ABSTRACT. Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chro-.
Plant Physiol. (1979) 63, 903-907 0032-0889/79/63/0903/05/$00.50/0
Isoenzymes of Pyruvate Kinase in Etioplasts and Chloroplasts' Received for publication September 6, 1978 and in revised form January 12, 1979
ROBERT J. IRELAND, VINCENZO DELUCA, AND DAVID T. DENNIS Department of Biology, Queen's University, Kingston, Ontario K7L 3N6 Canada Preparation of Total Leaf Pyruvate Kinase. Approximately 5 g of deribbed leaf tissue was cut up with scissors and homogenized for 30 s in a Polytron homogenizer (setting 5) in 15 ml of 10 mm imidazole (pH 7.1), containing 20%o (v/v) ethylene glycol, 2 mm DTT, and 2 g PVP. The homogenate was filtered through eight layers of cheesecloth and centrifuged at 25,000g for 10 min. The supematant was subjected to ion filtration chromatography on DEAE-Sephadex A-25 (7, 13). The gel was washed before use with NaOH, HC1, and EDTA (2) and then equilibrated with 10 mM imidazole (pH 7.1), containing 20%1o (v/v) ethylene glycol and 2 mM DTT. A column (2.5 x 70.0 cm) was poured and washed with the same buffer. The sample (12 ml) was applied and eluted with the same buffer containing 0.5 M KCI. The flow rate was 1.5 ml/min, and 3.5-ml fractions were collected. In some experiments, the filtered homogenate was precipitated with 50%1o ammonium sulfate, centrifuged, and the pellet resuspended in the column equilibration buffer prior to chromatography. Ammonium sulfate precipitation did not affect the elution profile of the two isoenzymes. Preparation of Pyruvate Kinase from Plastids. Approximately 40 g of castor bean leaf or etiolated pea leaf tissue was homogenized for 6 s in a Waring Blendor with 200 ml of 45 mm Mes buffer (pH 6.4), containing 0.33 M sorbitol, 10 mm NaCl, I mM MgC12, 1 mm MnCl2, 2 mm EDTA, 0.5 mm KH2PO4, 2 mM ascorbate, and 2 mm DTT. The homogenate was filtered through eight layers of cheesecloth and centrifuged at 3,000g for 30 s. The pellet was resuspended in 50 ml of the above buffer using a cotton swab, and again centrifuged at 3,000g for 30 s. The pellet was then resuspended in 12 to 15 ml of 10 mm imidazole (pH 7.1), containing 20%o (v/v) ethylene glycol and 2 mm DTT, using a Thomas hand homogenizer. After centrifugation at 12,000g for 10 min, the supernatant was chromatographed on a DEAE-Sephadex column as described above. Pyruvate Kinase Assay. The assay mixture for pyruvate kinase contained 0.5 mm ADP, 0.5 mm P-enolpyruvate, 10 mM MgCl2, 40 mM KC1, 0.1 mm NADH, 3 units lactate dehydrogenase, and 50 mM Tes-NaOH (pH 7.9) in a total volume of 1.0 ml. Enzyme activity was determined by following the decrease in A at 340 nm in a Gilford-modified Beckman DU spectrophotometer. It was sometimes necessary to correct the rate for a phosphatase activity by subtracting the activity found in the absence of ADP. All enzyme assays were at 25 C. Chemicals and Equipment. KCI concentrations were measured with a Radiometer CDM2f conductivity meter. Chemicals were obtained from Fisher or Sigma, and the imidazole was recrystallized from ethyl acetate before use.
ABSTRACT Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localzed in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity.
Fatty acid synthesis in developing castor bean endosperm occurs in the plastid2 fraction (20), and the entire pathway for the conversion of hexoses to long chain fatty acids is present in this organelle (10, 14, 18, 19). The enzymes of the glycolytic pathway are present in both the cytosol and plastids (13). Similarly, in jojoba beans, there appear to be two compartments for the synthesis of acetyl-CoA from hexoses (11). Recently, isoenzymes of pyruvate kinase (ATP: pyruvate P-transferase, EC 2.7.1.40), having different regulatory properties, have been isolated from the plastids and cytosol of developing castor bean endosperm (3, 4). Chloroplasts are known to synthesize fatty acids (15). Isolated chloroplasts will incorporate "4CO2 into fatty acids, suggesting that the enzymes for the conversion of P-glyceric acid to acetyl-CoA are present (8, 9). A similar conclusion was derived from the incorporation of 3H20 into fatty acids by chloroplasts (17). There also appear to be different pools and origins of acetyl-CoA in the leaf tissue (6). It has been suggested that pyruvate kinase is present in chloroplasts (1). Recently, a specific isoenzyme of P-fructokinase has been found in chloroplasts (5); when an enzyme occurs in two distinct compartments, isoenzymes are usually present ( 12). In this report it is shown that pyruvate kinase is present in both the cytosol and plastids in leaf tissue. The enzymes from these two sources can be separated by DEAE-ion filtration chromatography (7, 13), suggesting that isoenzymes of this activity are present in leaves.
MATERIALS AND METHODS Plant Materials. Castor bean plants, Ricinus communis L., variety Baker 296 (Baker Castor Oil Company, Plainview, Tex.) were grown in a greenhouse, and expanding leaves were harvested. RESULTS Pea plants, Pisum sativum L., variety Little Marvel (Stokes Seeds Pyruvate kinase from plastids is so unstable3 that it can only be Ltd., St. Catherines, Ontario) were grown in Vermiculite for 5 to detected when ethylene glycol and either DTT or 2-mercaptoeth7 days in total darkness, and their primary leaves harvested. anol are present during organelle breakage. Due to the sharp pH
'Supported by National Research Council of Canada Grant No. 5051.
2
The term proplastid has been used previously, but since these organelles are mature plastids specialized for fatty acid biosynthesis, and which will never develop into chloroplasts, the term plastid is more appropriate.
3 Partially purified pyruvate kinase from castor bean endosperm plastids has a half-life of approximately 3 min in the absence of dithiothreitol and
ethylene glycol or glycerol.
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IRELAND, DELUCA, AND DENNIS
optimum of the plastid enzyme, it is also necessary to perform the GDP, UDP, and IDP, at about half the rate obtained with ADP. assay at pH 7.9. In one experiment, spinach chloroplasts were PK2, however, has extremely low activity with nucleotides other isolated by sucrose density gradient centrifugation of broken than ADP. Two peaks of pyruvate kinase activity were obtained from protoplasts, and were found to contain pyruvate kinase. In all other experiments, plastids were prepared by gentle grinding and isolated chloroplasts on ion filtration chromatography (Fig. 3). differential centrifugation. These eluted at the same conductivities and showed the same Pyruvate Kinase from Castor Bean Leaves. Ion filtration chro- properties as PK, and PK2 obtained from a total leaf extract. The matography of a total extract of castor bean leaves produced two PK, peak is reduced compared with the total extract, but is still peaks of pyruvate kinase activity (Fig. 1). The first peak, PK1, fairly large. eluted at 3.5 mmho, and the second, PK2, at 8 mmho. The two Pyruvate Kinase from Pea Leaves. A total extract of etiolated isoenzymes have different pH profiles (Fig. 2). PK, has a broad pea leaves gave two peaks of pyruvate kinase activity when optimum from pH 6 to 9, whereas PK2 is much sharper, with an chromatographed on DEAE-ion filtration column (Fig. 4). These optimum at pH 8 and very little activity below pH 7. The two peaks eluted at the same conductivities as PK, and PK2 and had enzymes are also different in their ability to use alternative nu- properties similar to the isoenzymes from castor bean leaf. The cleotide substrates (Table I). At a nucleotide concentration of 0.5 first enzyme, PK,, had a broad pH profile and was able to use mM, PK, has maximal activity with ADP, but could also utilize alternate substrates, whereas the second enzyme, PK2, had a
PK1
E
0.02
15
/ PK2
/ 0
-
A/
co/\
10>E
10 >-
w 4
zI-I ~e 0.01 w
z
-
4 0.
45
35 FRACTION NO.
25
FIG. 1. Ion filtration chromatography of a total extract of castor bean leaves. (
): Pyruvate kinase activity; ----): conductivity.
0.02 LO
C-
PK2
E~~~~~~~~~~~~~~~~~~ C,)
0.01
P
1
Cf)
/K
W
I-J D~~~~
a.~~~ 6
7
8
pH FIG. 2. Effect of pH on activity of isoenzymes of pyruvate kinase, PK, (---) and PK2 (
9
) from separation shown in Figure 1.
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Plant Physiol. Vol. 63, 1979
TABLE
The relative efficiency of various nucleotides as phosphoryl acceptors in the pyruvate kinase reaction
I.
In the presence of ADP, peak 1 (PK1) gave a rate of 0.013 and peak 2 (PK ) a rate of 0.010 A340 /min/50pl. Relative Activity
Nucleotide (0.5mM)
Peak 1
Peak 2
ADP GDP IDP UDP
100 44 56 40
100 15 8 0
1-
15
E C3 r-
o
0.02
E
0
-4
10 E
cn z
0.01
5
0 z
w
0
CZ 4L
FIG. 3.
Ion
FRACTION NO. filtration chromatography of an extract from castor bean chloroplasts. (
): Pyruvate kinase activity;
E 0
.E
0
m
E Cv 4 Co 4
-
w
cn z
z 0 u
cL
20 FIG. 4.
Ion
30
FRACTION NO. filtration chromatography of a total extract of etiolated
40 pea
leaves.
---
-): conductivity.
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IRELAND, DELUCA, AND DENNIS
Plant Physiol. Vol. 63, 1979
narrow pH optimum and was less efficient at using nucleotides the column separation of PK, and PK2 indicates that PK2 is not other than ADP. Similarly, green pea leaves showed two peaks of a minor activity in the leaf and may represent 50% of the total pyruvate kinase activity on DEAE-ion filtration chromatography. activity. The elution profile of pyruvate kinase obtained from isolated etioplasts had only one peak of pyruvate kinase activity (Fig. 5) DISCUSSION which eluted at a similar conductivity to PK2 and possessed the These results show that both castor bean leaves and etiolated same sharp pH profile (Fig. 6) and inability to use alternate nucleotides. Unlike the extract from castor bean chloroplasts, pea leaves have two isoenzymes of pyruvate kinase with distinct etioplasts showed no contamination with PK, indicating that in properties. In each plant one isoenzyme is less negatively charged, this case PK, is confined to the cytosol. eluting at a lower conductivity from an ion filtration column, has Relative Amounts of PK1 and PK2 in Leaf Tissue. It is difficult a broad pH profile, and is able to function with nucleotides other to determine accurately the relative amounts of PK, and PK2 in than ADP. This isoenzyme has been designated PK,. The second leaf tissue because there is a large amount of phosphatase in the isoenzyme, PK2, has a narrow pH profile and uses alternate crude extracts which interferes with the assay. On the DEAE-ion nucleotides much less effectively. It has been shown that in filtration colunm this phosphatase elutes at a slightly higher etiolated pea leaves PK, is solely in the cytosol. It has not been conductivity than PK2 but there is considerable overlap in the possible to prove that PK2 is confined to the plastid because there peaks. There may also be differences in recoveries of PK, and PK2 is always some breakage of the organelles on extraction. In castor during the extraction procedure. An estimate of the peak areas on bean endosperm there is a correlation between the amount of PK2 15 Oft
//
o 0.008 _ .E
10 /
10
4 z
Y
/~~~~~
__
I-
w 4
_
Az
_
A
O
0
FRACTION NO. FIG. 5. Ion filtration chromatography of an extract from isolated etioplasts of pea
leaves.
0.01
E cm
E 0 v
4 cn z
0.005 I
I-
> 4 03
6
9 8 pH FIG. 6. Effect of pH on activity of etioplast pyruvate kinase activity from separation shown in Figure 5. 7
Plant Physiol. Vol. 63, 1979
ISOENZYMES OF PYRUVATE KINASE IN PLASTIDS
and ribulose- 1,5-bisP carboxylase (a plastid marker enzyme) found in the soluble phase and in the plastids (3, 14). In this case, therefore, it has been assumed that all PK2 activity is in the plastid. In the castor bean leaf a significant amount of PK1 sediments with the chloroplasts so that in this case PK1 appears not to be solely confined to the cytosol. The two isoenzymes are very similar in their properties to the two pyruvate kinase isoenzymes isolated from castor bean endosperm (3, 4). It is interesting to note that the plastid enzyme is inactive below pH 7, and fully active at pH 8. In the chloroplast, pyruvate kinase would be inactive in the dark, but fully active in the light, when stromal pH is increased to about pH 8 (16). Since it has also been demonstrated that fatty acid biosynthesis occurs in the light but not in the dark (17), it appears that chloroplast pyruvate kinase is a light-controlled regulatory enzyme at an early stage of chloroplast fatty acid biosynthesis. Acknowledgment-The authors would like to thank Dr. K. C. Woo for the isolation of spinach chloroplasts by density gradient centrifugation of broken protoplasts. LITERATURE CITED 1. BIRD IF, MJ CORNELIUS, TA DYER, AJ KEYS 1973 The purity of chloroplasts isolated in nonaqueous media. J Exp Bot 24: 211-215 2. BREWER JM, AJ PESCE, RB ASHWORTH 1974 Experimental Techniques in Biochemistry. Prentice-Hall, Englewood Cliffs, NJ, p 5 3. DELUCA V 1978 Pyruvate kinase isoenzymes in plants: purification and characterization from castor bean endosperm. M Sc thesis. Queen's University. Kingston. Ontario 4. DELUCA V, DT DENNIS 1978 Isoenzyme of pyruvate kinase in proplastids from developing castor bean endosperm. Plant Physiol 61: 1037-1039 5. KELLY GJ, E LATZKO 1977 Chloroplast phosphofructokinase. 1. Proof of phosphofructokinase
activity in chloroplasts. Plant Physiol 60: 290-295
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6. KENT SS 1977 On the metabolic relationship between the Calvin cycle and the tricarboxylic acid cycle. IV. A plant survey for anomalous acetyl coenzyme A. Plant Physiol 60: 274-276 7. KIRKEGAARD LE 1973 Gradient sievorptive chromatography. A focussing system for the separation of cell components. Biochemistry 12: 3627-3632 8. MURPHY DJ, RM LEECH 1977 Lipid biosynthesis from I'4Clbicarbonate, 12-"Clpyruvate and [1-'4Clacetate during photosynthesis by isolated spinach chloroplasts. FEBS Lett 77: 164168 9. MURPHY DJ, RM LEECH 1978 The pathway of ['4C]bicarbonate incorporation into lipids in isolated photosynthesizing spinach chloroplasts. FEBS Lett 88: 192-19% 10. NAKAMURA Y, M YAMADA 1974 Long chain fatty acid biosynthesis in developing castor bean seeds. 1. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system. Plant Cell Physiol 15: 37-48 11. OHLROGGE JB, MR POLLARD, PK STUMPF 1978 Studies on the biosynthesis of waxes by developing jojoba seed tissue. Lipids 13: 203-210 12. SCHNARRENBERGER C, M TETOUR, M HERBERT 1975 Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons. Plant Physiol 56: 836-840 13. Simcox PD, DT DENNIS 1978 Isoenzymes of the glycolytic and pentose phosphate pathway in proplastids from the developing endosperm of Ricinus communis. Plant Physiol 61: 871-877 14. SIMcox PD. EE REID, DT CANVIN, DT DENNIS 1977 Enzymes of the glycolytic and pentose phosphate pathways in proplastids from the developing endosperm of Ricinus communis L. Plant Physiol 59: 1128-1 132 15. STUMPF PK 1975 Biosynthesis of fatty acids in spinach chloroplasts. In T Galliard, El Mercer, eds, Recent Advances in the Chemistry and Biochemistry of Plant Lipids. Academic Press. New York, pp 95-113 16. WERDEN K, HW HELDT, M MILOVANCEV 1975 The role of pH in the regulation of carbon fixation in the chloroplast stroma, studies of CO2 fixation in the light and in the dark. Biochim Biophys Acta 396: 276-292 17. YAMADA M, Y NAKAMURA 1975 Fatty acid biosynthesis by spinach chloroplasts. II. The path from PGA to fatty acids. Plant Cell Physiol 16: 151-162 18. YAMADA M, Q USAMI 1975 Long chain fatty acid biosynthesis in developing castor bean seeds. IV. The synthetic system in proplastids. Plant Cell Physiol 16: 879-884 19. YAMADA M, Q USAMI, K NAKAJIMA 1974 Long chain fatty acid biosynthesis in developing castor bean seeds. II. Operation of the path from sucrose to long chain fatty acids in a subcellular particulate system. Plant Cell Physiol 15: 49-58 20. ZILKEY BF, DT CANVIN 1972 Localization of oleic acid biosynthesis in the proplastids of developing castor bean endosperm. Can J Bot 50: 323-326
