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NADP-specific malate dehydrogenase of green spinach leaf tissue
ARCHIVES
OF
BIOCHEMISTRY
AND
NADP-Specific
147, 156-164 (1971)
BIOPHYSICS
Malate
Dehydrogenase
of Green
Spinach
Leaf Tissue’ IRWIN Department
P. TING
of Biology, Received
University
VICTOR
AND
of California,
June 17, 1971; accepted
ROCHA’
Riverside, August
California
9.‘8609
12, 1971
An NADP-specific malate dehydrogenase was partially purified from spinach leaf tissue by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration. The protein appears to be localized in the stroma of intact chloroplasts. For activity, there is a requirement for a reagent such as dithiothreitol (DTT). Almost complete inactivation occurs after desalting by gel filtration or exhaustive dialysis. Reactivation occurs by incubation in 5 mM DTT. The completely activated prot,ein elutes from a calibrated Sephadex G-200 column with an apparent molecular weight of 110,000. An inactivated preparation elutes as three distinct peaks with estimated molecular weights of 29,000,42,000, and 75,060. Apparent K,,,‘s computed from hyperbolic kinetic data were: oxaloacetate = 0.03 mM, NADPH = 0.07 rnM, malate = 11.0 mM, and NADP = 0.04 mM. The data are of physiological interest since it appears that green spinach leaf tissue has MDH activity catalyzing OAA s MAL in all major subcellular compartments ; viz., NAD specific activity in microbodies, mitochondria, and in the cytosol, and NADP specific activity in the chloroplasts.
In 1969, Hatch and Slack (1) reported the existence of an NADP dependent malate dehydrogenase (i.e., an NADP requiring enzyme analogous to L-malate: NAD oxidoreductase, EC 1.1.1.37). Their investigations, designed to further elucidate the newly proposed Cq photosynthetic pathway (2), led to the conclusion that Cq plants (i.e., those with high photosynthetic efficiency and low COZ compensation points) have greater quantities of NADP specific malate dehydrogenase than those plants with low photosynthetic efficiencies and high COZ compensation points (i.e., CB plants). Their data suggested that NADP malate dehydrogenase of maize was localized in chloroplasts when investigated by nonaqueous methods (3). They suggested that the NADP-malate dehydrogenase of Cd plants participated in 1 Research was supported by NSF Grant GB25878 and the University of California Intramural Research Fund. 2 Present address: Department of Biology, University of California, San Diego.
malate formation in the Cq photosynthetic pathway. Later, Johnson and Hatch (4) characterized the NADP-malate dehydrogenase isolated from maize with respect to kinetic and some physical properties. Because of the report of the existence of NADP-malate dehydrogenase in spinach leaves (1) and our interest in malate dehydrogenase isoenzymes of spinach (5, 6) and other species (7, S), we isolated and characterized the spinach leaf NADP-malate dehydrogenase. In the present paper, we report on the preparation, activation, physical and kinetic properties, and the localization of NADP malate dehydrogenase of green spinach leaf tissue. MATERIALS Enzyme preparation leaves were purchased
AND
METHOD&Y
procedure. Fresh spinach at a local market, deribbed
a Abbreviations used in text are: MDH malate dehydrogenase, ME = malate enzyme 156
= or
NADP-SPECIFIC
MALATE
by hand, and throughly washed with distilled water. Four hundred g of washed tissue were thoroughly ground with a blendor in 500 ml of a 0.05 M Tris, pH 7.5 buffer containing 1 mM dithiothreitol and 1 mM EDTA. The homogenate was filtered through two layers of cheesecloth and centrifuged at 25,OOOg for 30 min. The latter supernatant fluid was brought to 20% saturation with solid ammonium sulfate. The 20% solution was centrifuged at 25,OOOg for 30 min and the supernatant fluid was brought to 65yo saturation with solid ammonium sulfate. The precipitate was pelleted by centrifugation at 25,000~ for 30 min. The pellet was suspended in 10 ml of 5 mM, pH 7.0, sodium phosphate buffer and dialyzed against 2 liters of the same buffer for about 18 hr. The dialyzed preparation (about 10 mg protein) was placed on a 1.5 X 15 cm DEAE-cellulose column (0.61 meg/ml) and eluted with a linear gradient of sodium phosphate buffer, pH 7.0 from 0.02 to 0.2 M (9). Five ml fractions were collected. Fractions with maximum activity were combined (about 50 ml) and precipitated between 20 and 65% ammonium sulfate as described above. After dialysis against 0.05 M tris buffer, pH 7.5, the preparation was activated by incubation in 5 mM dithiothreitol overnight. The activated preparation was placed on a 2.5 X 25 cm Sephadex G-200 column prepared in 0.05 M Tris buffer, pH 7.5, containing 5 mM DTT and 0.1 M KCI. The preparation was eluted with the above buffer. Three ml fractions were collected, the active fractions were combined, and precipitated between 20 and 65% ammonium sulfate. The 2&65W ammonium sulfate precipitate was suspended in 10 ml of 0.05 M Tris buffer, pH 7.5, and dialyzed against the same buffer. The dialyzed preparation was fully activated by incubation in 5 mM DTT. Dithiothreitol activation. Activation of the inactive or partially active protein was conducted by incubation at 0°C in various concentrations of dithiothreitol (4). Michaelis constant estimation. Michaelis constants (Km) were estimated with fully activated enzyme preparation at 25’. In the direction of oxaloacetate reduction to malate, the oxaloacetate constant was estimated at pH 7.5 at a constant NADPH concentration of 0.1 mM. The K, for NADP was estimated at pH 7.5 with an oxaloacetate concentration of 0.5 mM. In the direction of malate dehydrogenase (NADP) (decarboxylating), NADP = nicotinamide adenine dinucleotide phosphate, NADPH = reduced NADP, OAA = oxaloacetate, Tes = N-tris (hydroxymethyl) methyl-a-amino ethane sulfonic acid, DTT = dithiothreitol, Tris = tris (hydroxymethyl) aminomethane, DEAE = diethylaminoethyl.
DEHYDROGENASE
157
malate oxidation to oxaloacetate, Km’s were estimated at pH 8.5. With malate as the variable substrate, NADP was held constant at 0.5 mM; and with NADP as the variable, malate was constant at 10 mM. K,‘s were estimated from double reciprocal plots of initial rates versus variable substrate concentration (10). Gel fLItration and estimation of apparent molecular weight. Enzymic preparations (about 4 ml) containing approximately 3-5 mg/ml of protein were sieved through a 2.5 X 25 cm Sephadex G-200 column to estimate molecular weight (11). The elution buffer was 0.05 M Tris, pH 7.5, containing 0.1 M KCl. Inactivated NADP-malate dehydrogenase was passed through the column in the absence of dithiothreitol whereas the activated protein was eluted with Tris-KC1 buffer containing 5 mM DTT. Either 1.5 or 3 ml fractions were collected. The column was calibrated with equine heart cytochrome c (assumed molecular weight = 13,700; California Corporation for Biological Research, Los Angeles, California), yeast alcohol dehydrogenase (assumed molecular weight = 151,000; California Corporation for Biological Research), NADP-isocitrate dehydrogenase (assumed molecular weight = 64,000; California Corporation for Biochemical Research), beef liver catalase (assumed molecular weight = 225,000; Sigma Chemical Corporation, St. Louis, Missouri). Calibrating proteins were mixed with the experimental spinach leaf NADP-malate dehydrogenase for each column chromatographic run. During the purification procedures, the calibration proteins were left out of the preparation. Protein estimation. Protein profiles were estimated in column fractions by measuring the optical density at 280 mp and in enzyme preparations by the phenolic reagent method of Lowry et al.
02). pH optimum studies. pH optimum was determined in the direction of OAA reduction using 0.05 M buffers in a total volume of 3 ml. The oxaloacetate concentration was 0.5 mM and the NADPH was 0.1 mM. pH ranges used were 6.5 to 8.0 with TES, 7.5 to 9.5 with Tris, and 5.5 to 8.0 with sodium phosphate. In the direction of malate oxidation, 0.05 M Tris buffer was used with 10 mM L-malate and 0.5 mM NADP in a total volume of 3 ml. All assays were conducted in the presence of 5 mM DTT. Intracellular localization of NADP-malate dehydrogenase activity. One hundred g of fresh market spinach were thoroughly washed in distilled water, deribbed, and stored overnight in the dark to insure minimum starch. The washed tissue was minced finely with a chopper in 150 ml of medium containing 0.5 M sucrose, 1 mM EDTA, 1 mM 2-
158
TING
AND
mercaptoethanol, and 0.170 bovine serum albumin all in 0.05 M Tris buffer, pH 7.4. After mincing, the suspension was placed in a chilled blendor and ground at low speed twice for 3 sec. The homogenate containing intact chloroplasts, mitochondria, and microbodies was filtered through 8 layers of cheesecloth, and centrifuged at 250 g for 20 set to remove debris. The supernatant fraction was centrifuged first at 1000 g for 5 min and then at 3000 g for 15 min to obtain two pellets. The 1000 g and 3000 g pellets were separately layered on 4080% (w/v) (34 ml) linear sucrose-density gradients and centrifuged at maximum speed in a Beckman SW-27 bucket rotor for 3 hr. The gradients were fractionated into about 30 equal fractions. Organelles were localized within the gradient using the following marker enzymes: microbodies, catalase assayed by measuring the disappearance of H202 at 240 rnp (13) and glycolate oxidase assayed with o-dianisidine as described elsewhere (5) ; mitochondria, cytochrome oxidase assayed with reduced cytochrome c at’ 550 rnp (14) ; intact chloroplasts, NADP-glyceraldehyde 3-phosphate dehydrogenase assayed by coupling with phosphoglyceric acid kinase (15) and chlorophyll; broken or stripped chloroplasts lacking stroma, chlorophyll assayed by optical density measurements (5). The gradient fractions were incubated with 5 mM DTT for several hours prior to assay for NADP-malate dehydrogenase. The details of the organelle purification procedure are described elsewhere (5). Standard assay. NADP-malate dehydrogenase was assayed routinely by first incubating preparations in 5 mM dithiot,hreitol at 0” for several hours to insure complete activation. Assays were conducted at 25” in a total volume of 3 ml in pH 7.0, 0.05 M sodium phosphate buffer by measuring the oxidation of NADPH at 340 rnp with a spectrophotometer. Sufficient enzyme preparation was added to give an optical density change of about Q.l OD per minute. Stock solutions of oxaloacetate (1.0 mg/ml) and NADPH (1.4 mg/ml) were freshly prepared in the pH 7.0 phosphate buffer. The assay was conducted by pipetting into the spectrophotometer cuvette sufficient buffer to make a final volume of 3 ml, activated enzyme, and 0.2 ml NADPH (0.1 mM). The reaction was started with 0.2 ml of oxaloaeetate (0.5 mM). With fully activated enzyme, rates are linear for several minutes under these conditions. Except where noted in the text, linear rates were used to calculate activities. RESULTS
Purification precipitation
procedure. Ammonium sulfate procedures, and chromatog-
ROCHA
raphy on DEAEcellulose and Sephadex G-200 result in about a lOO-fold enrichment of NADP-malate dehydrogenase. Most of the purification takes place during DEAEcellulose chromatography with little more enrichment on the 2.5 X 25 cm Sephadex column (Figs. 1 and 2). About 50% of the starting activity was recovered during the procedure. The exact recovery is difficult to estimate because of the activation requirement of the enzyme. An accurate estimate of fully activated enzyme in a crude homogenate was not attempted and may not be feasible. The specific activity of the final preparation was 2.3 pmoles per min per mg protein (OAA -+ MAL). Dithiothreitol activation. Exhaustive dialysis or desalting by gel filtration through a Sephadex G-25 column (1 X 15 cm) tends to inachivate the protein. Reactivation can readily be accomplished by incubation in dilute dithiothreitol. Incubation of an enzyme preparation with 5 mM dithiothreitol
0
50 100 150 200 Elution Volume (ml)
Elution
Volume
250
(ml)
FIG. 1. DEAE-cellulose elution profile of protein (OD 280 rnp) (upper panel), NADP-malate dehydrogenase (NADP-MDH), NAD-malate dehydrogenase (NAD-MDH), and malate dehydrogenase (decarboxylating) (NADP) or malate enzyme (ME). A linear sodium phosphate gradient was used (P-gradient). Two malate enzyme (ME) peaks are resolved. The four NAD-MDH isoenzymes are partially resolved (see reference 5). A single peak with NADP-MDH activity was found.
NADP-SPECIFIC
MALATE
159
DEHYDROGENASE
.4 0 .3 -
i\
OD 180-l
‘.,
I ? 5
- 1.7
\
rNADP-MDH
- 1.5
\ \
/
\
:
- 1.3 a E
‘k
i
0
20
40 Elution
60 Volume
100
00
120
(ml)
profile of fully dithiothreitol activated NADP-MDH and optical FIG. 2. Sephadex G-200 elution density at 280 rnp giving an indication of protein. The material for gel filtration chromatography was obtained from the active NADP-MDH fractions shown in Fig. 1.
containing about 2-4 mg/ml of protein, which was inactivated by dialysis against 0.05 M Tris buffer, pH 7.5, results in complete reactivation in one to two hours (Fig. 3). The activation is exponential with time (Fig. 4) and the rate of activation increases with increasing concentration of dithiothreitol (Fig. 4). If an inactivated preparation is assayed in the presence of varying concentrations of DTT, the rates are exponential as shown in Fig. 4; however, if a linear rate is estimated for several concentrations of DTT, the activation as measured by the rate of reaction is almost linear between 0.5 mM and 50 mu (Fig. 5). Other sulfhydryl reagents, e.g., ,&mercaptoethanol and glutathione, are relatively ineffective when compared with DTT. No evidence was obtained for activation by prior incubation with NADPH, OAA, or malate. Gel filtration of active and inactive preparalions. When a DTT activated preparation is passed through a Sephadex G-200 column prepared with 0.05 M Tris buffer, pH 7.5, containing 0.1 M KC1 and 5 m&r DTT, the NADP-malate dehydrogenase activity elutes as a single, broad peak. Assuming a symmetrical peak, the estimated molecular weight is about 110,000 (Fig. 6). If a partially inactivated preparation is passed through a Senhadex column orenared simi-a
-1
larly to the above but without the DTT, the enzyme elutes as four distinct peaks (Fig. 6). Estimated molecular weights from the calibrated gel column were: Peak I = 29,000, Peak II = 42,000, Peak III = 75,000, and Peak IV = 110,000. The estimates are an average of two runs. If a completely inactivated preparation is passed through a column prepared in the same buffer as above except without DTT and each collected fraction incubated with 5 mM DTT, three
0- .6 * .4 ,E i .2 .E
./-
.
.
;(,. 0
, 50
100 Time
150 (min)
, 200
2 0
of NADP-MDH by incubaFIG. 3. Activation tion in 5 mM dithiothreitol at 0°C. An NADPMDH preparation containing about 4 mg protein per ml which was inactivated by exhaustive dialysis against 0.05 M Tris buffer, pH 7.5, was incubated with 5 mM DTT. At the times indicated, a sample was removed and assayed for NADP-MDH activity as indicated in the text under standard assay.
160
TING
AND
ROCHA
4.0
.4
2.0 3
0
*
/
50mM
1.0 0 0.8
0 2.2
:
;/
0.6 0.4
d
,
0.7
0.9
I
I
1.1
1.3
0.1 0
5
10 Time
15
20
0.5
25
log
IminI
lime
I 1.5
(mini
FIG. 4. Time course of NADP-MDH activation by dithiothreitol at 25”. Left: facsimile photometer recorder trace of optical density at 340 rnrc against time as the NADP-MDH activated by DTT. The activation is exponential as shown in the right panel in the log-log concentrations of DTT are shown.
enzymatically active peaks with estimated weights simiIar to Peaks I, II, and III are obtained. The 110,000 peak (Peak IV) was absent. One would assume from these data that the DTT activated preparation is in a form with an apparent molecular weight of 110,000. The inactivated enzyme has an apparent smaller size and exists in at least three forms; i.e., apparent sizes of about 29,000, 42,000, and 75,000. Each form can be activated since detection was after incubation of each fraction with 5 mM DTT. pH optimum. The optimum pH for assays in both catalytic directions was determined in 0.05 M buffers. In the direction of oxaloacetate reduction, when assayed at 0.5 mM oxaloacetate and 0.1 mM NADPH, the pH optimum was about 7.0 (Fig. 7). Relatively more activity was obtained with phosphate and Tris buffers than Tes. Hence, it was decided to use a pH 7.0,0.05 M sodium phosphate buffer in subsequent assays. In the opposite direction, i.e., the oxidation of malate, when assayed at 10 rnM lmalate and 0.5 mM NADP, the activity increased with increasing pH (Fig. 7). There was little difference between pH 8.5 and 9.0.
of spectroactivity is plot. Three
Hence subsequent assays in this direction were conducted in 0.05 M Tris buffer, pH 8.5. Michaelis constants. Apparent Michaelis constants (K,) for each substrate were deter-
7 -
~6_ ?s 2 ’ .i J 2 a. , Dithiothrcitol
ImMl
FIG. 5. Activation of inactivated NADP-MDH with dithiothreitol at 25°C as a function of DTT concentration. The experiment was conduct.ed by adding partially inactive enzyme, dithiothreitol in the concentration indicated, NADPH, and oxaloacetate to buffer and then following the time course of optical density change as NADPH was oxidized. Raw data were similar to the curves shown in Fig. 4. Assumed linear rates were estimated from the exponential traces and are pIotted in the graph as a function of DTT.
NADP-SPECIFIC
MALATE
161
DEHYDROGENASE
mined with a fully DTT activated preparation. For all four substrates or cofactors, the kinetics were apparently hyperbolic with no indication of sigmoidicity or significant substrate inhibition. The data could be fitted reasonably well to double reciprocal plots for estimation of Michaelis constants (Table 1). Graphical analyses of the four curves resulted in the following Km estimations: oxaloacetate = 0.03 mu, NADPH = 0.07 mu, I-malate = 11.0 mM, and NADP =
0.04 mM. 0
20
40 Elution
60 Volume
100
a0
The rate of reaction in the direction of oxaloacetate reduction is much greater than the reverse reaction. Estimation of an apparent equilibrium constant at pH 8.5 and 25°C using initial rates and known molarities of substrates resulted in a value of about 2 x 103 (OAA + NADPH + MAL + NADP) . Intracellular localization. When intact chloroplasts, broken chloroplasts, mitochondria, and microbodies were purified from spinach leaf tissue, analysis indicated that NADP-malate dehydrogenase was associated with intact chloroplasts (Fig. 8). Because the ratio of enzymic activity to chlorophyll is the same (0.01 A OD/min/mg chlorophyll) in both the 1000 g (chloroplast enriched) and 3000 g (mitochondria en-
120
(ml)
FIG. 6. Gel filtration of completely activated and partially inactivated (by dialysis against0.05 M Tris buffer, pH 7.5) NADP-MDH on a 2.5 X 25 cm Sephadex G-200 column. Upper panel: Activated (Act) and partially inactivated (Inact) NADPH-MDH profiles. Lower panel : calibration of column with enzymes of known molecular weights. BD = blue dextran (void volume of column), CAT = catalase, ADH = alcohol dehydrogenase, IDH = NADP isocitrate dehydrogenase, cyto c = cytochrome c (see text for details). The data indicate that the fully activated enzyme elutes with an estimated molecular weight of 110,000 and the partially inactivated form elutes as four peaks with assumed molecular weights of 29,000, 42,000, 75,060, and 110,000.
1.0
1.0 .
A-
.9 .8 f ‘G .-
.7 - 013
f 4 .6 a .t G .5U e
OAA+MAL
\
.4 -
1.
.3 .2 I 5.5
I
I 6.5
I
I 7.5
I
I 6.5
PH
FIG. 7. pH optimum curves for spinach chloroplast of OAA + MAL direction. Right panel: pH optimum
I
I 9.5
7.5
8.0
a.5
9.0
PH
NADP-MDH activity. Left panel: of MAL + OAA direction.
pH optimum
TING
162
AND
ROCHA
the oxidation of malate to oxaloacetate. Because of the central role of NADP in chloroplast metabolism, the NADP dependDEHYDROGENASE OF SPINACH LEAF TISSUK~ ent malate dehydrogenase may be of sigSubstrate Km (md) nificance. Recent available evidence indicates that, although the chloroplast,s may 0.03 Oxaloacetate 11.0 have NAD (16, 17) no NAD malate dehyI-malate 0.07 NADPH drogenase is present (5, 18). 0.04 NADP The biological functions of the main malate dehydrogenases of spinach leaf tissue a See text for details of methods. are not clear. It seems reasonabIe, however, to assume that the mit,ochondrial-NAD riched) pellet fractions, NADP-MDH is malate dehydrogenase functions in citric acid assumed to be associated only wit,h the intact cycle activity. The microbody-NAD malate chloroplasts and not with mitochondria or dehydrogenase may function in photorespimicrobodies. The lack of appreciable activity ration (19) which occurs in leaf microbodies in the broken or stripped chloroplasts (i.e., (or peroxisomes) and which is pronounced those without outer membranes and stroma) in spinach leaf tissue. Previously, we have suggests that the NADP-malate dehydrospeculated that the soluble-NAD malate genase is a chloroplast stromal enzyme. No dehydrogenase coupled with P-enolpyruvate evidence was obtained in these experiments carboxylase functions in dark or nonphotoregarding the possible presence of a soluble synthetic CO2 fixation (8). The chloroplastform of the enzyme. NADP malate dehydrogenase could function in the photosynthesis of malate as envisioned DISCUSSION for Ch photosynthetic plants (2). Malate The data presented in this paper suggest may be a product of photosynthesis in spinthat green spinach leaf tissue has a specific ach and spinach chloroplasts but probably protein which catalyzes the reversible reducnot an initial product (20). Knowledge of tion of oxaloacetate to malate using NADPH the extent to which the latter is valid must as the redox cofactor. AS suggested by Johnawait further experimentation. son and Hatch (4), based on their studies The existence of powerful dehydrogenase with maize, the systematic name would be activity in all major compartments suggests L-malate: NADP oxidoreductase. Slack et al. an hypothesis similar to that of Williamson (3) presented data based on nonaqueous et al. (21). They suggested that dehydrochloroplast isolation techniques, indicating genases, such as malate dehydrogenase, localization within the mesophyll chloromaintain specific redox potentials within plasts of maize. Our data, presented here, their respective compartments. Furtherindicate that the NADP-malate dehydromore, the malate dehydrogenases of the genase of green spinach leaf tissue is localized mitochondria, microbodies, chloroplasts, and in the stroma of intact chloroplasts. cytosol could function in “hydrogen shutThe finding of NADP malate dehydrotles” between or among compartments as genase activity in leaf tissue and demonsuggested by Delbruck et al. (22) and Kaplan stration of localization in chloroplasts is of (23) for mitochondria and cytosol and by interest and significance. Previous data have Heber and Santarius (17) for chloroplasts indicated at least three NAD-malate dehyand cytosol. drogenase isoenzymes in spinach leaf tissue Kinetic properties of the NADP-malate each localized in different subcellular comdehydrogenase of spinach are quite similar partments, viz., mitochondrial, microbody, to the kinetic properties of the NAD-malate and the soluble phase of the cell or cytosol. Hence the data suggest that each major sub- dehydrogenases. At pH 7.5, the apparent cellular compartment in spinach leaf tissue Michaelis constants for oxaloacetate are has a specific protein catalyzing the reduc- 0.06, 0.04, and 0.04 mM for the soluble-, t’ion of oxaloacetate to malate and perhaps mitochondrial-, and microhody-NAD malTABLE
MICHAELIS
I
CONSTANTS (Km) FOR NAl)P-M2~~2\~~
NADP-SPECIFIC
MALATE
163
DEHYDROGENASE
10009 --
6
600-
E
;500.
2.5 . .-c
:.4 "p n g .3 a
NADP-,,
F -4oo=
MDH
.
i\
z.2 rrc 0 .l
U
0
300
.4 z .3;; . .-c
E ;200 E
E
.2; 0 a E
,. c :
L 100 0
.l
it
U
: 0 c G
5
10 Fraction
15
20 Number
25
0 1
FIG. 8. Subcellular localization of NADP-MDH activity in spinach leaf chloroplasts. Upper panel: 1000 g pellet enriched in intact chloroplasts purified by sucrose density gradient centrifugation. Lower panel: 3ooO g pellet enriched in mitochondria and microbodies purified on sucrose density gradients. Chl = chlorophyll, CA = catalase, CO = cytochrome oxidase. The first chlorophyll peak (fractions #21-23) represents stripped chloroplasts and the second chlorophyll peak (fractions 115-16) represents primarily intact chloroplasts in the 1060 g pellet and primarily mitochondria in the 3600 g pellet. The CA peak represents microbodies. The graph indicates NADP-MDH localization in intact chloroplasts.
dehydrogenases respectively (24) ate whereas for the NADP enzyme it was 0.03 mM. For NADH, the soluble, mitochondrial, and microbody forms had apparent Km’s of 0.02, 0.05, and 0.02 mM (24) whereas the NADP form had an NADPH K,,, of 0.07 mM. The dithiothreitol requirement for enzymic activity of NADP-malate dehydrogenase is presently not understood. Johnson and Hatch (4) reported that the maize leaf enzyme was activated in situ by light and
inactivated by darkness. Their inactivated enzyme was activated by dithiotreitol plus MgCL. Furthermore, their data indicated that the enzyme eluted from a Sephadex G-200 column as two or perhaps three peaks. Although our Sephadex elution profiles are somewhat different, multiple peaks were apparent. In our experiments, the fully activated protein eluted as a single, relatively sharp peak. The inactivated protein eluted as three distinct peaks with apparent molec-
164
TING
AND
ular sizes of 29,000 (Peak I) 42,000 (Peak II), and 75,000 (Peak III) molecular weight. The fully activated protein elutes with an apparent molecular weight of 110,000. It is tempting to speculate that the fully activated protein is composed of one Peak I unit and two Peak II units, and Peak III is made up of a Peak I and a Peak II unit. The exact nature of the protein and Sephadex peaks must await further experimentation. It should be made clear, however, that each peak can be activated by incubation in DTT. The question of an artifact is difficult to answer without complete purification. Unpublished results of experiments conducted at the Photosynthesis and Photorespiration Conference held in Canberra, 1970, with Hilary Johnson supported our initial observations that NADP malate dehydrogenase was associated with spinach leaf chloroplasts and that no transhydrogenase or NADPH phosphatase activities were present in the preparation. The data presented in this paper, although not conclusive, do not suggest that the NADP malate dehydrogenase is an artifact. In summary, spinach leaf tissue apparently has a malate dehydrogenase protein requiring NADPH rather than NADH. The protein appears to be localized in the stroma of chloroplasts and has kinetics somewhat similar to the NAD-malate dehydrogenases localized in other subcellular compartments of spinach leaf tissue. ACKNOWLEDGMENTS The expert technical assistance of Miss Shirley A. Reed during this investigation is acknowledged. Discussions with Hilary Johnson and some preliminary experiments conducted with her in the laboratories of M. D. Hatch and N. K. Boardman, CSIRO Plant Industry, Canberra, are also acknowledged.
ROCHA REFERENCES 1. HATCH, M. D., AND SLACK, C. R., Biochem. Biophys. Res. Commun. 34, 589 (1969). 2. HATCH, M. D., AND SLACK, C. R., Annu. Rev. Plant Physiol. 21, 141 (1970). 3. SLACK, C. R., HATCH, M. D., AND GOODCHILD, D. J., Biochem. J., 114, 489 (1969). 4. JOHNSON, H. S., .AND HATCH, M. D., Biochem. J. 119, 273 (1970). 5. ROCHA, V., AND TING, I. P., Arch. Biochem. Biophys. 140, 398 (1970). 6. ROCHA, V., AND TING, I. P., Plant Physiol., 46, 754 (1970). 7. MUKERJI, S. K., AND TING, I. P., Arch. Biothem. Biophys. 131, 336 (1969). 8. TING, I. P., Arch. Biochem. Biophys. 126, 1 (1968). 9. SNYDER, L. R., Chromatog. Rev. 7, 32 (1965). 10. LINEWEAVER, H., AND BURK, D., J. Amer. Chem. Sot. 66,658 (1934). 3, 723 (1964). 11. ACKERS, G. K., Biochemistry, 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 13. MALHLY, A., AND CHANCE, B., Methods Biothem. Anal. 1,357 (1954). 14. SIMON, E. W., Biochem. J. 69,67 (1958). 15. HEBER, U., PON, D. G., AND HEBER, M., Plant Physiol. 36, 355 (1963). 16. HARVEY, M. J., AND BROWN, A. P., Biochim. Biophys. Acta. 173, 116 (1969). 17. HEBER, U. W., AND SANTARIUS, K. A., Biochim. Biophys. Acta 109, 390 (1965). 18. TOLBERT, N. E., OESER, A., YAMAZAKI, R. K., HOGEMAN, R. H., AND KISAKI, T., Plant Physiol. 44, 135 (1969). 19. JACKSON, W. A., AND VOLK, R. J., Annu. Rev. Plant Physiology 21,385 (1970). 20. HOLM-HANSEN, O., PON, N. G., NISHIDA, R., MOSES, V., AND C.~LVIN, M., Physiol. Plant. 12, 475 (1959). 21. WILLIAMSON, D. H., LUND, P., .&ND KREBS, H. A., Biochem. J. 103,514 (1967). 22. DELBRUCK, A., ZEBE, E., AND B&HER, T. H., Biochem. 2.331,273 (1959). 23. KAPLAN, N. O., Bacterial. Rev. 27, 155 (1963). 24. ROCHA, V., AND TING, I. P., Arch. Biochem. Biophys. 147, 114 (1971).
OF
BIOCHEMISTRY
AND
NADP-Specific
147, 156-164 (1971)
BIOPHYSICS
Malate
Dehydrogenase
of Green
Spinach
Leaf Tissue’ IRWIN Department
P. TING
of Biology, Received
University
VICTOR
AND
of California,
June 17, 1971; accepted
ROCHA’
Riverside, August
California
9.‘8609
12, 1971
An NADP-specific malate dehydrogenase was partially purified from spinach leaf tissue by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration. The protein appears to be localized in the stroma of intact chloroplasts. For activity, there is a requirement for a reagent such as dithiothreitol (DTT). Almost complete inactivation occurs after desalting by gel filtration or exhaustive dialysis. Reactivation occurs by incubation in 5 mM DTT. The completely activated prot,ein elutes from a calibrated Sephadex G-200 column with an apparent molecular weight of 110,000. An inactivated preparation elutes as three distinct peaks with estimated molecular weights of 29,000,42,000, and 75,060. Apparent K,,,‘s computed from hyperbolic kinetic data were: oxaloacetate = 0.03 mM, NADPH = 0.07 rnM, malate = 11.0 mM, and NADP = 0.04 mM. The data are of physiological interest since it appears that green spinach leaf tissue has MDH activity catalyzing OAA s MAL in all major subcellular compartments ; viz., NAD specific activity in microbodies, mitochondria, and in the cytosol, and NADP specific activity in the chloroplasts.
In 1969, Hatch and Slack (1) reported the existence of an NADP dependent malate dehydrogenase (i.e., an NADP requiring enzyme analogous to L-malate: NAD oxidoreductase, EC 1.1.1.37). Their investigations, designed to further elucidate the newly proposed Cq photosynthetic pathway (2), led to the conclusion that Cq plants (i.e., those with high photosynthetic efficiency and low COZ compensation points) have greater quantities of NADP specific malate dehydrogenase than those plants with low photosynthetic efficiencies and high COZ compensation points (i.e., CB plants). Their data suggested that NADP malate dehydrogenase of maize was localized in chloroplasts when investigated by nonaqueous methods (3). They suggested that the NADP-malate dehydrogenase of Cd plants participated in 1 Research was supported by NSF Grant GB25878 and the University of California Intramural Research Fund. 2 Present address: Department of Biology, University of California, San Diego.
malate formation in the Cq photosynthetic pathway. Later, Johnson and Hatch (4) characterized the NADP-malate dehydrogenase isolated from maize with respect to kinetic and some physical properties. Because of the report of the existence of NADP-malate dehydrogenase in spinach leaves (1) and our interest in malate dehydrogenase isoenzymes of spinach (5, 6) and other species (7, S), we isolated and characterized the spinach leaf NADP-malate dehydrogenase. In the present paper, we report on the preparation, activation, physical and kinetic properties, and the localization of NADP malate dehydrogenase of green spinach leaf tissue. MATERIALS Enzyme preparation leaves were purchased
AND
METHOD&Y
procedure. Fresh spinach at a local market, deribbed
a Abbreviations used in text are: MDH malate dehydrogenase, ME = malate enzyme 156
= or
NADP-SPECIFIC
MALATE
by hand, and throughly washed with distilled water. Four hundred g of washed tissue were thoroughly ground with a blendor in 500 ml of a 0.05 M Tris, pH 7.5 buffer containing 1 mM dithiothreitol and 1 mM EDTA. The homogenate was filtered through two layers of cheesecloth and centrifuged at 25,OOOg for 30 min. The latter supernatant fluid was brought to 20% saturation with solid ammonium sulfate. The 20% solution was centrifuged at 25,OOOg for 30 min and the supernatant fluid was brought to 65yo saturation with solid ammonium sulfate. The precipitate was pelleted by centrifugation at 25,000~ for 30 min. The pellet was suspended in 10 ml of 5 mM, pH 7.0, sodium phosphate buffer and dialyzed against 2 liters of the same buffer for about 18 hr. The dialyzed preparation (about 10 mg protein) was placed on a 1.5 X 15 cm DEAE-cellulose column (0.61 meg/ml) and eluted with a linear gradient of sodium phosphate buffer, pH 7.0 from 0.02 to 0.2 M (9). Five ml fractions were collected. Fractions with maximum activity were combined (about 50 ml) and precipitated between 20 and 65% ammonium sulfate as described above. After dialysis against 0.05 M tris buffer, pH 7.5, the preparation was activated by incubation in 5 mM dithiothreitol overnight. The activated preparation was placed on a 2.5 X 25 cm Sephadex G-200 column prepared in 0.05 M Tris buffer, pH 7.5, containing 5 mM DTT and 0.1 M KCI. The preparation was eluted with the above buffer. Three ml fractions were collected, the active fractions were combined, and precipitated between 20 and 65% ammonium sulfate. The 2&65W ammonium sulfate precipitate was suspended in 10 ml of 0.05 M Tris buffer, pH 7.5, and dialyzed against the same buffer. The dialyzed preparation was fully activated by incubation in 5 mM DTT. Dithiothreitol activation. Activation of the inactive or partially active protein was conducted by incubation at 0°C in various concentrations of dithiothreitol (4). Michaelis constant estimation. Michaelis constants (Km) were estimated with fully activated enzyme preparation at 25’. In the direction of oxaloacetate reduction to malate, the oxaloacetate constant was estimated at pH 7.5 at a constant NADPH concentration of 0.1 mM. The K, for NADP was estimated at pH 7.5 with an oxaloacetate concentration of 0.5 mM. In the direction of malate dehydrogenase (NADP) (decarboxylating), NADP = nicotinamide adenine dinucleotide phosphate, NADPH = reduced NADP, OAA = oxaloacetate, Tes = N-tris (hydroxymethyl) methyl-a-amino ethane sulfonic acid, DTT = dithiothreitol, Tris = tris (hydroxymethyl) aminomethane, DEAE = diethylaminoethyl.
DEHYDROGENASE
157
malate oxidation to oxaloacetate, Km’s were estimated at pH 8.5. With malate as the variable substrate, NADP was held constant at 0.5 mM; and with NADP as the variable, malate was constant at 10 mM. K,‘s were estimated from double reciprocal plots of initial rates versus variable substrate concentration (10). Gel fLItration and estimation of apparent molecular weight. Enzymic preparations (about 4 ml) containing approximately 3-5 mg/ml of protein were sieved through a 2.5 X 25 cm Sephadex G-200 column to estimate molecular weight (11). The elution buffer was 0.05 M Tris, pH 7.5, containing 0.1 M KCl. Inactivated NADP-malate dehydrogenase was passed through the column in the absence of dithiothreitol whereas the activated protein was eluted with Tris-KC1 buffer containing 5 mM DTT. Either 1.5 or 3 ml fractions were collected. The column was calibrated with equine heart cytochrome c (assumed molecular weight = 13,700; California Corporation for Biological Research, Los Angeles, California), yeast alcohol dehydrogenase (assumed molecular weight = 151,000; California Corporation for Biological Research), NADP-isocitrate dehydrogenase (assumed molecular weight = 64,000; California Corporation for Biochemical Research), beef liver catalase (assumed molecular weight = 225,000; Sigma Chemical Corporation, St. Louis, Missouri). Calibrating proteins were mixed with the experimental spinach leaf NADP-malate dehydrogenase for each column chromatographic run. During the purification procedures, the calibration proteins were left out of the preparation. Protein estimation. Protein profiles were estimated in column fractions by measuring the optical density at 280 mp and in enzyme preparations by the phenolic reagent method of Lowry et al.
02). pH optimum studies. pH optimum was determined in the direction of OAA reduction using 0.05 M buffers in a total volume of 3 ml. The oxaloacetate concentration was 0.5 mM and the NADPH was 0.1 mM. pH ranges used were 6.5 to 8.0 with TES, 7.5 to 9.5 with Tris, and 5.5 to 8.0 with sodium phosphate. In the direction of malate oxidation, 0.05 M Tris buffer was used with 10 mM L-malate and 0.5 mM NADP in a total volume of 3 ml. All assays were conducted in the presence of 5 mM DTT. Intracellular localization of NADP-malate dehydrogenase activity. One hundred g of fresh market spinach were thoroughly washed in distilled water, deribbed, and stored overnight in the dark to insure minimum starch. The washed tissue was minced finely with a chopper in 150 ml of medium containing 0.5 M sucrose, 1 mM EDTA, 1 mM 2-
158
TING
AND
mercaptoethanol, and 0.170 bovine serum albumin all in 0.05 M Tris buffer, pH 7.4. After mincing, the suspension was placed in a chilled blendor and ground at low speed twice for 3 sec. The homogenate containing intact chloroplasts, mitochondria, and microbodies was filtered through 8 layers of cheesecloth, and centrifuged at 250 g for 20 set to remove debris. The supernatant fraction was centrifuged first at 1000 g for 5 min and then at 3000 g for 15 min to obtain two pellets. The 1000 g and 3000 g pellets were separately layered on 4080% (w/v) (34 ml) linear sucrose-density gradients and centrifuged at maximum speed in a Beckman SW-27 bucket rotor for 3 hr. The gradients were fractionated into about 30 equal fractions. Organelles were localized within the gradient using the following marker enzymes: microbodies, catalase assayed by measuring the disappearance of H202 at 240 rnp (13) and glycolate oxidase assayed with o-dianisidine as described elsewhere (5) ; mitochondria, cytochrome oxidase assayed with reduced cytochrome c at’ 550 rnp (14) ; intact chloroplasts, NADP-glyceraldehyde 3-phosphate dehydrogenase assayed by coupling with phosphoglyceric acid kinase (15) and chlorophyll; broken or stripped chloroplasts lacking stroma, chlorophyll assayed by optical density measurements (5). The gradient fractions were incubated with 5 mM DTT for several hours prior to assay for NADP-malate dehydrogenase. The details of the organelle purification procedure are described elsewhere (5). Standard assay. NADP-malate dehydrogenase was assayed routinely by first incubating preparations in 5 mM dithiot,hreitol at 0” for several hours to insure complete activation. Assays were conducted at 25” in a total volume of 3 ml in pH 7.0, 0.05 M sodium phosphate buffer by measuring the oxidation of NADPH at 340 rnp with a spectrophotometer. Sufficient enzyme preparation was added to give an optical density change of about Q.l OD per minute. Stock solutions of oxaloacetate (1.0 mg/ml) and NADPH (1.4 mg/ml) were freshly prepared in the pH 7.0 phosphate buffer. The assay was conducted by pipetting into the spectrophotometer cuvette sufficient buffer to make a final volume of 3 ml, activated enzyme, and 0.2 ml NADPH (0.1 mM). The reaction was started with 0.2 ml of oxaloaeetate (0.5 mM). With fully activated enzyme, rates are linear for several minutes under these conditions. Except where noted in the text, linear rates were used to calculate activities. RESULTS
Purification precipitation
procedure. Ammonium sulfate procedures, and chromatog-
ROCHA
raphy on DEAEcellulose and Sephadex G-200 result in about a lOO-fold enrichment of NADP-malate dehydrogenase. Most of the purification takes place during DEAEcellulose chromatography with little more enrichment on the 2.5 X 25 cm Sephadex column (Figs. 1 and 2). About 50% of the starting activity was recovered during the procedure. The exact recovery is difficult to estimate because of the activation requirement of the enzyme. An accurate estimate of fully activated enzyme in a crude homogenate was not attempted and may not be feasible. The specific activity of the final preparation was 2.3 pmoles per min per mg protein (OAA -+ MAL). Dithiothreitol activation. Exhaustive dialysis or desalting by gel filtration through a Sephadex G-25 column (1 X 15 cm) tends to inachivate the protein. Reactivation can readily be accomplished by incubation in dilute dithiothreitol. Incubation of an enzyme preparation with 5 mM dithiothreitol
0
50 100 150 200 Elution Volume (ml)
Elution
Volume
250
(ml)
FIG. 1. DEAE-cellulose elution profile of protein (OD 280 rnp) (upper panel), NADP-malate dehydrogenase (NADP-MDH), NAD-malate dehydrogenase (NAD-MDH), and malate dehydrogenase (decarboxylating) (NADP) or malate enzyme (ME). A linear sodium phosphate gradient was used (P-gradient). Two malate enzyme (ME) peaks are resolved. The four NAD-MDH isoenzymes are partially resolved (see reference 5). A single peak with NADP-MDH activity was found.
NADP-SPECIFIC
MALATE
159
DEHYDROGENASE
.4 0 .3 -
i\
OD 180-l
‘.,
I ? 5
- 1.7
\
rNADP-MDH
- 1.5
\ \
/
\
:
- 1.3 a E
‘k
i
0
20
40 Elution
60 Volume
100
00
120
(ml)
profile of fully dithiothreitol activated NADP-MDH and optical FIG. 2. Sephadex G-200 elution density at 280 rnp giving an indication of protein. The material for gel filtration chromatography was obtained from the active NADP-MDH fractions shown in Fig. 1.
containing about 2-4 mg/ml of protein, which was inactivated by dialysis against 0.05 M Tris buffer, pH 7.5, results in complete reactivation in one to two hours (Fig. 3). The activation is exponential with time (Fig. 4) and the rate of activation increases with increasing concentration of dithiothreitol (Fig. 4). If an inactivated preparation is assayed in the presence of varying concentrations of DTT, the rates are exponential as shown in Fig. 4; however, if a linear rate is estimated for several concentrations of DTT, the activation as measured by the rate of reaction is almost linear between 0.5 mM and 50 mu (Fig. 5). Other sulfhydryl reagents, e.g., ,&mercaptoethanol and glutathione, are relatively ineffective when compared with DTT. No evidence was obtained for activation by prior incubation with NADPH, OAA, or malate. Gel filtration of active and inactive preparalions. When a DTT activated preparation is passed through a Sephadex G-200 column prepared with 0.05 M Tris buffer, pH 7.5, containing 0.1 M KC1 and 5 m&r DTT, the NADP-malate dehydrogenase activity elutes as a single, broad peak. Assuming a symmetrical peak, the estimated molecular weight is about 110,000 (Fig. 6). If a partially inactivated preparation is passed through a Senhadex column orenared simi-a
-1
larly to the above but without the DTT, the enzyme elutes as four distinct peaks (Fig. 6). Estimated molecular weights from the calibrated gel column were: Peak I = 29,000, Peak II = 42,000, Peak III = 75,000, and Peak IV = 110,000. The estimates are an average of two runs. If a completely inactivated preparation is passed through a column prepared in the same buffer as above except without DTT and each collected fraction incubated with 5 mM DTT, three
0- .6 * .4 ,E i .2 .E
./-
.
.
;(,. 0
, 50
100 Time
150 (min)
, 200
2 0
of NADP-MDH by incubaFIG. 3. Activation tion in 5 mM dithiothreitol at 0°C. An NADPMDH preparation containing about 4 mg protein per ml which was inactivated by exhaustive dialysis against 0.05 M Tris buffer, pH 7.5, was incubated with 5 mM DTT. At the times indicated, a sample was removed and assayed for NADP-MDH activity as indicated in the text under standard assay.
160
TING
AND
ROCHA
4.0
.4
2.0 3
0
*
/
50mM
1.0 0 0.8
0 2.2
:
;/
0.6 0.4
d
,
0.7
0.9
I
I
1.1
1.3
0.1 0
5
10 Time
15
20
0.5
25
log
IminI
lime
I 1.5
(mini
FIG. 4. Time course of NADP-MDH activation by dithiothreitol at 25”. Left: facsimile photometer recorder trace of optical density at 340 rnrc against time as the NADP-MDH activated by DTT. The activation is exponential as shown in the right panel in the log-log concentrations of DTT are shown.
enzymatically active peaks with estimated weights simiIar to Peaks I, II, and III are obtained. The 110,000 peak (Peak IV) was absent. One would assume from these data that the DTT activated preparation is in a form with an apparent molecular weight of 110,000. The inactivated enzyme has an apparent smaller size and exists in at least three forms; i.e., apparent sizes of about 29,000, 42,000, and 75,000. Each form can be activated since detection was after incubation of each fraction with 5 mM DTT. pH optimum. The optimum pH for assays in both catalytic directions was determined in 0.05 M buffers. In the direction of oxaloacetate reduction, when assayed at 0.5 mM oxaloacetate and 0.1 mM NADPH, the pH optimum was about 7.0 (Fig. 7). Relatively more activity was obtained with phosphate and Tris buffers than Tes. Hence, it was decided to use a pH 7.0,0.05 M sodium phosphate buffer in subsequent assays. In the opposite direction, i.e., the oxidation of malate, when assayed at 10 rnM lmalate and 0.5 mM NADP, the activity increased with increasing pH (Fig. 7). There was little difference between pH 8.5 and 9.0.
of spectroactivity is plot. Three
Hence subsequent assays in this direction were conducted in 0.05 M Tris buffer, pH 8.5. Michaelis constants. Apparent Michaelis constants (K,) for each substrate were deter-
7 -
~6_ ?s 2 ’ .i J 2 a. , Dithiothrcitol
ImMl
FIG. 5. Activation of inactivated NADP-MDH with dithiothreitol at 25°C as a function of DTT concentration. The experiment was conduct.ed by adding partially inactive enzyme, dithiothreitol in the concentration indicated, NADPH, and oxaloacetate to buffer and then following the time course of optical density change as NADPH was oxidized. Raw data were similar to the curves shown in Fig. 4. Assumed linear rates were estimated from the exponential traces and are pIotted in the graph as a function of DTT.
NADP-SPECIFIC
MALATE
161
DEHYDROGENASE
mined with a fully DTT activated preparation. For all four substrates or cofactors, the kinetics were apparently hyperbolic with no indication of sigmoidicity or significant substrate inhibition. The data could be fitted reasonably well to double reciprocal plots for estimation of Michaelis constants (Table 1). Graphical analyses of the four curves resulted in the following Km estimations: oxaloacetate = 0.03 mu, NADPH = 0.07 mu, I-malate = 11.0 mM, and NADP =
0.04 mM. 0
20
40 Elution
60 Volume
100
a0
The rate of reaction in the direction of oxaloacetate reduction is much greater than the reverse reaction. Estimation of an apparent equilibrium constant at pH 8.5 and 25°C using initial rates and known molarities of substrates resulted in a value of about 2 x 103 (OAA + NADPH + MAL + NADP) . Intracellular localization. When intact chloroplasts, broken chloroplasts, mitochondria, and microbodies were purified from spinach leaf tissue, analysis indicated that NADP-malate dehydrogenase was associated with intact chloroplasts (Fig. 8). Because the ratio of enzymic activity to chlorophyll is the same (0.01 A OD/min/mg chlorophyll) in both the 1000 g (chloroplast enriched) and 3000 g (mitochondria en-
120
(ml)
FIG. 6. Gel filtration of completely activated and partially inactivated (by dialysis against0.05 M Tris buffer, pH 7.5) NADP-MDH on a 2.5 X 25 cm Sephadex G-200 column. Upper panel: Activated (Act) and partially inactivated (Inact) NADPH-MDH profiles. Lower panel : calibration of column with enzymes of known molecular weights. BD = blue dextran (void volume of column), CAT = catalase, ADH = alcohol dehydrogenase, IDH = NADP isocitrate dehydrogenase, cyto c = cytochrome c (see text for details). The data indicate that the fully activated enzyme elutes with an estimated molecular weight of 110,000 and the partially inactivated form elutes as four peaks with assumed molecular weights of 29,000, 42,000, 75,060, and 110,000.
1.0
1.0 .
A-
.9 .8 f ‘G .-
.7 - 013
f 4 .6 a .t G .5U e
OAA+MAL
\
.4 -
1.
.3 .2 I 5.5
I
I 6.5
I
I 7.5
I
I 6.5
PH
FIG. 7. pH optimum curves for spinach chloroplast of OAA + MAL direction. Right panel: pH optimum
I
I 9.5
7.5
8.0
a.5
9.0
PH
NADP-MDH activity. Left panel: of MAL + OAA direction.
pH optimum
TING
162
AND
ROCHA
the oxidation of malate to oxaloacetate. Because of the central role of NADP in chloroplast metabolism, the NADP dependDEHYDROGENASE OF SPINACH LEAF TISSUK~ ent malate dehydrogenase may be of sigSubstrate Km (md) nificance. Recent available evidence indicates that, although the chloroplast,s may 0.03 Oxaloacetate 11.0 have NAD (16, 17) no NAD malate dehyI-malate 0.07 NADPH drogenase is present (5, 18). 0.04 NADP The biological functions of the main malate dehydrogenases of spinach leaf tissue a See text for details of methods. are not clear. It seems reasonabIe, however, to assume that the mit,ochondrial-NAD riched) pellet fractions, NADP-MDH is malate dehydrogenase functions in citric acid assumed to be associated only wit,h the intact cycle activity. The microbody-NAD malate chloroplasts and not with mitochondria or dehydrogenase may function in photorespimicrobodies. The lack of appreciable activity ration (19) which occurs in leaf microbodies in the broken or stripped chloroplasts (i.e., (or peroxisomes) and which is pronounced those without outer membranes and stroma) in spinach leaf tissue. Previously, we have suggests that the NADP-malate dehydrospeculated that the soluble-NAD malate genase is a chloroplast stromal enzyme. No dehydrogenase coupled with P-enolpyruvate evidence was obtained in these experiments carboxylase functions in dark or nonphotoregarding the possible presence of a soluble synthetic CO2 fixation (8). The chloroplastform of the enzyme. NADP malate dehydrogenase could function in the photosynthesis of malate as envisioned DISCUSSION for Ch photosynthetic plants (2). Malate The data presented in this paper suggest may be a product of photosynthesis in spinthat green spinach leaf tissue has a specific ach and spinach chloroplasts but probably protein which catalyzes the reversible reducnot an initial product (20). Knowledge of tion of oxaloacetate to malate using NADPH the extent to which the latter is valid must as the redox cofactor. AS suggested by Johnawait further experimentation. son and Hatch (4), based on their studies The existence of powerful dehydrogenase with maize, the systematic name would be activity in all major compartments suggests L-malate: NADP oxidoreductase. Slack et al. an hypothesis similar to that of Williamson (3) presented data based on nonaqueous et al. (21). They suggested that dehydrochloroplast isolation techniques, indicating genases, such as malate dehydrogenase, localization within the mesophyll chloromaintain specific redox potentials within plasts of maize. Our data, presented here, their respective compartments. Furtherindicate that the NADP-malate dehydromore, the malate dehydrogenases of the genase of green spinach leaf tissue is localized mitochondria, microbodies, chloroplasts, and in the stroma of intact chloroplasts. cytosol could function in “hydrogen shutThe finding of NADP malate dehydrotles” between or among compartments as genase activity in leaf tissue and demonsuggested by Delbruck et al. (22) and Kaplan stration of localization in chloroplasts is of (23) for mitochondria and cytosol and by interest and significance. Previous data have Heber and Santarius (17) for chloroplasts indicated at least three NAD-malate dehyand cytosol. drogenase isoenzymes in spinach leaf tissue Kinetic properties of the NADP-malate each localized in different subcellular comdehydrogenase of spinach are quite similar partments, viz., mitochondrial, microbody, to the kinetic properties of the NAD-malate and the soluble phase of the cell or cytosol. Hence the data suggest that each major sub- dehydrogenases. At pH 7.5, the apparent cellular compartment in spinach leaf tissue Michaelis constants for oxaloacetate are has a specific protein catalyzing the reduc- 0.06, 0.04, and 0.04 mM for the soluble-, t’ion of oxaloacetate to malate and perhaps mitochondrial-, and microhody-NAD malTABLE
MICHAELIS
I
CONSTANTS (Km) FOR NAl)P-M2~~2\~~
NADP-SPECIFIC
MALATE
163
DEHYDROGENASE
10009 --
6
600-
E
;500.
2.5 . .-c
:.4 "p n g .3 a
NADP-,,
F -4oo=
MDH
.
i\
z.2 rrc 0 .l
U
0
300
.4 z .3;; . .-c
E ;200 E
E
.2; 0 a E
,. c :
L 100 0
.l
it
U
: 0 c G
5
10 Fraction
15
20 Number
25
0 1
FIG. 8. Subcellular localization of NADP-MDH activity in spinach leaf chloroplasts. Upper panel: 1000 g pellet enriched in intact chloroplasts purified by sucrose density gradient centrifugation. Lower panel: 3ooO g pellet enriched in mitochondria and microbodies purified on sucrose density gradients. Chl = chlorophyll, CA = catalase, CO = cytochrome oxidase. The first chlorophyll peak (fractions #21-23) represents stripped chloroplasts and the second chlorophyll peak (fractions 115-16) represents primarily intact chloroplasts in the 1060 g pellet and primarily mitochondria in the 3600 g pellet. The CA peak represents microbodies. The graph indicates NADP-MDH localization in intact chloroplasts.
dehydrogenases respectively (24) ate whereas for the NADP enzyme it was 0.03 mM. For NADH, the soluble, mitochondrial, and microbody forms had apparent Km’s of 0.02, 0.05, and 0.02 mM (24) whereas the NADP form had an NADPH K,,, of 0.07 mM. The dithiothreitol requirement for enzymic activity of NADP-malate dehydrogenase is presently not understood. Johnson and Hatch (4) reported that the maize leaf enzyme was activated in situ by light and
inactivated by darkness. Their inactivated enzyme was activated by dithiotreitol plus MgCL. Furthermore, their data indicated that the enzyme eluted from a Sephadex G-200 column as two or perhaps three peaks. Although our Sephadex elution profiles are somewhat different, multiple peaks were apparent. In our experiments, the fully activated protein eluted as a single, relatively sharp peak. The inactivated protein eluted as three distinct peaks with apparent molec-
164
TING
AND
ular sizes of 29,000 (Peak I) 42,000 (Peak II), and 75,000 (Peak III) molecular weight. The fully activated protein elutes with an apparent molecular weight of 110,000. It is tempting to speculate that the fully activated protein is composed of one Peak I unit and two Peak II units, and Peak III is made up of a Peak I and a Peak II unit. The exact nature of the protein and Sephadex peaks must await further experimentation. It should be made clear, however, that each peak can be activated by incubation in DTT. The question of an artifact is difficult to answer without complete purification. Unpublished results of experiments conducted at the Photosynthesis and Photorespiration Conference held in Canberra, 1970, with Hilary Johnson supported our initial observations that NADP malate dehydrogenase was associated with spinach leaf chloroplasts and that no transhydrogenase or NADPH phosphatase activities were present in the preparation. The data presented in this paper, although not conclusive, do not suggest that the NADP malate dehydrogenase is an artifact. In summary, spinach leaf tissue apparently has a malate dehydrogenase protein requiring NADPH rather than NADH. The protein appears to be localized in the stroma of chloroplasts and has kinetics somewhat similar to the NAD-malate dehydrogenases localized in other subcellular compartments of spinach leaf tissue. ACKNOWLEDGMENTS The expert technical assistance of Miss Shirley A. Reed during this investigation is acknowledged. Discussions with Hilary Johnson and some preliminary experiments conducted with her in the laboratories of M. D. Hatch and N. K. Boardman, CSIRO Plant Industry, Canberra, are also acknowledged.
ROCHA REFERENCES 1. HATCH, M. D., AND SLACK, C. R., Biochem. Biophys. Res. Commun. 34, 589 (1969). 2. HATCH, M. D., AND SLACK, C. R., Annu. Rev. Plant Physiol. 21, 141 (1970). 3. SLACK, C. R., HATCH, M. D., AND GOODCHILD, D. J., Biochem. J., 114, 489 (1969). 4. JOHNSON, H. S., .AND HATCH, M. D., Biochem. J. 119, 273 (1970). 5. ROCHA, V., AND TING, I. P., Arch. Biochem. Biophys. 140, 398 (1970). 6. ROCHA, V., AND TING, I. P., Plant Physiol., 46, 754 (1970). 7. MUKERJI, S. K., AND TING, I. P., Arch. Biothem. Biophys. 131, 336 (1969). 8. TING, I. P., Arch. Biochem. Biophys. 126, 1 (1968). 9. SNYDER, L. R., Chromatog. Rev. 7, 32 (1965). 10. LINEWEAVER, H., AND BURK, D., J. Amer. Chem. Sot. 66,658 (1934). 3, 723 (1964). 11. ACKERS, G. K., Biochemistry, 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 13. MALHLY, A., AND CHANCE, B., Methods Biothem. Anal. 1,357 (1954). 14. SIMON, E. W., Biochem. J. 69,67 (1958). 15. HEBER, U., PON, D. G., AND HEBER, M., Plant Physiol. 36, 355 (1963). 16. HARVEY, M. J., AND BROWN, A. P., Biochim. Biophys. Acta. 173, 116 (1969). 17. HEBER, U. W., AND SANTARIUS, K. A., Biochim. Biophys. Acta 109, 390 (1965). 18. TOLBERT, N. E., OESER, A., YAMAZAKI, R. K., HOGEMAN, R. H., AND KISAKI, T., Plant Physiol. 44, 135 (1969). 19. JACKSON, W. A., AND VOLK, R. J., Annu. Rev. Plant Physiology 21,385 (1970). 20. HOLM-HANSEN, O., PON, N. G., NISHIDA, R., MOSES, V., AND C.~LVIN, M., Physiol. Plant. 12, 475 (1959). 21. WILLIAMSON, D. H., LUND, P., .&ND KREBS, H. A., Biochem. J. 103,514 (1967). 22. DELBRUCK, A., ZEBE, E., AND B&HER, T. H., Biochem. 2.331,273 (1959). 23. KAPLAN, N. O., Bacterial. Rev. 27, 155 (1963). 24. ROCHA, V., AND TING, I. P., Arch. Biochem. Biophys. 147, 114 (1971).