ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 503-510, 1979

NADP-Malic SUM10

ASAMI,2

Enzyme from KAZUAKI

Maize Leaf: Purification

INOUE, AND

Research

Irzstitdefbl-

Biocher~~ical

KEIJI MATSUMOTO, T. AKAZAWA

Regulation, Xagoya Received

and Properties1

School qf’Agriculture, 464, Japatz December

AKIRA Nagoya

Unicersity,

MURACHI, Chikusa,

1, 1978

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C, pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 pmol malate decarboxylatedimg proteinimin at pH 8.4. The enzyme protein had a sedimentation coefficient (sZo.,,) of 9.7 and molecular weight of 2.27 x lo:’ in sucrose density gradient centrifugation, and molecular weight of 2.26 x loj calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 x lo1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO,proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K,,, (malate), 0.4 mM; K,,, (NADP), 17.6 PM; K,,, (Mg”), 0.11 mM. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48”C, and the activation energy was calculated to be 9500 calimol.

The unique dimorphic leaf structure as the combined action of PEP-carboxylase well as the compartmentalized photosyn(EC 4.1.1.31) and of NADP-malate dehythetic enzyme machinery in the C, plants have drogenase (EC 1.1.1.82) is transported to now been well established (1,2). In the leaf tisthe bundle sheath cells (1, 2, 4, 5). Subsue of maize which is commonly classified as sequently malic enzyme (L-malate:NADP NADP-malic enzyme (ME)3 type among oxidoreductase, decarboxylating, EC 1.1. three major classes of C, plants (l-3), malic 1.40) decarboxylate,s the malate molecule acid which is formed in mesophyll cells by (Eq. HI), malate + NADP * This is paper No. 48 in the series “Structure and Function of Chloroplast Proteins.” The research is supported in part by the research grants from the Ministry of Education of Japan (No. 310413), the Toray Science Foundation (Tokyo), and the Nissan Science Foundation (Tokyo). ’ Recipient of Postdoctoral Fellowship of the Japan Society for the Promotion of Science (JSPS), 1978. :I Abbreviations used: DTE, Dithioerythritol; PCMB, p-chloromecuribenzoate; PEP, P-enolpyruvate; RuP,, ribulose-1,5-bisphosphate; TCA, trichloroacetic acid, ME, malic enzyme; Hepes, 4-(2-Hydroxyethyl)-lpiperazineethanesulfonic acid; Tricine, N-Tris(hydroxymethyl)methylglycine; buffer A, 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM EDTA, 5 mM MgCl,, and 10 mM p-mercaptoethanol; buffer B, same as A but mercaptoethanol replaced by 1 ITIM DTE.

pyruvate

* + CO, + NADHP,

HI

and the liberated CO, (HCO,)) is refixed by RuP, carboxylase (EC 4.1.1.39); both enzymes are exclusively localized in the bundle sheath chloroplasts (4-9). It is thus conceivable that the interaction between malic enzyme and RuP,-carboxylase plays a crucial role in the mechanism of CO, concentration, a unique feature of C, photosynthesis. Several investigators have attempted to isolate the malic enzyme from C, plants (10, ll), and the present work reports for the first time the full characterization of NADPmalic enzyme from maize leaf. 503

0003-9861/79/060503-08$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

504

ASAMI MATEIALS

AND

METHODS

Materials The following reagents were used throughout the investigation, Boehringer-Mannheim: NADP, DTE, Na-pyruvate, yeast alcohol dehydrogenase (EC l.l.l.l), cow liver catalast (EC 1.11.1.16), bovine erythrocyte carbonic anhydrase (EC 4.2.1.1), and cow muscle lactate dehydrogenase (EC 1.1.1.27). Pharmacia: DEAE-Sephadex A-50 and Blue-Sepharose CL-6B. Seikagaku Kogyo Company Ltd. (Tokyo): hydroxylapatite and DEAE-cellulose. Wako Chemical Company Ltd. (Osaka): Na-L-malate. Sigma Chemical Company: NADPH, Tris, Hepes, and Tricine. All other chemicals were of reagent grade.

Assay

Method of Malic

Enzyme

The standard assay for malic enzyme activity was carried out spectrophotometrically using a Gilford Model 250 spectrophotometer. The reaction mixture for measuring the forward reaction (malate decarboxylation) contained the following components: Tris-HCl (pH 8.0) (during the purification step) or Hepes-NaOH (pH 7.1, 7.6) or Tricine-NaOH (pH 7.6-8.9) or glytine-NaOH (pH 9.2-10.4), 50 mM; EDTA, 2.5 mM; MgCl,, %mM; NADP, 0.4 mM; L-malate, 25 mM; DTE, 5 mM; and enzyme solution in a total volume of 0.5 ml. The reaction was carried out at 25°C and the absorbance increase at 340 nm was measured. For quantitating the pyruvate formed in the malate decarboxylation reaction, the following procedures were employed. At the end of reaction 50 ~1 of 10% TCA solution was added to the reaction mixture; after 3 min 50 ~1 of saturated NaHCO, solution was added, followed by the addition of 0.1 mM NADH and 0.3 unit of lactate dehydrogenase, and the absorbance decrease at 340 nm was measured. In the backward reaction (pyruvate carboxylation) the reaction mixture contained the following components: Tricine-NaOH (pH 8.4), 100 mM; EDTA, 2.5 mM; MgCl,, 7.5 mM; NADPH, 0.15 mM; pyruvate, 25 mM; HCO,or CO,, 2 mM; DTE, 5 mM; and enzyme solution in a total volume of 0.5 ml. Then the absorbance decrease at 340 nm was measured at the incubation temperature of 10°C. To distinguish the active molecular species between “CO,” and “HCO,-” in the enzyme reaction, the experimental procedure of Cooper et al. (12) was employed.

Isolation

and Purijkation

of Malic Enzyme

Step I: Extraction and (NH,),SO, (SO-70% tion)fractionation. Maize plants (Zea lnays cv. Cross Bantam) were grown in a green house 36°C). Freshly harvested young leaves (50 g, at the 3- to 4-week growth stage, from which

saturaGolden (28 to wet wt) central

ET AL. veins were removed, were immediately pulverized in a mortar using liquid N,. Approximately 100 ml of ice-chilled 100 mM Tris-HCl buffer (pH 7.5, containing 0.1 mM EDTA, 5 mM MgCl,, 5 mM DTE, 2.5% Polyclar AT, and 0.5% isoascorbate), was added to the freshly ground powder, and the mixture was further thoroughly ground for 20 min at 4°C. Immediately after filtration of the resulting homogenate through four layers of cheesecloth and centrifugation of the filtrate at 10,000 g for 10 min, the supernatant fluid was applied to a column of Sephadex G-25 which had been equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM EDTA, 5 mM MgCl,, and 10 mM P-mercaptoethanol (buffer A). DTE solution was added to the eluate so as to reach a final concentration of 1 mM. Then solid (NH&SO, was added, and the precipitate collected at 50-70% saturation was stored at -80°C. This step was repeated 14 times, so that an (NH&SO, cake was obtained from 700 g of leaf tissue. Step II: DEAE-cellulose columrc chromatography. The 50-70% (NH&SO, precipitate was dissolved in buffer B (the same composition as buffer A except that p-mercaptoethanol was replaced by 1 mM DTE) and applied to a column of Sephadex A-25, which had been equilibrated with the above buffer. The eluate was further applied to a column of DEAE-cellulose (4.2 x 34 cm), which had been equilibrated with buffer B. After washing the column with 400 ml of the above buffer containing 0.1 M KCl, the enzyme was eluted with 1600 ml of the same buffer with a linear gradient of 0.1-0.25 M KCl. The malic enzyme fraction (elution peak at 0.13 M KCl) was concentrated by 70% saturation of (NH&SO,, followed by passage through a small column of Sephadex G-25 equilibrated with the above buffer B. Step III: DEAE-Sephadex A-50 co1 umn chromatography. The Sephadex G-25 eluate was applied to a column of DEAE-Sephadex A-50 (2.5 x 21 cm), equilibrated with buffer B, and eluated by 1000 ml of the same buffer with a gradient of 0.1-0.3 M KCl. The enzymically active component (elution peak at 0.16 M KCl) was concentrated again by (NH&SO, saturation at 70%. The (NH,),SO, precipitable fraction was dissolved in buffer B and passed through a column of Sephadex G-25, equilibrated with 50 mM Naphosphate buffer (pH 7.2) containing 0.1 mM EDTA, 5 mM MgCl,, and 1 mM DTE. Step IV: Hydroxylapatite column chromatography. The Sephadex G-25 eluate was applied to a column (2.5 x 10.5 cm) of hydroxylapatite equilibrated with Na-phosphate buffer. After eluting with 100 ml of the above buffer, the enzyme was further fractionated with 400 ml of the same buffer with a gradient of 40- 150 mM Na-phosphate. The enzymically active fractions (elution peak at 100 mM) were concentrated by both Amicon Diaflo (UM-10) and collodion membrane bag, and then applied to a small column of Sephadex G-25, equilibrated with 20 mM Na-phosphate buffer (pH 7.2).

MAIZE

LEAF NADP-MALIC

Step V: Blue-Sepharose CL-6B column chromatography. The above eluate was then applied to a column (1.6 x 10.8 cm) of Blue-Sepharose CL-GB, equilibrated with 20 mM Na-phosphate buffer (pH 7.2) (see Fig. 1). As shown in Fig. 1, most of the malic enzyme was eluted by the above buffer containing ‘75 mM KCl, and the elevation of the KC1 concentration caused elution of the residual enzyme fraction. The initially eluting enzyme fractions were concentrated in a collodion membrane bag up to ca. 3 mgiml and stored at 0°C. The enzyme preparation was found to be stable under this condition, without any loss of the activity for at least 1 month.

Sucrose Density Gradient Centrifugation Sucrose density gradient centrifugation was performed according to the method of Martin and Ames (13) in a Beckman Spinco preparative ultracentrifuge using an SW 65 Ti rotor. The gradient was made from 2.25 ml of each of 5% and 20% sucrose (w/v) dissolved in 50 mM Tris-HCl buffer (pH 7.5)-0.1 mM EDTA. Alcohol dehydrogenase and catalase were included as the internal marker proteins. The centrifugation was carried out at a rotor speed of 32,000 rpm for 17 h at 3X, and at the end of centrifugation aliquots of the fractions were used for the assays of alcohol dehydrogenase by Racker (14) and catalase by Chance and Mahley (15).

Sedimentation Equilibrium

Centrifugation

This was carried out following the procedure described by Yphantis (16), using a Hitachi UCA-1A analytical ultracentrifuge. The sample preparation of malic enzyme (0.9 mg/ml) was exhaustively dialyzed against 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 mM EDTA, 0.5 mM DTE, and 0.1 M NaCl. Centrifugation was done at a rotor speed of 11,450 rpm for 30 h at 20°C. For the molecular weight estimation of the en-

TABLE PURIFICATION OF NADP-MALIC

Purification step (1) Crude extract (supernatant) 50-70% (NH&SO, fraction (II) DEAE-cellulose chromatography (III) DEAE-Sephadex A-50 chromatography (IV) Hydroxylapatite chromatography (V) Blue-Sepharose CL-6B chromatography n Units: Fmol NADP reduced/min (pH 8.0).

ENZYME

505

PURIFICATION

zyme molecule, a partial specific volume of 0.74 was used.

Polyacrylamide

Gel Electrophoresis

The standard polyacrylamide gel (7.5% gel porosity) electrophoresis was carried out following the method described by Davis (17), and the electrophoresis at different gel concentrations (4, 5, 6, and 7.5%) was carried out according to Hedrick and Smith (18). Sodium dodecyl sulfate-gel electrophoresis was conducted according to the method described by Weber and Osborn (19); for determining the molecular u-eight of the monomeric form of malic enzyme, six standard proteins were included.

Analysis of Protein Content Protein content was analyzed by method of Lowry

et al. (20) using serum albumin as a standard. RESULTS

Purity of Malic Enzyme and Molecular Weight

Results of the enzyme purification are summarized in Table I. The final enzyme preparation obtained after passage through a column of Blue-Sepharose CL-6B (Fig. 1) was purified 80-fold from the initial crude extract. The polyacrylamide gel electrophoresis (7.5%) of the enzyme sample showed a single protein band, which exactly coincided with the zymogram pattern as shown in the inset of the figure. Use of gels at five different gel concentrations gave the same results (picture not shown). In sodium I ENZYME FROM MAIZE LEAF

Total protein (mg)

Total units”

Specific activity (units/mg protein)

Purification

Recovery (%)

7370 1164 142 42.4 13.5 5.3

6550 3545 2706 1447 796 277

0.89 3.06 19.1 34.1 59.0 71.1

1 3.4 21.5 38.3 66.3 79.9

100 54.1 41.3 22.0 12.2 5.8

506

ASAMI ET AL.

Blue

Sepharose

CL- 66

1

1))

. . $ 2 06

Volume

of

Eluate

(ml)

FIG. 1. El&ion profile of malic enzyme in Blue-Sepharose CL-6B column chromatography. EXperimental procedures are described under Materials and Methods. Inset photographs: Polyacrylamide gel electrophoresis patterns of purified malic enzyme fraction (shadowed in the figure). (a) Standard gel (7.5%) electrophoresis. (b) Zymogram pattern after immersing the gel piece in the reaction mixture (pH 8.0) of assaying NADP-malic enzyme (see text) containing 0.025% nitroblue tetrazolium and 0.0025% phenazinemethosulfate. (c) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme protein was shown to give a single band, with a molecular weight of 6.3 x 104(Fig. 2). By the procedure of Martin and Ames (15) for sucrose density gradient centrifugation, using two standard protein molecules, i.e., alcohol dehydrogenase (s*~,,~, 7.4) and catacoeffilase (s~~,~,-,11.3), the sedimentation cient (s~,,,~~)of malic enzyme was determined to be 9.7 (Fig. 3); this corresponds to a molecular weight of 2.27 x 105. Results of sedimentation equilibrium centrifugation experiments gave the molecular weight of the enzyme as 2.26 x105 (Fig. 4). The linear slope of (radial distanceY vs log (Ay) indicates that the enzyme protein obtained is homogeneous. From the overall results it is likely that the native form of the maize leaf malic enzyme is comprised of four subunits of identical or very similar size. An extinction coefficient of 0.87 for a 0.1%

02

04

08

Mobility

FIG. 2. Molecular weight estimation by sodium dodecyl sulfate-gel electrophoresis. For experimental details see the text. The following standard protein samples of the known molecular weights were used: phosphorylase a (9.4 x lo?, bovine serum albumin (6.8 x 104), catalase (6.0 x 104), ovalbumin (4.3 x 104), aldolase (4.0 x 104), and alcohol dehydrogenase (3.7 x 10”).

MAIZE

LEAF NADP-MALIC

ENZYME

507

PURIFICATION

Stoichiometq

L 0

20

10 Fraction

M

Number

FIG. 3. Sucrose density gradient centrifugation. For experimental details see the text. Enzyme sample solution, 0.1 ml, containing maize leaf malic enzyme (ME) (0.03 mg), catalase (sZO.,,,11.3) (0.08 mg), and alcohol dehydrogenase (ADH) (s~~,~, 7.4) (0.05 mg), was layered on top of the sucrose gradient. Rotor speed was 32,000 rpm (11 h at 3°C).

solution of pure malic enzyme at 278 nm and an absorbance coefficient of 1.98 x 105. M-‘. cm-’ were determined.

I

It was found that the maize leaf malic enzyme activity was absolutely dependent on NADP, and NAD being totally inactive, Mgz+ was essential.. At equilibrium, starting from 50 pmol of malate and excess NADP in the assay mixture of pH 8.4 as described under Materials and Methods, it was found that the formation of 49.1 pmol NADPH was accompanied by the formation of 49.0 In order to determine pm01 pyruvate. whether CO, or HCO,- was the actual molecular species in the enzyme reaction, the effect of carbonic anhydrase was tested in the reverse react.ion (pyruvate carboxylation). As clearly seen from the results in Fig. 5, the oxidation of NADPH in the presence of HCOap was much faster than that in Con. The prior addition of carbonic anhydrase to the CO, reaction mixture resulted in the faster progress of the reaction, suggesting that HCO,- was the active molecular species involved. Therefore, it is likely that the maize leaf malic enzyme catalyzes the following enzyme reaction (Eq. [2]). malate + NADP+ + HCO,-

+ OH- c pyruvate + NADPH

+ H+

PI

E f

L

I 38

FIG. 4. Sedimentation equilibrium centrifugation of maize leaf malic enzyme. Centrifugation was performed according to the method of Yphantis (16) as described under Materials and Methods, and the plots were made from the centrifugation patterns obtained.

i

FIG. 5. Progress curve for reductive carboxylation of pyruvate catalyzed by maize leaf malic enzyme. Reaction was started at zero time by adding 2 mM HCO:,or CO, at 10°C in the presence or absence of cow erythrocyte carbonic anhydrase (CA.) (1 mg) according to the procedures described by Cooper et al. (12). Other experimental details for the enzyme assays are described under Materials and Methods.

508

ASAMI ET AL.

Ejrect oj’ Temperature

Enzyme activity was measured using the standard assay system at temperatures ranging from 13 to 63°C and the maximal activity was observed at 53°C (Fig. 7). From a linear Arrhenius plot of the data, in a temperature range of 13 to 48”C, the activation energy of the reaction was calculated to be 9500 calimol. EjTect of SH-Reagents and Cyanide

Although neither 1 mM iodoacetate nor 1 oxidized glutathione exhibited any inhibitory effect on the enzyme activity, 1 mM PCMB showed a strong inhibition (97%). However, the inhibition caused by PCMB could be reversed (70%) by postincubation (3 min) of the treated enzyme with 5 mM DTE. A similar observation was reported by the pigeon liver NADP-malic enzyme (22) as well as by the enzyme from CAM plant (23). Cyanide (1 mM) exhibited 40% inhibition of the enzyme activity, and azide (1 mM) also showed a slight (12%) inhibitory effect. mM

FIG. 6. Effect of pH on activity of maize leaf malic enzyme at various concentrations of malate and Mg’+. The same reaction mixture as that described under Materials and Methods was used except for the following: 50 mM Hepes-NaOH (0) for pH 7.1, 7.6; Tricine-NaOH (0) for pH 7.6-8.9; glycine-NaOH (a) for pH 9.2-10.4. Concentrations of malate and Mg2+ were: (A) 25 mM; 25 mM; (B) 1 tTIM; 1 mM; and (C) 1 mM; 5 mM, respectively.

Optimal pH

As shown in Fig. 6, in the presence of 25 mM malate, 25 mM MgC12, and 0.4 lllM NADP, the optimal pH of the enzyme reaction was 8.4 at which point the specific activity was 101 pmol malate decarboxylated/mg proteitimin. However, in agreement with the previous observation by Johnson and Hatch (21), there occurred a shift in the optimal pH of the reaction with changes in malate/MgCl, concentration ratios. At a fixed concentration of NADP, and with 1 mM each of malate and MgC&, optimal pH was 8.4, whereas by the elevating MgCl, concentration to 5 InM it was found that pH optimum shifted to 7.8. K, Values I

I 20

From the results of the enzyme activity measurements at pH 8.4 and in the presence of excess constituent substrate components (25 mM malate, 25 mM MgCL, and 0.4 mM NADP), the following kinetic constants were obtained: K, (malate), 0.4 InM; K, (NADP), 17.6 PM, and K, (MgCl,), 0.11 InM.

I

.

40

Temperature

I

I

60

1 ‘C I

FIG. 7. Effect of temperature on activity of maize leaf malic enzyme. The same reaction mixture as that described under Materials and Methods was used except that 50 mM Tricine-NaOH buffer (pH 8.4) was used. The inset figure shows the Arrhenius plot of the data in a temperature range of 13 to 48°C.

MAIZE

LEAF

NADP-MALIC

DISCUSSION

Among many kinds of C, plants, maize is classified as a typical malate-forming (NADP-ME type) plant. In the organization of carbon assimilation in these plants, the malic enzyme reaction, coupled with RuP,carboxylase, is considered to have an important function. To understand the precise mechanism of interaction of the two enzymes purification is essential. This paper appears to be the first report concerning the isolation of the pure NADP-malic enzyme employing a stepwise purification, including Blue-Sepharose column chromatography at the final step (cf: Table I). The enzyme protein so obtained was judged to be homogeneous and its physicochemical properties were examined. In the NADP-ME type C, plants, malic enzyme is one of the major enzyme constituents and it is often claimed that on the basis of chlorophyll or protein the specific activity of malic enzyme in maize leaf tissue is quite high (5, 6). From the results presented in Table I, it can be estimated that the enzyme comprises 1.2% of the total soluble leaf protein. One can postulate that there exists a close intermolecular interaction between malic enzyme and RuP,-carboxylase during the step of the malate decarboxylation and refixation of CO, in the bundle sheath chloroplasts of C, plants. Dalziel and Londesborough (24) had shown that in the reaction catalyzed by NADP malic enzyme from wheat germ CO, is the reactive species in the pyruvate carboxylation reaction; however, we have now found that HCO,- rather than CO, is released from malate by the maize leaf malic enzyme. It has been demonstrated that RuP,-carboxylase from spinach leaf utilizes CO, as the substrate (25). Although there is no experimental proof available indicating that the same mechanism operating in the RuP,-carboxylase reaction in C, plants, it has been shown that the structural makeup of the maize leaf RuP,carboxylase molecule is similar to the spinach enzyme (8,9). Therefore, it is likely that the role of malic enzyme in maize leaf is to furnish HCO,into the bundle sheath

ENZYME

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PURIFICATION

chloroplasts from rnalate molecules transmesophyll ported from the surrounding cells. The question occurs whether there is carbonic anhydrase activity in the bundle sheath chloroplasts to help the interaction between malic enzyme and RuP,-carboxylase. It has been reported that the activity of carbonic anhydrase in maize leaf, in particular in the bundle sheath cell, is low (26, 27). It is thus conceivable that the high concentration of HC03-(CO,)& situ can be supplied by malic enzyme without the aid of carbonic anhydrase. At the same time we infer that the shift in the pH optimum as a function of malate/Mg*+ ratio may have a significant role in the regulatory nature of the enzyme reaction in the C, photosynthesis (see Fig. 6). Experimental results concerning the detailed kinetic properties of the purified maize leaf malic enzyme in relation to its regulatory properties will be reported in an accompanying paper. ACKNOWLEDGMENTS The authors are very grateful to Dr. K. Asada, Kyoto University, for conducting the sedimentation equilibrium analysis and to Dr. Elizabeth F. Neufeld for critical reading of the manuscript and giving valuable comments. REFE.RENCES 1. HATCH, M. D., AND OSMOND, C. B. (1976) in Encyclopedia of Plant Physiology, New Series (Stocking, E. R., and Heber, U, eds.), Vol. 3, pp. 143-184, Springer-Verlag, Berlin. 2. HATCH, M. D. (1976) in Plant Biochemistry (Bonner, J., and Varner, J. E., eds.), pp. 797-844, Academic Press, New York. 3. HATCH, M. D., KAGA~A, T., AND CRAIG, S. (1975) Aust. J. Plant Physiol. 2, 111-128. 4. SLACK, C. R., HATCH, M. D., AND GOODCHILD, D. J. (1969) Biochem. J. 114, 489-498. 5. ANDREWS, T. J., JOHNSON, H. S., SLACK, C. R., AND HATCH, M. D. (1971) Phytochemistry 10, 2005-2013. 6. BERRY, J. A., DOWNTON, W. J. S., AND TREGUNNA, E. B. (1970) Canad. J. Bat. 48, 777786. 7. HUBER, S. C., HALL, T. C., ANDEDWARDS, G. E. (1976)

Plant

Physiol.

57, 730-733.

8. HATTERSLEY, P. W., WATSON, L., AND OSMOND, C. B. (1977)Aust J. PlantPhysiol. 4,523-539.

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9. MATSUMOTO, K., NISHIMURA, RI., ANDAKAZAWA, T. (1977) Plant Cell Physiol. 18, 1281-1290. 10. PERSANOV, V. M., VORONOVA, E. A., OPARINA, L. A., AND KARPILOV, Yu. S. (1976) Biokhiruhiya 41, 921-925. 11. HATCH, M. D., AND MAU, S. L. (1977) Arch. Biothem. Biophys. 179, 361-369. 12. COOPER, T. G., TCHEN, T. T., WOOD, H. G., AND BENEDICT, C. R. (1968) J. Biol. C&m. 243, 3857-3863. 13. MARTIN, R. G.,AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 14. RACKER, E. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 500-503, Academic Press, New York. 15. CHANCE, B., AND MAEHLEY (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 764-775, Academic Press, New York. 16. YPHANTIS, D. A. (1964)Biochernistry 3,297-317. 17. DAVIS, B. J. (1971) Ann. New York Acad. Sci. 121, 404-427. 18. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164.

ET AL. 19. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 20. LOWRY, 0. H., ROSEBROUGH, J. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193, 265-275. 21. JOHNSON, H. S., AND HATCH, M. D. (1970) Biochern. J. 119, 273-280. 22. CHANG, G.-G., AND Hsu, R. Y. (1977) Biochernistry 16, 311-320. 23. BRANDON, P. C., AND VAN BOEKEL-MOL, T. N. (1973) Eur. J. Biochem. 35, 223-230. 24. DALZIEL, K., AND LONDESBROUGH, J. C. (1968) Biochena. J. 110, 223-230. 25. COOPER, T. G., FILMER, D., WISHNICK, M., AND LANE, M. D. (1969) J. Biol. Chem. 244, 1081-1083. 26. GRAHAM, D., ATKINS, C. A., REED, M. L., PATTERSON, B. D., AND SMILLIE, R. M. (1971) in Photosynthesis and Photorespiration (Hatch, M. D., Osmond, C. B., and Slayter, R. O., eds.), pp. 267-274, Wiley-Interscience, New York. 27. POINCELOT, 340.

R. P. (1972)

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50, 336-