Plant Physiol.

(1972) 49, 117-123

Malate Dehydrogenase Isoenzymes from Cotton Leaves

MOLECULAR WEIGHTS Received for publication July 20, 1971

STEPHEN A. O'SULLIVAN AND RANDOLPH T. WEDDING Department of Biochemistry, University of California at Riverside, Riverside, California 92502
ABSTRACT Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf extracts appear to be charge isomers with a molecular weight of approximately 60,000. A fifth malate dehydrogenase isoenzyme found in leaf extracts has a molecular weight of approximately 500,000. Under appropriate conditions it is possible to form this high molecular weight isoenzyme from at least one of the smaller isoenzymes. In addition, malate dehydrogenase isoenzymes of approximately 700,000 and 130,000 molecular weight have been observed under some conditions, although these isoenzymes do not appear in the crude cotton leaf preparations. The relationship of this heterogeneity with respect to size and to the discrepancies in the number and size of malate dehydrogenase isoenzymes reported from plant tissues may be significant.

been suggested (21, 28, 29) as well as a role in salt uptake by plants (27). The considerable confusion regarding malate dehydrogenase isoenzymes outlined above and the interest which would attach to a better understanding of these isoenzymes prompted this study of the malate dehydrogenase isoenzymes of the cotton plant. Isoenzyme is here broadly defined to include all proteins with malate dehydrogenase activity.

In 1968 only two or three malate dehydrogenase isoenzymes had been detected in plants by a variety of techniques (23). Since then more isoenzymes of malate dehydrogenase have been found in plants, and there is considerable variation in the number which have been reported for the same species. These range from three (25) to twelve (22) in maize, from two (4) to ten (24) in wheat, three (28) or four (21) in spinach, and in certain ornamental plants only one malate dehydrogenase enzyme has been detected (12). In cotton, at least three malate dehydrogenase isoenzymes have been reported (8, 27), but with crude extracts of cotton plants the exact number of isoenzymes could not be ascertained (27). In addition to this variation in the number of malate dehydrogenase isoenzymes which have been reported even from the same plant, a number of workers have found changes in malate dehydrogenase isoenzymes during plant development (5, 12, 14, 15, 27), or that the isoenzymes are localized in specific cell organelles (3, 10, 19, 21, 22, 25, 28), or that they have different molecular weights (5, 11, 15, 25). Malate dehydrogenase isoenzymes have also been used as genetic markers with plants (4, 19, 22, 24), apparently without regard for the difficulty of obtaining reproducible identification of the isoenzymes. One source of interest in the study of isoenzymes is the possible correlation of physiological function with individual isoenzymes. The belief that such a separation of functions may exist for plant malate dehydrogenase stems from the observation that malate exists in two pools in the plant cell (18). Unique metabolic roles in different parts of the plant cell have

MATERIALS AND METHODS Malate dehydrogenase (E.C. 1 . 1 .1 . 37) isoenzymes were obtained from the leaves of cotton (Gossypium hirsutum L., var. Delta Pine 16). Plants were grown in a temperaturecontrolled glasshouse (29 C maximum, 21 C min.) under natural light. Leaves from one group of 6- to 8-week-old plants were generally used. Leaves were excised, and the petioles were placed in distilled water for transport to the laboratory. Extraction commenced within 20 min. Extraction and Ammonium Sulfate Precipitation. For extraction, petioles were removed, 10 g of leaf blades were weighed and counted. The leaves were rolled and cut into pieces approximately 0.5 X 0.5 cm with a scissors. All succeeding operations were performed in the cold at 0 to 4 C. The chopped leaves were placed in an 80-ml container of a Lourdes homogenizer and sprinkled with 5.0 g of Polyclar AT. To this was added 40 ml of extraction buffer consisting of 50 mm tris-HCl, 10 mm mercapthoethanol and 1 mM EDTA at pH 7.5. Homogenization consisted of three periods of 10 sec at 60 v with 20-sec intervals. The ground slurry was squeezed through a nylon bag with a 25 X 35 threads/cm2 mesh and an additional 0.5 g of Polyclar AT was added to the filtrate. This Polyclar and all debris were removed by centrifugation at 5,000g for 10 min at 0 C. Ammonium sulfate precipitation was performed on the supernatant suspension after adjustment to pH 7.0. Sufficient salt to bring the solution to 30% saturation was added over a 20-min period, followed by stirring for 20 min. The precipitate collected by centrifugation was discarded. The percentage of saturation of ammonium sulfate was raised to 80%, and the precipitate was collected by centrifugation at 1 2,000g for 20 min. The fraction precipitating between 30 and 80% ammonium sulfate, which was shown by preliminary experiments to contain all the malate dehydrogenase isoenzymes and 95% of the malate dehydrogenase activity, was resuspended in 50 mm tris-HCI buffer, pH 7.5, containing 10 mM mercapthoethanol. This suspension was centrifuged at 10,000g for 20 min, and the clear supernatant fluid constituted the crude cotton leaf enzyme preparation. Assays for malate dehydrcgenase were carried out at 340 nm in a double beam recording spectrophotometer and a 1-cm light path at 27 C as previously described (26). The assay
117

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O'SULLIVAN AND WEDDING

Plant Physiol. Vol. 49, 1972

mixture contained 50 mm tris-HCl buffer, pH 9.2; neutralized sodium malate, 25.0 mM; DPN adjusted to pH 5.5, 1.0 mM; and the reaction was initiated by the addition to 10 A1 of enzyme. One unit of malate dehydrogenase is defined as an amount of enzyme which brings about the appearance of 1.0 ,umole of DPNH per min. Gel Filtration. The column, Sephadex G-200, Blue dextran, and the reference proteins used for the gel filtration were obtained from Pharmacia Ltd. The column, 1.5 X 90 cm, was poured with deaerated Sephadex G-200 in 50 mm tris-HCl buffer, pH 7.5, containing 0.5 M KCI and was washed with 3 liters of this buffer before each chromatographic run. Samples were applied as recommended by the makers, following and succeeded by an application of 10% (w/v) sucrose prepared in the eluting buffer. Upward flow was used, and 4-ml fractions were collected in a Gilson fraction collector at 4 C. The Sephadex G-200 column was calibrated with aldolase (mol wt 158,000), chymotrypsin (mol wt 75,000), ovalbumin (mol wt 45,000), and ribonuclease (mol wt 13,700). Blue dextran was used to determine the void volume of the column. Samples of enzyme preparation (2 ml) containing 50 to 100 units of malate dehydrogenase activity were applied to the column and a flow rate of 6 ml/hr was maintained by adjusting the buffer reservoir. The column effluent was monitored by a recording UV monitor, and the absorbance at 280 nm was measured in a Beckman model DB-G spectrophotometer. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis was carried out at pH 8.3 essentially according to the method of Davis (7) using gels of different acrylamide concentrations as described by Hedrick and Smith (13). A Buchler polyacrylamide gel electrophoresis apparatus (Polyanalyst Model 3-1750) was used in association with a Buchler power supply, and the gel columns and lower buffer chamber were maintained at 0 to 4 C by circulation of ice water. The running gel was 0.5 x 7.0 cm and was polymerized with ammonium persulfate. The spacer gel was omitted and 10 mM tris-50 mm glycine buffer, pH 8.3, was used for electrophoresis. The sample was mixed with bromphenol blue and added under the electrode buffer to the top of the running gel. Electrophoresis at a current of 3 to 4 ma per tube was continued for 2 hr by which time the bromphenol blue marker front had migrated to within 0.5 cm of the end of the gel. Following electrophoresis, the gels were loosened with a nichrome wire under water and removed from the glass columns. The position of the dye front was marked bv inserting a small piece of wire into the gel (13). Protein bands were detected by staining with either 1% (w/v) amido black in 7% (v/v) acetic acid (7) or with onetwentieth dilution of 1% (w/v) coomassie blue, warmed to 60 C, in 12.5% (w/v) trichloroacetic acid (6). The protein stained gels were washed free of excess dye with 7% (v/v) acetic acid or with 10% (w/v) trichloroacetic acid respectively until the bands were clear. Malate dehydrogenase activity was detected by incubating the gels in the dark at 37 C in a detection medium prepared essentially as described by Fine and Costello (9) and containing neutralized malic acid, 0.83 mM, DPN, adjusted to pH 5.5, 0.18 mM, nitrotetrazolium blue, 0.1 mg/ml; and phenazine methosulfate 5 jtg/ml, prepared in 10 mM tris60 mm glycine buffer, pH 8.3. Colored bands were allowed to develop in the dark, generally for 45 min, and then washed several times with water. The position of each band appearing on all gels and that of the dye front was recorded to within 0.05 cm by means of a transparent ruler. The relative mobility of each band was

calculated and plots made of the log of relative mobility versus gel concentration (13). The slopes of the lines were obtained by fitting the log of relative mobility against gel concentration by means of a linear regression analysis program using a Wang Model 700 calculator. Semipreparative Polyacrylamide Gel Electrophoresis. For semipreparative gel electrophoresis, one sample was run simultaneously on twelve 7% (w/v) acrylamide gels with conditions identical to those described above. Following electrophoresis, representative gels were stained for malate dehydrogenase activity, and the other gels were placed in a Petri dish at 0 C. When the malate dehydrogenase stains had developed in the incubated gels, they were removed, washed and placed beside the unstained gels with the top of the gels, and the position of the bromphenol blue front in line. Regions equivalent to the stained bands were then cut from the unstained gels with a sharp blade. These equivalent band regions were pooled and macerated with a glass rod in either 1 ml of 50 mm tris-HCl-10 mm mercaptoethanol buffer, pH 7.5, or in 1 ml of 50 mm tris-0.3 M glycine buffer, pH 8.3.
RESULTS
In early experiments using polyacrylamide disc gel electrophoresis for separation of malate dehydrogenase enzymes, it was found that the number of isoenzymes detected was somewhat variable. Preliminary experiments revealed the fact that the bands can best be distinguished when between 0.01 and 0.1 unit of malate dehydrogenase activity was applied to each gel. Concentrations above 0.1 unit led to blurring of the stain, and lower concentrations of total malate dehydrogenase revealed only a single band towards the anode. When total activity in this range is applied to each gel, five malate dehydrogenase isoenzymes are reproducibly found in cotton leaf preparations. The distribution of these five isoenzymes (A, B, C, D, and E) is shown in Figure 1 a, where isoenzyme A is the most negatively charged under the conditions used and so has moved furthest towards the anode in this 7% acrylamide gel. Isoenzyme E gives a faint thin band with crude cotton leaf preparations and remains close to the origin under normal electrophoresis conditions in a 7% gel. In addition to these five generally observed malate dehydrogenase isoenzymes, the cotton leaf preparations uniformly contained a material which produced a red band in each gel (indicated by arrow in Fig. 1). The red color of this band was readily distinguishable from the blue formazan deposits given by the substrate-specific malate dehydrogenase isoenzymes. The fact that this red band is nonspecific is indicated by controls in which the red band was produced when malate was absent from the staining mixture. Electrophoretic Determination of Molecular Size of Malate Dehydrogenase Isoenzymes. Using the technique of Hedrick and Smith (13), in which proteins are separated by electrophoresis in gels of varying acrylamide concentration, it is possible to distinguish between charge and size isomers and to estimate the molecular weight of the proteins. The application of this technique to 30 to 80% ammonium sulfate precipitates of cotton leaf extracts is illustrated in Figure 2. Here are shown plots of log Rm1 versus percentage of gel for the five malate dehydrogenase isoenzymes detected in these extracts, plus two other characteristic bands which appear in these gels. Malate dehydrogenase isoenzymes A, B, C, and D appear to be of the same molecular size and to differ only in net charge
' Rm is the ratio of the distance moved by the protein band to the distance moved by bromphenol blue included in the sample

when applied to the gel.

Plant Physiol. Vol.

49, 1972
-pm

MALATE DEHYDROGENASE ISOENZYMES

119

G
E
E

.:.

B A

"

FIG. 1. Polyacrylamide gel electrophoresis of dehydrogenase isoenzymes in 7% acrylamide gels. a i:Cotton leaf extract; b: material excluded from Sephadex G-200 (not concentrated); c: material excluded from Sephadex G-200 (concentrated by precipitation with 80% saturation ammonium s5 ulfate); d: reelectrophoresis of isoenzyme A, showing additional formation of isoenzymye E. Experimental details are described in the text. Different locations of isoenzymes are due to different migration times, and identification is on the basis of Rm value.

slope, and calculated in the same way, its estimated molecular weight is 427,000 + 36,000. Confirmation of this estimate for the molecular weight of isoenzyme E is given by the fact that its slope is consistently closely parallel to that for the nonspecific red band, which is apparently (see below) identiE cal with ribulose diphosphate carboxylase, which has a molecular weight of approximately 500,000. Identity of Nonspecific Red Band. Other reports (14) have indicated the possibility that the nonspecific red band might be due to fraction 1 protein. To check this possibility, crystalline ribulose diphosphate carboxylase from spinach leaves (kindly provided by Dr. N. G. Pon) was run on polyacrylamide disc gels under the conditions used for cotton leaf extracts. When these gels were stained with the detection mixture used for malate dehydrogenase, a red band appeared at the same Rm value as that found for the red band in cotton leaf extracts. When subjected to electrophoresis on gels of varying acrylamide concentrations, the plot of log mobility versus percentage of gel concentration gave the same slope as the nonspecific red band in cotton leaf extracts. When the purified ribulose diphosphate carboxylase was added to cotton leaf extracts and separated by disc gel electrophoresis, only one red band appeared. These observations led to the conclusion A that the nonspecific red band in cotton leaf extracts is ribulose diphosphate carboxylase or fraction 1 protein. To eliminate the possibility that the large molecular weight malate dehydrogenase isoenzyme (E) which migrated close to ribulose diphosphate carboxylase was an artifact due to binding of some of the smaller isoenzymes to Fraction 1 protein, purified ribulose diphosphate carboxylase was mixed with + malate dehydrogenase isoenzymes A, B, and C which had been separated from E by gel filtration on Sephadex G-200. for malate dehydrogenase activity these cottc leaf malate When stained gels h red but formazan deposit ad u no in the soe the e band, ofraa vicinity c)ncetoalafshowed eoi ntevcnt
of the ribulose diphosphate carboxylase. Another nonspecific reaction, which has been previously observed when staining for malate dehydrogenase activity on polyacrylamide gels, is the appearance of sharply defined areas which do not show any background color and which have been termed "anti bands" (12). The molecular weight of one of these anti bands (AB, Fig. 2) was calculated to be ap-

by the similar slopes for these lines. The line for the fifth of the malate dehydrogenase isoenzymes has a larger negative slope, which is significantly different (p < 0.01) from those of the other isoenzymes. The use of the slopes of the log Rm versus p ercentage of gel plots in the estimation of molecular weighi ts under the conditions used in these experiments is illustrat ed in Figure 3. where the slopes of such lines for a series of s tandard proteins are plotted against their known molecu lar weights. The equation fcr the line of this plot (molecul ar weight = -112,012-2,724,805 X slope) provides the meaIns for calculating the molecular weights of the malate dehydirogenase isoas indicated

proximately 15,000. Gel Filtration of Malate Dehydrogenase Isoenzymes. Another method of classifying large molecules such as enzymes according to their molecular size is offered by gel filtration (1). When the 30 to 80% saturation ammonium sulfate precipitate of cotton leaf extract is separated on a column of Sephadex G-200, malate dehydrogenase activity is recovered in three adjoining fractions as shown by the solid line of Figure 4. The first fraction showing malate dehydrogenase activity (fraction I of Fig. 4) contained 2% of the total activity applied to the column and, under the conditions used in Figure 4, did not have the characteristics of a peak. This first fraction was included in the exclusion volume from the column. The second fraction (fraction II of Fig. 4) also contained approximately 2% of the activity applied to the column and, under the conditions used in Figure 4, was eluted as an initial shoulder of the main peak of malate dehydrogenase activity (fraction III of Fig. 4). This third fraction of malate dehydrogenase activity contained approximately 50 to 60% of the activity applied to the column and formed a distinct peak. Calibration of the same column with a series of standard proteins (2, 3. 4, and 5 of Fig. 4) indicated that the predominant peak of malate dehydrogenase activity (fraction III of Fig. 4) corresponded to a molecular weight of approximately 65,000 as shown by the inset in Figure 4 which relates log molecular weight with elution volume.

enzymes.

By this method it was found that the four prin cipal malate dehydrogenase isoenzymes (A, B. C. and D), wvhich appear to differ only in net ch3rge, have molecular weighlts of approximately 60,000. Analysis of variance performed crn the slopes of Hedrick-Smith plots for these isoenzvmes madle over a period of more than 6 months revealed no significanit differences between their slopes. It is therefore assumed thzat they have the same molecular weight, which on the basis of the mean slope for all four isoenzymes (-0.06275) woul d, using the standard equation for molecular weight versuis slope given above, indicate a molecular weight of 59,000 + 5 ,100. Malate dehydrogenase isoenzyme E has a significantly lairger negative

120

O'SULLIVAN AND WEDDING

II
I

O 0-

~~C
I

Plant Physiol. Vol.

49, 1972

II

AB

II 4

I 8

II 10

I
12

Gel Concentration (%)

FIG. 2. Mobility of the five principal malate dehydrogenase isoenzymes from cotton leaf extracts in gels of differing acrylamide concentrations (13). In addition to malate dehydrogenase isoenzymes A, B, C, D, and E, a nonspecific red band (RB) and an "anti band" (AB) are shown. Experimental conditions described in the text.

o10
-2 0

present, but that isoenzyme D was clearly absent from this fraction. When fraction II of the eluate of malate dehydrogenase activity from the Sephadex column was subjected to gel electrophoresis the small isoenzymes A, B, and C were clearly present, but a new and previously unobserved isoenzyme was also revealed. This new malate dehydrogenase isoenzyme is designated as isoenzyme F. On the basis of Hedrick-Smith plots
were
(see

-10

s 6/ _ 4_<

Fig. 5),

this

enzyme

has a molecular

by

slope of -0.089, of 130,000. Isoenzyme F migrated to an Rm similar to that of isoenzyme D in gels of 7 to 10% acrylamide concentration. This fact does not (17) prove that the net charge of F is the same as that of D, but the similar Rm values may be indicative.
a
may

weight,

as indicated

1
o
10

20

30

40

disappearance of isoenzyme D and the appearance of F The during gel filtration is interesting, but it is impossible to
determine whether this have

been

due

to

the

denatura-

Molecular *\eight

(x

10-4)

FIG of Thecrlationshi thesare ofeHe. 1: ick-mith weigh t of standard proteins. ovalbumi; plots to 3o.molecular 2: bovinte serum albumin; 3: pig heart malate dehydrogenase; 4: yeast glucose-6-phosphate dehydrogenase; 5: lactate dehydrogenase; 6: aldlolase; 7: catalase; 8: ferritin; 9: urease; 10: ribulose diphosphate carboxylase. All proteins, with the exception of number 10, were c:ommercial preparations.

tion of isoenzyme D, or whether isoenzyme D may have been converted to F. If the latter had occurred, this might account
for the 40 to 50% loss of total enzyme

activity
of

on

the column.

Isoenzyme E, with a molecular weight approximately 500,000, was found in the exclusion volume of the Sephadex
G-200 column (fraction
I), and gave the most intense stain

Pollyacrylamide gel electrophoresis of the predominant peak of m;alate dehydrogenase activity from the Sephadex G-200 colunin (fraction III) revealed the presence of only the small (65,0(D0) charge isomers of malate dehydrogenase. Neither the latrger isoenzyme (E) nor the nonspecific red band could be de tected in this fraction. Identification of the individual small malate dehydrogenase isoenzymes present was somewhat difficualt because of blurring of the stained bands, but on the basis of intercepts of Hedrick-Smith plots, it was determined

that rnalate dehydrogenase isoenzymes A, B, and possibly C

precipitation with 80% saturation ammonium sulfate, four other malate dehydrogenase isoenzymes in addition to isoenzyme E were observed by polyacrylamide gel electrophoresis (Fig. lc). The molecular weights of the four additional isoenzymes were found, by Hedrick-Smith plots (Fig. 6) to correspond approximately to 700,000 (G), 120,000 (F) and two of 60,000 which were identified on the basis of their HedrickSmith plot intercepts as A and B. That such a wide range of molecular sizes of malate dehydrogenase isoenzymes was found in the exclusion volume of a Sephadex G-200 column suggests that the smaller molecular

when this fraction was subjected to polyacrylamide gel electrophoresis (Fig. I b). When this fraction was concentrated by

Plant Physiol. Vol. 49, 1972

0.5
-

MALATE DEHYDROGENASE ISOENZYMES

121

Cu

0.

E 0.4
0

20

@n Li
m
w

10d
Co

0.4
w

et
Lo

0.2-

I10

4.

em
o-,

0 L.
14-

I4V- m

Volume (ml)
FIG. 4. Gel filtration of standard proteins and cotton leaf extracts on Sephadex G-200. Shown are the elution volume of blue dextran, 1 and a series of standard proteins; 2: aldolase; 3: chymotrypsin; 4: ovalbumin; and 5: ribonuclease A. The solid line shows the elution profile of malate dehydrogenase activity from cotton leaf extract. The elution volumes of malate dehydrogenase activity included in fractions I, II, and III are indicated below the ordinate (see text).
1I

2 1-

2
x4
0

I
0

I
4

10

12

Gel Concentration (%)

FIG. 5. Polyacrylamide gel electrophoresis of traction II from Sephadex G-200 gel filtration of cotton leaf extract. Hedrick-Smith plot (13) shows isoenzyme B (mol. wt. 60,000) and new isoenzyme F (mol. wt. 120,000),

weight isoenzymes had arisen during the chromatography or concentration by dissociation of the larger molecular weight species, since the smaller molecules originally present in the sample should have been found in subsequent fractions.

The possibility of interconversion of the various molecular sizes of malate dehydrogenase isoenzymes is given some credibility by a previous report (2) that re-electrophoresis of a high molecular weight material with malate dehydrogenase activity

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O'SULLIVAN AND WEDDING

Plant Physiol. Vol. 49, 1972

T
2

0 0
-

09
OL

(Z
RB

0ol
0

I
I I

-J

10

12

Gel Concentration (%)

FIG. 6. Polyacrylamide gel electrophoresis of fraction I from Sephadex G-200 gel filtration of cotton leaf extract after concentration by ammonium sulfate precipitation. Fraction I is shown to contain isoenzymes A and B (mol. wt. 60,000); isoenzyme E (mol. wt. 500,000); nonspecific red band (RB-ribulose diphosphate carboxylase, mol. wt. 500,000); and new isoenzymes F (mol. wt. 120,000) and G (mol. wt. 700.000).
gave rise to the original high molecular weight material together with a malate dehydrogenase isoenzyme of approximately 65,000 mol wt. These observations led to attempts to bring about such interconversions. Homogeneity and Interconversion of Isoenzymes. When the five principal isoenzymes (A, B, C, D, and E) from cotton leaf extracts were separated on polyacrylamide gel by the semipreparative electrophoretic technique described under "Materials and Methods," and extracted from the gels in 50 mm tris-HCl buffer at pH 7.5 containing 10 mm mercaptoethanol, re-electrophoresis of the five isoenzymes separately resulted in their migration to their original Rm values as single isoenzyme bands. The activity of the malate dehydrogenase isoenzymes separated in this way decreased rapidly on storing at 0 C. After several days, re-electrophoresis led to smearing on the gels so that the presence of additional isoenzyme bands could not be ascertained. Attempts were made to bring about the interconversion of one isoenzyme to another by freezing and thawing, acidifying and neutralizing or by heating for 1 min at 60 C the isoenzymes separated by semipreparative gel electrophoresis. None of these attempts were clearly successful in producing a change in the number or identity of isoenzymes detected on subsequent gel electrophoresis. When buffers other than tris-HCl were used for extraction of the isoenzymes from the gel after the original separation, some of the original five isoenzymes disappeared, but only one buffer resulted in the appearance of additional isoenzymes. When isoenzyme A was extracted in 50 mm tris-0.3 M glycine buffer, pH 8.3, re-electrophoresis revealed the presence of both the original isoenzyme A and isoenzyme E of approximately 500,000 molecular weight (Fig. ld). Further interconversions of one isoenzyme size to another was observed when isoenzymes B and C, extracted in 50 mm tris-0.3 M glycine, pH 8.3, were subjected to re-electrophoresis. In each case, an isoenzyme of higher molecular weight was found in addition to the original 60,000 mol wt isoenzyme. In these cases Hedrick-

Smith plots indicated that the new isoenzymes lecular weight in the range 120,000 to 170,000.

were

of

a mo-

DISCUSSION Malate dehydrogenase isoenzymes of varying molecular weights have been reported from a variety of plants. Habig and Racusen (11) reported two forms of malate dehydrogenase from wax bean leaves with molecular weights of 69,000 and 275,000. The larger form was dissociable to active units of 69,000 molecular weight. A large molecular weight (about 145,000) malate dehydrogenase was also reported from soybean leaves (5) and the glyoxysomal malate dehydrogenase isoenzyme from castor bean plants were found to exist, "in some sort of complex or aggregate" (3). In duckweed, malate dehydrogenase isoenzymes with molecular weights estimated as 34,000, 68,000, and 136,000 were found. There were differences in the responses of these three forms to activation by Ca2+, and the relative distribution found in plants appeared to be controlled by the level of calcium in the medium on which the plants were grown. In this case, the authors concluded that there was no evidence that the changes in the properties of the various malate dehydrogenase isoenzymes was due to the synthesis of distinct isoenzymes (15). The majority of reports of malate dehydrogenases from plants, however, indicate that even when several isoenzymes are found, they are of a size in the range 60.000 to 70,000 mol wt. Gel filtration studies of malate dehydrogenase from potato, cauliflower, and milkweed (20) and from maize extracts (16) indicate malate dehydrogenase isoenzymes of similar molecular size. In Vicia faba, when malate dehydrogenase isoenzymes were found by gel filtration to be of higher molecular weight than 70,000, the interpretation was that these were due to malate dehydrogenase activity associating itself with other proteins (2). Although the malate dehydrogenase isoenzyme of 500,000 mol wt (E) found here may be unique to cotton, there is some

Plant Physiol. Vol. 49, 1972

MALATE DEHYDROGENASE ISOENZYMES

LITERATURE CITED

123

malate dehydrogenases with different fractions of cell organelles could represent a fractionation of malate dehydrogenases on the basis of differing sizes. It is interesting that malate dehydrogenase isoenzymes associated with organelle fractions migrate in starch gel electrophoresis in the sequence, measured from the cathode towards the anode, microbody malate dehydrogenase, mitochondrial malate dehydrogenase, and cytoplasmic malate dehydrogenase (19, 21, 22, 28). In the present work it has been shown that the order in which different sizes of malate dehydrogenase isoenzymes migrate on 7 to 10% polyacrylamide gels at pH 8.3 is a reflection of their molecular size and that this order, as measured from the cathode toward the anode, includes malate dehydrogenases of molecular size 700,000 (G), 500,000 (E), 130,000 (F) and four charge isomers (D, C, B and A) which migrate furthest towards the anode. We have also found some indications that apparent differences in the isoenzyme composition of cellular organelle fractions may be an artifact of different total amounts of malate dehydrogenase activity associated with different fractions. When equivalent total amounts of malate dehydrogenase activity are applied to the gels, these differences in zymogram patterns may tend to disappear. Whereas cotton leaf malate dehydrogenase isoenzymes could represent an atypical situation, there is presently little reason to think that this is the case. Rather it seems probable that in other plant extracts different malate dehydrogenase bands separated by gel electrophoresis may represent mixtures of charge and size isomers of the enzyme which are at least to some degree interconvertible. There seems little doubt that some interconversion could result as an artifact of the extraction and electrophoretic or gel filtration process so that great care should be exercised in comparisons of malate dehydrogenase isoenzymes from different plants, or even from organelles of the same plant until it is certain that technical procedures may not be in some measure responsible for the observed results.

weight malate dehydrogenase which failed to migrate under conditions of electrophoresis or enzyme activity associated with mitochondrial fragments which were unable to penetrate the gel" (10). The present work shows that malate dehydrogenase isoenzyme E remains close to the origin under usual 7 to 10% polyacrylamide gel electrophoresis conditions. A band in similar position on 7.5% polyacrylamide gels has also been noted in barley (29) and in wheat (14), where it was thought to be "apparently a non-specific stain of protein aggregate that occurs in the leaf extracts of many plants." The high molecular weight malate dehydrogenase from cotton leaf extracts (isoenzyme E) gives a faint band when the total activity of malate dehydrogenase in the extract applied to the gel is maintained between 0.01 and 0.1 unit. Malate dehydrogenase isoenzyme bands which are "fainter than any other band," have been reported for the malate dehydrogenase associated with the glyoxysomal fractions of corn, and these also remain close to the origin in starch gel electrophoresis at pH 8.0 (22). In other cases (19, 21, 22, 28), malate dehydrogenase isoenzymes which remain close to the origin in starch gel electrophoresis at pH values between 7.0 and 8.0 have been associated with the glyoxysomal or microbody fractions of plant cells, but there has been no report that the association of
our

reason to believe that similar malate dehydrogenase isoenzymes may occur in other plants. A malate dehydrogenase isoenzyme from barley extracts which remained at the interface of a polyacrylamide gel was reported to be "either a high molecular

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