glyoxysomes for substrate in this tissue. The mitochondria receive succinate from the glyoxylate cycle and perhaps malate (2, 16). The mitochondria have no significant supply of acetyl-CoA since it is reserved for the glyoxylate cycle. The subcellular distribution of NADP isocitrate dehydrogenase includes the cytosol, the mitochondria, the glyoxysomes, and a large unidentified fraction. This latter fraction, after sedimenting at low centrifugal force was obtained at a lower density than mitochondria in a gradient. This fraction cannot represent unbroken cells since neither 6-phosphogluconate dehydrogenase nor fumarase was associated with it. The plastids appear to lack isocitrate dehydrogenase. Cooper and Beevers (2) originally detected isocitrate dehydrogenase in glyoxysomes but found most of the activity in cytosol fractions as reported here. This is the only known COPproducing enzyme in the glyoxysomes. The cr-ketoglutarate produced may be metabolized further in the mitochondria or it may serve to accept amino groups from aspartate yielding oxalacetate in the glyoxysomes (16). In any case, the result would be to divert carbon from the glyoxylate cycle. The presence of NADP(H) in the isolated glyoxysomes lends emphasis to the isocitrate dehydrogenase therein. However, the function of NADPH generated in the glyoxysomes is unclear. It would not be expected to penetrate the membrane. Enzymes that would support a metabolic shuttle analogous to that suggested for NADH (16) have not been described in glyoxysomes. The NADPH could be involved in the reduction of a flavoprotein and cytochrome P-420 in the glyoxysome membrane reported in the following article (23), also detected as NADPH cytochrome c reductase (10). The NADP+ in the plastids would seem to be involved in the pentose shunt since 6-phosphogluconate dehydrogenase is located there as well. The glucose-&phosphate dehydrogenase is, however, found in the cytosol (18). No malic enzyme was detected. NADPH generated in the plastids would be used to support fatty acid syn-

MITOCHONDRIA.

AND

PLASTIDS

279

thesis (21). Both NAD(H) and NADP(H) have previously been detected in chloroplasts (13). The production of NADPH in the cytosol may serve mixed-function oxidase activities in the ER (22). The overall impression is that each subcellular compartment contains NADP+ along with at least one enzyme capable of reducing it. REFERENCES 1. BERNOFSKY, C., AND SWAN, M. (1973) And Bioch.em 53,452-458. 2. COOPER, T. G., AND BEEVERS, H. (1969) J. Btil Chem. 244, 3507-3515. 3. COOPER, T. G., AND BEEVERS, H. (1969) J. Bid Chem. 244, 3514-3520. 4. DONALDSON, R. P. (1976) Plant Physid 57, 510515. 5. DONALDSON, R. P. (1979) Plant Physid 63, (suppl.), 5. 6. DONALDSON, R. P. (1981) Plant Physid 67 (suppl.), 92. 7. DONALDSON, R. P., AND BEEVERS, H. (1977) Plant PhystiL 59, 259-263. 8. DONALDSON, R. P., AND BEEVERS, H. (1978) Pmt&m 97, 317-327. 9. DONALDSON, R. P., TOLBERT, N. E., ANLI SCHNARRENBERGER, C. (1972) Arch Biochem. Biophys. 152,199-215. 10. DONALDSON, R. P., TALLY, R. E., YOUNG, 0. A., AND BEEVERS, H. (1981) Plant PhysioL 67,2125. 11. FRITSCH, H., AND BEEVERS, H. (1979) Plant PhysioL 63, 687-691. 12. GREGOR, H. D. (1977) And Biochem 82,255-257. 13. HARVEY, M. J., AND BROWN, A. P. B&him Biophys Acta 172,116-K%. 14. LAZAROW, P. B. (1978) J. BtiL Chem 253, 15221528. 15. LOWRY, 0. H., ROBERTS, N. R., AND KAPPHAHN, J. I. (1957) J. Bid Chem. 244,1047-1064. 16. METTLER, I. J., AND BEEVERS, H. (1980) Plant Physid 66,555-560. 17. MIFLIN, B. J., AND BEEVERS, H. (1974) Plant Physid 53,870-874. 18. NISHIMURA, M., AND BEEVERS, H. (1979) Plant PhysioL 64,31-37. 19. NISSELBAUM, J. S., AND GREEN, S. (1969) Anal. Bioc?t.em 27, 212-217. 20. SIMCOX, P. D., REID, E. E., CANVIN, D. T., AND DENNIS, D. T. (1977) PZunt PhysioL 59, 11281132. 21. VICK, B., AND BEEVERS, H. (1978) P&t Physid 62, 173-178. 22. YOUNG, 0.. AND BEEVERS, H. (1976) Phytochemistry 15.379-385. 23. HICKS, D. B., AND DONALDSON, R. P. (1982) Arch Biochem Biophys. 215,280-288.