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On methods for the isolation of mitochondria from etiolated corn shoots
Plant Science Letters, 11 (1977) 99--104 © Elsevier/North-Holland Scientific Publishers Ltd.
99
ON METHODS FOR THE ISOLATION OF MITOCHONDRIA FROM ETIOLATED CORN SHOOTS
D.A. DAY* and J.B. HANSON
Department of Botany, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received July 26th, 1977) (Revision received and accepted October 4th, 1977)
SUMMARY
Mitochondria were isolated from etiolated corn shoots by two methods, one involving gradient purification and the other by centrifuging through a 0.6 M sucrose "cushion". There were no major differences in State 3 or State 4 malate oxidation rates, respiratory control ratios, ADP:O ratios, outer membrane integrity, or swelling rates in salt solutions. The slightly superior quality of cushion mitochondria probably reflects the shorter time required for isolation. Corn mitochondria isolated by both methods showed passive swelling in KC1 solutions but this does not appear to reflect anything abnormal about membrane integrity.
INTRODUC~ON
The integrity and purity of plant mitochondrial suspensions have received considerable attention in the literature during the past decade [1--3]. In attempts to achieve high quality, homogeneous preparations, lengthy isolation procedures, often involving density gradient centrifugation, have been developed [1,3]. While these methods may result in the isolation of intact and pure organelles, it is not always expedient to use them. The present study compares two methods of isolating mitochondria from etiolated corn shoots and shows that a second low speed centrifugation step, and the use of a 0.6 M sucrose "cushion" during washing, eliminate the need for gradient purification. Both
* Present address: Department of Biology, University of California, Los Angeles, Calif. 90024, U.S.A. Abbreviations: BSA, bovine serum albumin; EGTA, ethyleneglycol bis-(~-aminoethylether)N,N'-tetraacetic acid; TES, N-tris-(hydroxymethyl)methyl-2-aminoethanesulphonicacid.
I00 cushion and gradient mitochondria are permeable to high concentrations of KCI. MATERIALS A N D M E T H O D S Corn seedlings(Zea mays L., W F 9 × M14) were grown in the dark at 29°C on paper toweling saturated with a 0.1 M CaCl2 solution.Mitochondria were isolated from 3 or 4.day old etiolatedshoots essentiallyby the method of Hanson [4]. Approximately 100 g of tissuewas ground in an ice-coldmortar with 200 ml of 0.4 M sucrose, 50 m M KH2PO~, 5 m M E G T A and 0.1% B S A (the medium being adjusted to p H 7.6 with KOH). The homogenate was squeezed through 4 layers of cheesecloth,centrifugedat 1000 g for 10 rain, and then at 12 000 g for 10 rain to collectthe mitochondria. The pelletwas resuspended in 0.4 M sucrose and again centrifugedat 1000 g for 10 rain. The resultant supernatant was treated in one of the following manners: (1) for "cushion" mitochondria it was underlaid with 20 ml of 0.6 M sucrose and centrifuged at 10 000 g for 20 rain (the pelletbeing resuspended in 1 ml of 0.25 M sucrose + 0.1% BSA); (2) for "gradient" mitochondria it was layered on top of the discontinuous sucrose gradientdescribed by Douce et al. [3], although TES replaced phosphate buffer, and centrifugedfor 60 rain at 41 000 g in a Beckman L5 ultracentrifuge(SW 27.1 rotor).The fractionfound at the 1.2--1.45 M sucrose layerswas collectedwith a syringe whose tip had been bent, and carefully diluted over a 30-rain period by dropwise addition of 10 m M TES buffer (pH 7.2) and 0.1% BSA. The dilutedmitochondria were centrifuged at 10 000 g for 10 rain and resuspended in 0.25 M sucrose + 0.1% BSA. All operations were carried out at 2---4°C. Oxygen consumption was measured as described elsewhere [4] in 4.0 ml of standard reaction medium which consisted of 0.25 M sucrose, 10 m M TES buffer, 5 m M MgCI2, 5 m M KH2PO4 and 0.1% BSA, adjusted to p H 7.2 with KOH. Mitochondrial protein was approximately 1.5 m g per vessel. Swelling was monitored by following absorbance changes at 520 n m in a Hitachi model 100-10 spectrophotometer. Influx of osmotically activesolute results in swelling of the mitochondria and a subsequent decrease in light scatteringcausing a drop in ak/sorbance [5,6]. Cytochrome c reduction was measured by following an increase in absorbance at 550 nm, in a Hitachi model 100-10 spectrophotometer with cuvettes of i c m lightpath at room temperature. Mitochondria (approximately I m g protein) were added to 3 ml of standard reactionmedium which also contained 5 ~ M cytochrome c and 1 m M KCN; the reaction was initiatedby adding 10 m M succinate. Protein was estimated by the method of Lowry et aL [7] using bovine serum albumin (fractionV) as the standard. RESULTS A N D DISCUSSION After gradient centrifugationas described by Douce et al. [3] the bulk of the mitochondria (determined biochemically and by electron microscopy) were
101 TABLE I OXYGEN CONSUMPTION BY " C U S H I O N " AND " G R A D I E N T " CORN SHOOT MITOCHONDRIA Oxygen uptake was measured as described in Materials and Methods; malate (5 raM) was the substrate and 5 mM glutamate and 5 mM KH2PO 4 were included in the reaction medium. Rates are expressed as nmol 02 • rain -I • m g "1 protein.
*Base rate State 3 State 4 RCR ADP/O
Cushion Mitochondria
Gradient Mitochondria
Expt. 1
Expt. 2
Expt.1
Expt~ 2
37 110 22 5.0 2.5
44 132 25 5.3 2.7
40 90 23 4.2 2.8
20 72 18 4.0 2.8
*Base rate is that with substrate alone. State 3 respiration refers to the rate o f 02 uptake in the presence of ADP, while state 4 refers to the rate upon depletion of ADP, i.e., when all ADP has been phosphorylated [21 ]. RCR = respiratory control ratio. ADP/O ratios are a measure of Pi esterified [21].
at the 1.2--1.45 M sucrose interface, with a very small band at the 1.8 M interface. Electron micrographs of gradient and cushion mitochondria were indistinguishable (not shown). Both types appeared to be more than 90% intact mitochondria with clearly visible outer membranes and with only occasional proplastids and membrane fragments. It appeared that the two low speed centrifugations and the rapid centrifugation through the 0.6 M sucrose cushion had mimicked the effects of gradient centrifugation. Meng and Vanderhoef [8] give a typical micrograph and bacterial counts for soybean seedling mitchondria isolated by the cushion method.
TABLE II CYTOCHROME c REDUCTION BY CORN SHOOT MITOCHONDRIA Assay conditions are described in Materials and Methods; rates are expressed as nmol cyt c • rain -i • m g protein -1 . Deoxycholate (0.05%) was used to disrupt the mitochondria.
Control Detergent -treated
Cushion Mitochondria
Gradient Mitochondria
Expt. 1
Expt. 2
Expt. 1
Expt. 2
10.5 151.5
12.0 155.3
18.5 121.2
19.0 160.1
102
A.
B.
/Mi,0
/M,,0
KCl
KCl
Swelling
"~-
j to.o 0D520
Fig. 1. Swelling of corn shoot mitochondria in KCI and a m m o n i u m phosphate. Swelling was measured as described in Materials and Methods, Mitochondria (approximately 1 rng protein) were added to 3 ml of either 1 5 0 r a m KCI or 150 mM a m m o n i u m phosphate which contained 10 mM TES buffer (pH 7.2), 0.1% BSA and 5 ~M antimycin A. Numbers o n traces refer to initial swellingrate~ expressed as AAs=0 ~ I st rain-' *mg -1 protein. A "cushion" mitochondria; B "gradient" mitochondria.
Respiratory parameters of gradient and cushion mitochondria oxidizing malate are shown in Table I. State 3 rates and respiratory control ratios were
slightly"higher in cushion mitochondria and A D P : O ratioswere slightlyhigher in gradient mitochondria. Respiratory control and A D P : O ratiosare considered indicative of inner mitochondrial membrane integrity(i.e.,permeability to I-r) and preservation of energy-linked functions [9,10]. By these criteriathe preparations were quite intact and much alike. Outer membrane integritywas tested by measuring cytochrome c reduction [3,11]. If the outer membranes are damaged (or disrupted with detergent), the cytochrome has access to the inner membrane succinate-cytochrome c reductase. As shown in Table II, both cushion and gradient mitochondria
103
preparations show only low levels of activity until treated with detergent. Assuming that all outer membranes were disrupted by detergent, the preparations show 85 to 93% intact outer membranes. Passive swelling in salt solutions at neutral pH is sometimes regarded as indicative of membrane integrity [12]. Both preparations of corn mitochondria showed modest swelling rates in 150 mM KC1, the gradient mitochondria swelling somewhat faster (Fig. 1). Swelling in KCI is rate limited by K+ entry as evidenced by the large increase in rate with valinomycin [13,14] ; we confirmed this for these mitochondria (data not shown). Hence, both preparations have a sufficiently large permeability coefficient for chloride that chloride influx creates a potential for K÷ influx, and the rate of swelling reflects K÷ permeability. Clearly, a finite permeability exists but there is no evidence that it adversely affects energy-linked ATP formation (Table I). Although not shown here, we investigated with corn mitochondria reports that oligomycin can induce chloride transport in mammalian mitochondria [15] and that in erythrocytes chloride enters via the pyruvate carrier [16]. Neither oligomycin nor a-cyano-4-OH-cinnamic acid, an inhibitor of pyruvate transport in corn mitochondria [17], had any effect on passive swelling in KC1 or on subsequent active contraction upon addition of NADH. Swelling in ammonium phosphate is driven by the penetration of NH3 which creates a pH gradient for phosphate transport [5]. The greater swelling rate of gradient mitochondria (Fig. 1) suggests a larger NH3 permeability coefficient or a greater Pi/OH- antiporter activity. Again, there is no evidence that these permeability properties affect the efficiency of oxidative phosphorylation. In summary, the mitochondria isolated by the two methods described here differed only slightly in purity and integrity. The "cushion" procedure seems to mimic the inherently more exacting "gradient" procedure, and it requires less time, which in itselfcan be a factor in the integrity of isolated mitochondria. Both preparations show swelling in KCI solutions, but we see no evidence that this indicates an abnormal condition. Swelling in KCI is Subject to variables of concentration and p H [18], but this does not obscure the basic observation that isolated corn mitochondria demonstrate chloride permeability. In general, plant cell membranes and thylakoid membranes are characterized by rapid chloride transport, and it does not seem abnormal that avenues for chloride penetration of the inner mitochondrial membrane should exist. Corn mitochondria suspended in 100 m M KCI as osmoticant (instead of sucrose) exhibit good P:O ratios and respiratory control [19,20], indicating that chloride permeability poses no functional problems.
ACKNOWLEDGEMENT This research was supported by the U.S. Energy Research and Development Administration (El1-1-790).
104 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
J.E. Baker, L.G. Elfvin, J.B. Biale and S.I: Honda, Plant Physiol., 43 (1968) 2001. M. Plmnicar, W.D. Bonnet and B.T. Storey, Plant Physiol., 42 (196"/) 366. R. Douce, E.L. Chrktenmen and W.D. Bonner, Biochim. Biophys. Acts, 275 (1972) 148. J.B. Hanson, Plant Physiol., 50 (1972) 347. J.B. Chappell and K.N. I4A~hoff, in E.C. Slater, Z. Koninga and L. Woljczak (Eds.), Biochemistry of Mitochondria, Elsevier, Amsterdam, 1966, p. 293. A.R. Overman, G.H. Lorimer and R.J. Miller, Plant Physiol., 45 (1970) 126. O.H. Lowry, N.J. Rosebrongh, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.L. Meng and L.N. Vanderhoef, Plant Physiol., 50 (1972) 298. B. Chance, W.D. Bonner and B.T. Storey, Armu. Rev. Plant Physiol., 19 (1968) 310. H. Ikuma, Annu. Rev. Plant Physiol., 23 (1972) 419. D.A. Day and J.T. Wiskich, Plant Physiol., 58 (1974) 104. A. DeSantis, G. Borraccino, O. Arrigoni and F. Palmieri, Plant Cell Physiol., 16 (1976) 911. R.H. Wil~on, J. Dever, W. Harper and R. Fry, Plant Cell Physiol., 13 (1972) 1103. B.I. Kirk and J.B. Hanson, Plant Physiol., 51 (1973) 357. N. Ariel and Y. Avi-Dor, Biochem. J., 136 (1973) 911. A.P. Halestrap, Biochem. J., 156 (19"/6) 193. D.A. Day and J.B. Hanson, Plant Physiol., 59 (1977.) 630. C.D. Stoner and J.B. Hanson, Plant Physiol., 41 (1965) 255. J.B. Hanson and R.J. Miller, proc. Natl. Acad. Sci. USA, 58 (1967) 727. D.G. Kenefiek and J.B. Hammn, in Isotopes in Plant Nutrition and Physiology, International Atomic Enar~y Al~ency, Vienna, 1967, p. 27. B. Chance and G.R. Williams, Adv. Enzymol., 17 (1956) 65.
99
ON METHODS FOR THE ISOLATION OF MITOCHONDRIA FROM ETIOLATED CORN SHOOTS
D.A. DAY* and J.B. HANSON
Department of Botany, University of Illinois, Urbana, Ill. 61801 (U.S.A.) (Received July 26th, 1977) (Revision received and accepted October 4th, 1977)
SUMMARY
Mitochondria were isolated from etiolated corn shoots by two methods, one involving gradient purification and the other by centrifuging through a 0.6 M sucrose "cushion". There were no major differences in State 3 or State 4 malate oxidation rates, respiratory control ratios, ADP:O ratios, outer membrane integrity, or swelling rates in salt solutions. The slightly superior quality of cushion mitochondria probably reflects the shorter time required for isolation. Corn mitochondria isolated by both methods showed passive swelling in KC1 solutions but this does not appear to reflect anything abnormal about membrane integrity.
INTRODUC~ON
The integrity and purity of plant mitochondrial suspensions have received considerable attention in the literature during the past decade [1--3]. In attempts to achieve high quality, homogeneous preparations, lengthy isolation procedures, often involving density gradient centrifugation, have been developed [1,3]. While these methods may result in the isolation of intact and pure organelles, it is not always expedient to use them. The present study compares two methods of isolating mitochondria from etiolated corn shoots and shows that a second low speed centrifugation step, and the use of a 0.6 M sucrose "cushion" during washing, eliminate the need for gradient purification. Both
* Present address: Department of Biology, University of California, Los Angeles, Calif. 90024, U.S.A. Abbreviations: BSA, bovine serum albumin; EGTA, ethyleneglycol bis-(~-aminoethylether)N,N'-tetraacetic acid; TES, N-tris-(hydroxymethyl)methyl-2-aminoethanesulphonicacid.
I00 cushion and gradient mitochondria are permeable to high concentrations of KCI. MATERIALS A N D M E T H O D S Corn seedlings(Zea mays L., W F 9 × M14) were grown in the dark at 29°C on paper toweling saturated with a 0.1 M CaCl2 solution.Mitochondria were isolated from 3 or 4.day old etiolatedshoots essentiallyby the method of Hanson [4]. Approximately 100 g of tissuewas ground in an ice-coldmortar with 200 ml of 0.4 M sucrose, 50 m M KH2PO~, 5 m M E G T A and 0.1% B S A (the medium being adjusted to p H 7.6 with KOH). The homogenate was squeezed through 4 layers of cheesecloth,centrifugedat 1000 g for 10 rain, and then at 12 000 g for 10 rain to collectthe mitochondria. The pelletwas resuspended in 0.4 M sucrose and again centrifugedat 1000 g for 10 rain. The resultant supernatant was treated in one of the following manners: (1) for "cushion" mitochondria it was underlaid with 20 ml of 0.6 M sucrose and centrifuged at 10 000 g for 20 rain (the pelletbeing resuspended in 1 ml of 0.25 M sucrose + 0.1% BSA); (2) for "gradient" mitochondria it was layered on top of the discontinuous sucrose gradientdescribed by Douce et al. [3], although TES replaced phosphate buffer, and centrifugedfor 60 rain at 41 000 g in a Beckman L5 ultracentrifuge(SW 27.1 rotor).The fractionfound at the 1.2--1.45 M sucrose layerswas collectedwith a syringe whose tip had been bent, and carefully diluted over a 30-rain period by dropwise addition of 10 m M TES buffer (pH 7.2) and 0.1% BSA. The dilutedmitochondria were centrifuged at 10 000 g for 10 rain and resuspended in 0.25 M sucrose + 0.1% BSA. All operations were carried out at 2---4°C. Oxygen consumption was measured as described elsewhere [4] in 4.0 ml of standard reaction medium which consisted of 0.25 M sucrose, 10 m M TES buffer, 5 m M MgCI2, 5 m M KH2PO4 and 0.1% BSA, adjusted to p H 7.2 with KOH. Mitochondrial protein was approximately 1.5 m g per vessel. Swelling was monitored by following absorbance changes at 520 n m in a Hitachi model 100-10 spectrophotometer. Influx of osmotically activesolute results in swelling of the mitochondria and a subsequent decrease in light scatteringcausing a drop in ak/sorbance [5,6]. Cytochrome c reduction was measured by following an increase in absorbance at 550 nm, in a Hitachi model 100-10 spectrophotometer with cuvettes of i c m lightpath at room temperature. Mitochondria (approximately I m g protein) were added to 3 ml of standard reactionmedium which also contained 5 ~ M cytochrome c and 1 m M KCN; the reaction was initiatedby adding 10 m M succinate. Protein was estimated by the method of Lowry et aL [7] using bovine serum albumin (fractionV) as the standard. RESULTS A N D DISCUSSION After gradient centrifugationas described by Douce et al. [3] the bulk of the mitochondria (determined biochemically and by electron microscopy) were
101 TABLE I OXYGEN CONSUMPTION BY " C U S H I O N " AND " G R A D I E N T " CORN SHOOT MITOCHONDRIA Oxygen uptake was measured as described in Materials and Methods; malate (5 raM) was the substrate and 5 mM glutamate and 5 mM KH2PO 4 were included in the reaction medium. Rates are expressed as nmol 02 • rain -I • m g "1 protein.
*Base rate State 3 State 4 RCR ADP/O
Cushion Mitochondria
Gradient Mitochondria
Expt. 1
Expt. 2
Expt.1
Expt~ 2
37 110 22 5.0 2.5
44 132 25 5.3 2.7
40 90 23 4.2 2.8
20 72 18 4.0 2.8
*Base rate is that with substrate alone. State 3 respiration refers to the rate o f 02 uptake in the presence of ADP, while state 4 refers to the rate upon depletion of ADP, i.e., when all ADP has been phosphorylated [21 ]. RCR = respiratory control ratio. ADP/O ratios are a measure of Pi esterified [21].
at the 1.2--1.45 M sucrose interface, with a very small band at the 1.8 M interface. Electron micrographs of gradient and cushion mitochondria were indistinguishable (not shown). Both types appeared to be more than 90% intact mitochondria with clearly visible outer membranes and with only occasional proplastids and membrane fragments. It appeared that the two low speed centrifugations and the rapid centrifugation through the 0.6 M sucrose cushion had mimicked the effects of gradient centrifugation. Meng and Vanderhoef [8] give a typical micrograph and bacterial counts for soybean seedling mitchondria isolated by the cushion method.
TABLE II CYTOCHROME c REDUCTION BY CORN SHOOT MITOCHONDRIA Assay conditions are described in Materials and Methods; rates are expressed as nmol cyt c • rain -i • m g protein -1 . Deoxycholate (0.05%) was used to disrupt the mitochondria.
Control Detergent -treated
Cushion Mitochondria
Gradient Mitochondria
Expt. 1
Expt. 2
Expt. 1
Expt. 2
10.5 151.5
12.0 155.3
18.5 121.2
19.0 160.1
102
A.
B.
/Mi,0
/M,,0
KCl
KCl
Swelling
"~-
j to.o 0D520
Fig. 1. Swelling of corn shoot mitochondria in KCI and a m m o n i u m phosphate. Swelling was measured as described in Materials and Methods, Mitochondria (approximately 1 rng protein) were added to 3 ml of either 1 5 0 r a m KCI or 150 mM a m m o n i u m phosphate which contained 10 mM TES buffer (pH 7.2), 0.1% BSA and 5 ~M antimycin A. Numbers o n traces refer to initial swellingrate~ expressed as AAs=0 ~ I st rain-' *mg -1 protein. A "cushion" mitochondria; B "gradient" mitochondria.
Respiratory parameters of gradient and cushion mitochondria oxidizing malate are shown in Table I. State 3 rates and respiratory control ratios were
slightly"higher in cushion mitochondria and A D P : O ratioswere slightlyhigher in gradient mitochondria. Respiratory control and A D P : O ratiosare considered indicative of inner mitochondrial membrane integrity(i.e.,permeability to I-r) and preservation of energy-linked functions [9,10]. By these criteriathe preparations were quite intact and much alike. Outer membrane integritywas tested by measuring cytochrome c reduction [3,11]. If the outer membranes are damaged (or disrupted with detergent), the cytochrome has access to the inner membrane succinate-cytochrome c reductase. As shown in Table II, both cushion and gradient mitochondria
103
preparations show only low levels of activity until treated with detergent. Assuming that all outer membranes were disrupted by detergent, the preparations show 85 to 93% intact outer membranes. Passive swelling in salt solutions at neutral pH is sometimes regarded as indicative of membrane integrity [12]. Both preparations of corn mitochondria showed modest swelling rates in 150 mM KC1, the gradient mitochondria swelling somewhat faster (Fig. 1). Swelling in KCI is rate limited by K+ entry as evidenced by the large increase in rate with valinomycin [13,14] ; we confirmed this for these mitochondria (data not shown). Hence, both preparations have a sufficiently large permeability coefficient for chloride that chloride influx creates a potential for K÷ influx, and the rate of swelling reflects K÷ permeability. Clearly, a finite permeability exists but there is no evidence that it adversely affects energy-linked ATP formation (Table I). Although not shown here, we investigated with corn mitochondria reports that oligomycin can induce chloride transport in mammalian mitochondria [15] and that in erythrocytes chloride enters via the pyruvate carrier [16]. Neither oligomycin nor a-cyano-4-OH-cinnamic acid, an inhibitor of pyruvate transport in corn mitochondria [17], had any effect on passive swelling in KC1 or on subsequent active contraction upon addition of NADH. Swelling in ammonium phosphate is driven by the penetration of NH3 which creates a pH gradient for phosphate transport [5]. The greater swelling rate of gradient mitochondria (Fig. 1) suggests a larger NH3 permeability coefficient or a greater Pi/OH- antiporter activity. Again, there is no evidence that these permeability properties affect the efficiency of oxidative phosphorylation. In summary, the mitochondria isolated by the two methods described here differed only slightly in purity and integrity. The "cushion" procedure seems to mimic the inherently more exacting "gradient" procedure, and it requires less time, which in itselfcan be a factor in the integrity of isolated mitochondria. Both preparations show swelling in KCI solutions, but we see no evidence that this indicates an abnormal condition. Swelling in KCI is Subject to variables of concentration and p H [18], but this does not obscure the basic observation that isolated corn mitochondria demonstrate chloride permeability. In general, plant cell membranes and thylakoid membranes are characterized by rapid chloride transport, and it does not seem abnormal that avenues for chloride penetration of the inner mitochondrial membrane should exist. Corn mitochondria suspended in 100 m M KCI as osmoticant (instead of sucrose) exhibit good P:O ratios and respiratory control [19,20], indicating that chloride permeability poses no functional problems.
ACKNOWLEDGEMENT This research was supported by the U.S. Energy Research and Development Administration (El1-1-790).
104 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
J.E. Baker, L.G. Elfvin, J.B. Biale and S.I: Honda, Plant Physiol., 43 (1968) 2001. M. Plmnicar, W.D. Bonnet and B.T. Storey, Plant Physiol., 42 (196"/) 366. R. Douce, E.L. Chrktenmen and W.D. Bonner, Biochim. Biophys. Acts, 275 (1972) 148. J.B. Hanson, Plant Physiol., 50 (1972) 347. J.B. Chappell and K.N. I4A~hoff, in E.C. Slater, Z. Koninga and L. Woljczak (Eds.), Biochemistry of Mitochondria, Elsevier, Amsterdam, 1966, p. 293. A.R. Overman, G.H. Lorimer and R.J. Miller, Plant Physiol., 45 (1970) 126. O.H. Lowry, N.J. Rosebrongh, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.L. Meng and L.N. Vanderhoef, Plant Physiol., 50 (1972) 298. B. Chance, W.D. Bonner and B.T. Storey, Armu. Rev. Plant Physiol., 19 (1968) 310. H. Ikuma, Annu. Rev. Plant Physiol., 23 (1972) 419. D.A. Day and J.T. Wiskich, Plant Physiol., 58 (1974) 104. A. DeSantis, G. Borraccino, O. Arrigoni and F. Palmieri, Plant Cell Physiol., 16 (1976) 911. R.H. Wil~on, J. Dever, W. Harper and R. Fry, Plant Cell Physiol., 13 (1972) 1103. B.I. Kirk and J.B. Hanson, Plant Physiol., 51 (1973) 357. N. Ariel and Y. Avi-Dor, Biochem. J., 136 (1973) 911. A.P. Halestrap, Biochem. J., 156 (19"/6) 193. D.A. Day and J.B. Hanson, Plant Physiol., 59 (1977.) 630. C.D. Stoner and J.B. Hanson, Plant Physiol., 41 (1965) 255. J.B. Hanson and R.J. Miller, proc. Natl. Acad. Sci. USA, 58 (1967) 727. D.G. Kenefiek and J.B. Hammn, in Isotopes in Plant Nutrition and Physiology, International Atomic Enar~y Al~ency, Vienna, 1967, p. 27. B. Chance and G.R. Williams, Adv. Enzymol., 17 (1956) 65.