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Factors affecting the oxidation of glycerol-1-phosphate by insect flight-muscle mitochondria
J_ Insect PhysioL, 1969, Vol. IS, pp. 367 to 372. Pergamon Press. Printed in Great Britain
FACTORS AFFECTING THE OXIDATION OF GLYCEROL-l-PHOSPHATE BY INSECT FLIGHT-MUSCLE MITOCHONDRIA J. Woodstock Chemical
F. DONNELLAN Agricultural Enzymology,
and R. B. BEECHEY
Research Centre ‘Shell’ Research
(Received
10 October
and Milstead Laboratory of Ltd., Sittingboume, Kent 1968)
Abstract-The oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria isolated from a variety of insects has been studied. Low levels of calcium ions, lo-’ M, and strontium ions, 10W5 M, act as heterotropic aliosteric effecters of the oxidation by both mitochondria and sub-mitochondrial particles. L-Glycerol-l-phosphate also acts as a homotropic effector in the absence of calcium and strontium ions. The preparation is described of submitochondrial particles, EGTA particles, which do not show allosteric behaviour. The site of action of calcium and strontium ions is discussed. INTRODUCTION
THE INHIBITION by ethylenediamine tetra acetate (EDTA) of the oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria from Musca domestica and the relief of this inhibition by 5 mM concentrations of divalent metal ions has been reported (ESTABROOKand SACKTOR, 1958). A later study (HAXFORD and CHAPPELL, 1967) has shown that micromolar concentrations of Ca2+ are sufficient to induce rapid rates of oxidation of L-glycerol-l-phosphate and respiratory control in flight-muscle mitochondria isolated from Calliphora vomitoria, by acting as a co-operative heterotropic allosteric effector. This report confirms that Ca2T play a key role in the oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria isolated from a number of insects and that the allosteric site involved in L-glyceroll-phosphate oxidation is located on the mitochondrial L-glycerol-l-phosphate dehydrogenase molecule (E.C. 1.1.99.5). The preparation of two types of submitochondrial particles is described; EGTA particles effectively oxidize L-glyceroll-phosphate independently of the Ca2+ concentration, whereas calcium particles show a marked dependence upon Ca2+. MATERIALS
AND METHODS
Flight-muscle mitochondria were isolated from Sarcophaga barbata, Pieris brassicae, Apis mellifera, Schistocerca gregaria, and Musca domestica, using the Nagarse proteinase method (CHANCE and HAGIHARA, 1961). The final mitochondrial peIlet was suspended in a cold solution containing 0.25 M sucrose, 5 mM Tris-chloride, and 1 mM ethyleneglycol-bis-(aminoethyl)-tetra acetate (EGTA), pH 74. 367
368
J. F. DOWELLAW.ND R. B. BEECHEY
The protein concentration was approximately 10 mg/ml. Submitochondriaf particles were prepared by subjecting a mitochondrial suspension (5 mg of protein/ ml) to the output of a 100 W MSE ultrasonicator for 2 min. The supematant, after centrifuging for 1.2 x lO”gjmin, was recentrifuged for 2 x 106g/min to yield a pellet of submitochondrial particles which we call EGTA particles. Calcium particles were prepared by the same procedure save that CaCl, was added to the mitochondrial suspension before sonication to give a buffered concentration of 10-B M free calcium ions (PORTZEHLet aE., 1964). Oxygen consumption was measured at 25°C using an oxygen electrode inserted into a 2 ml Perspex cell. RESULTS
AND DISCUSSION
We have found that for the oxidation of L-glycerol-l-phosphate by flightmuscle mitochondria, isolated from Sarcophaga barbata, Pi& brassicae, Apis me&f&a, Schistocerca gregaria, and _iMzrscadome&a, normal Michaelis-Menten kinetics are observed in the presence of lo-’ M free Ca2+. In the absence of Ca2+ allosteric kinetics are observed. (See Table 1 and Fig. 1, curves a and b.) The variation of mitochondrial respiration rates with the concentration of L-glycerol-lphosphate in the presence and absence of lo-’ M Ca2+ is essentially the same as reported for CaZZiphora zomitoria by HANSFORDand CHAPPELL (1967). The data illustrated in Fig. 2 show that Sr2+ will replace Ca2’, but the concentrations required are one hundredfold greater. Magnesium ions are ten thousandfold less effective than Ca2+; the concentration of Mg2+ could not be increased in this experiment because of the inadequate buffering capacity of the EDTA-Mg2+ system used. At higher concentrations of L-glycerol-l-phosphate, the requirement of flight-muscle mitochondria for either Ca”+, Sr2+, or Mg2+ to achieve a maximum rate of oxygen consumption disappears (Fig. 1, curve b). Calcium particles also show a marked difference in their affinity for L-glycerol-lphosphate dependent upon the presence or absence of Ca2+. In the presence of lo-‘M Ca2+ (Fig. 1, curve c) the oxidation of L-glycerol-l-phosphate follows normal Michaelis-Menten kinetics; I& = 1.8 mM & 0.28, 10 assays. However, in the absence of Ca*+ (Fig. 1, curve d), the variation of oxygen consumption with L-glycerol-l-phosphate follows a sigmoid curve. The mean L-glycerol-l-phosphate concentration giving 50 per cent of the maximum rate of oxygen consumption was 4.9 mM + 1.4, 10 assays. The pattern of response to Ca2+, Sr”+, and h’lg2+ is identical with that shown by intact mitochondria. This indicates that the allosteric rate-limiting factor is identical in both intact mitochondria and calcium particles. The absence of a similar effect of Ca2+ on the oxidation of either pyruvic acid or proline by intact flight-muscle mitochondria and on the oxidation of NADH by calcium particles limits the site’ of action of Ca2+ to the L-glycerol-l-phosphate dehydrogenase protein. The possibility of Ca a+ affecting an L-glycerol-l-phosphate porter (MITCHELL, 1966) is eliminated by the presence of allosteric kinetics in the calcium particles, where the cristal membrane is inside out (LEE and ERXSTER, 1966). The effects of Caaf on the oxidation of L-glycerol-I-phosphate by EGTA
-
50
194
7.5
194 194 194 -
144 184
2.5 5
10 15 20 30
+ lo-’ M Caa+
97 101 101 101 -
91
-
64 78
13 48 132 184 194 -
+ lo-’ M Ca’+
No Ca2+
-
59 90 93 -
42
9 26
No Ca*+-
Schistocerca gregaria
-
510 510 510 -
510
22s 410
+ lo-’ M Ca*-‘-
+ IO-’ M Ca2+ 29 50 57 58 58 58 -
No Cae +. 15 45 90 150 320 440 -
Musca domestica
27
6 11 18 36
5
2 3
No Caa+
Pieris brassicae
L-GLYCEROL-~-PHOSPHATE BY FLIGHT-MUSCLE MITOCHONDRIA
An uncoupling agent, 4,5,6,7-tetrachloro-Z-trifluoromethylbenzimidazole
Mitochondrial suspension (0.5-Z mg protein) was added to 154 mM KC1 containing 10 mM phosphate and 2 mM KGTA, pH 7.1. (1 mpM)-was also present (BEECHEY, 1966). Oxygen uptake was followed as the L-glycerol-l-phosphate concentration was increased, in the presence and absence of IO--’ M Ca2+. The respiration rates were corrected for endogenous respiration which in no’case exceeded 5 n-moles O,/min per mg protein.
-
Apis mellifera
Concentration of L-glyceroll-phosphate (mM)
OF
Mitochondrial respiration rate (n-moles O,/min per mg protein)
TABLE ~---EFFECT OF Ca’+ ON THE OXIDATION
g g g j:
E 8
k
&
s ;! i; 2
2
E E z
5
; $ z
t
E
370
J. F. DONNELLANANDR. B. BEECHEY
particles are marginal (see Fig. 1, curves e and f). The oxidation usually follows Michaelis-Menten kinetics both in the presence of lo-’ M Ca2+ (KJ1 = 4.0 mM f l-47, 15 assays) and the absence of Ca2+ (K-l1 = 5.6 mM + 1.1, 15 assays). The L-glycerol-l-phosphate dehydrogenase activity present in Triton X-l 14 solubilized extracts of both EGTA and calcium particles showed little response to the presence or absence of Ca2+ (Kx = 4.8 mM f l-35, 8 assays in the presence of lo-’ M Ca2+ and KLV = 4.9 mM? 1.5, 8 assays, in the absence of Ca2+). Solubilization by Triton X-114 in the presence and absence of Ca a+ had no effect on these results.
FIG. 1. Oxidation of L-glycerol-l -phosphate by mitochondria and either calcium or EGTA particles isolated from Sarcophuga barbatu flight-muscle. Mitochondrial or particle suspension was added to 154 mM KC1 containing 10 mM phosphate, 2 n-&l EGTA, 1 mM KCN, and 0.25 mg phenazine methosulphate, pH 7.1. Oxygen uptake was followed as the concentration of L-glycerol-l-phosphate was increased in the presence and absence of lo-’ M free Ca?+; curve a, mitochondria (0.4 mg of protein) plus Ca*+; curve b, mitochondria (O-4 mg of protein) EGTA only; curve c, calcium particles (O-53 mg of protein) plus Ca2+; curve d, calcium particles (0.53 mg of protein) EGTA only; curve e, EGTA particles (0.53 mg of protein) plus Ca2+; curve f, EGTA particles (0.53 mg of protein) EGTA only.
The simplest explanation for these observations is as follows: In intact flightmuscle mitochondria, L-glycerol-l-phosphate is acting as a homotropic effector and Cat+ as a heterotropic effector. When mitochondria are sonicated in the presence of 10m6 M Ca 2+ the dehydrogenase molecule is in the polymeric form and this conformation is retained when the cristal membrane vesiculates forming calcium particles. In this conformation the polymer is able to undergo depolymerization and polymerization in response to the absence or presence of Ca2+ or L-glycerol-l-phosphate. However, when the mitochondria are sonicated in the presence of EGTA (Ca”T concentration < lob9 RI) the L-glycerol-l-phosphate dehydrogenase molecule is monomeric. On formation of the submitochondrial
GLYCEROL-1 -PHOSPHATE OXIDATIOK BY FLIGHT-MUSCLE MITOCHONDRIA
371
FIG. 2. Effect of CaZ+, Sr*+, and Mg2+ on the oxidation of 2.5 mM L-glycerol-lphosphate by flight-muscle mitochondria isolated from Sarcophaga barbutu. Mitochondrial suspension (0.58 mg protein) was added to 154 mM KC1 containing 10 mM phosphate, 2 mM EDTA, 1 m,uhl 4,5,6,7-tetrachloro-2&fluoromethylbenzimidazole and 2.5 mM L-glycerol-l-phosphate, pH 7.1. Oxygen uptake was followed as the level of divalent metal ion was increased by addition of the appropriate volume of 200 mM CaCle (O), SrCl, (O), or MgCI, (m).
particles the new membrane monomers in the presence curves e and f in Fig. 1. Preliminary experiments and guinea-pig, show that similarly by Ca2+. Acknowledgement-We
conformation is such that the interaction between the of Ca2+ is severely restricted, hence the hyperbolic with skeletal muscle mitochondria, isolated from rat the oxidation of L-glycerol-l-phosphate is affected
would
like to thank Mr.
D. W. JENNER for skilled assistance.
REFERENCES BEECHEY R. B. (1966) The uncoupling of respiratory-chain phosphorylation by 4,5,6,7tetrachloro-2-trifluoromethylbenzimidazole. Biochem. J. 98, 284-289. CH.LUCE B. and HAGIHARA B. (1961) Direct spectroscopic measurements of interaction of components of the respiratory chain with ATP, ADP, phosphate and uncoupling agents.
Proc. 5th int. Congr. Biochem. 5, 3-33. ESTABROOK R. W. and SACKTOR B. (1958) a-Glycerophosphate oxidase of flight muscle mitochondria. J. biol. Chem. 233, 1014-1019. HANSFORD R. G. and CHAPPELL J. B. (1967) The effect of Ca2+ on the oxidation of glycerol Biochem. Biophys. Res. Comm. 27, phosphate by blowfly flight-muscle mitochondria.
686-692.
372
J. F. DONNELLANANDR. B. BJZECHEY
LEE C. P. and ERNSTER L. (1966) The energy-linked nicotinamide nucleotide transhydrogenaae reaction: its characteristics and its use as a tool for the study of oxidative phosphorylation. BBA Library 7, 218-234. MITCHELL P. (1966) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 41, 445-502. PORTZEHLI-I., CALDWELLP. C., and R&GG J. C. (1964) The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591.
FACTORS AFFECTING THE OXIDATION OF GLYCEROL-l-PHOSPHATE BY INSECT FLIGHT-MUSCLE MITOCHONDRIA J. Woodstock Chemical
F. DONNELLAN Agricultural Enzymology,
and R. B. BEECHEY
Research Centre ‘Shell’ Research
(Received
10 October
and Milstead Laboratory of Ltd., Sittingboume, Kent 1968)
Abstract-The oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria isolated from a variety of insects has been studied. Low levels of calcium ions, lo-’ M, and strontium ions, 10W5 M, act as heterotropic aliosteric effecters of the oxidation by both mitochondria and sub-mitochondrial particles. L-Glycerol-l-phosphate also acts as a homotropic effector in the absence of calcium and strontium ions. The preparation is described of submitochondrial particles, EGTA particles, which do not show allosteric behaviour. The site of action of calcium and strontium ions is discussed. INTRODUCTION
THE INHIBITION by ethylenediamine tetra acetate (EDTA) of the oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria from Musca domestica and the relief of this inhibition by 5 mM concentrations of divalent metal ions has been reported (ESTABROOKand SACKTOR, 1958). A later study (HAXFORD and CHAPPELL, 1967) has shown that micromolar concentrations of Ca2+ are sufficient to induce rapid rates of oxidation of L-glycerol-l-phosphate and respiratory control in flight-muscle mitochondria isolated from Calliphora vomitoria, by acting as a co-operative heterotropic allosteric effector. This report confirms that Ca2T play a key role in the oxidation of L-glycerol-l-phosphate by flight-muscle mitochondria isolated from a number of insects and that the allosteric site involved in L-glyceroll-phosphate oxidation is located on the mitochondrial L-glycerol-l-phosphate dehydrogenase molecule (E.C. 1.1.99.5). The preparation of two types of submitochondrial particles is described; EGTA particles effectively oxidize L-glyceroll-phosphate independently of the Ca2+ concentration, whereas calcium particles show a marked dependence upon Ca2+. MATERIALS
AND METHODS
Flight-muscle mitochondria were isolated from Sarcophaga barbata, Pieris brassicae, Apis mellifera, Schistocerca gregaria, and Musca domestica, using the Nagarse proteinase method (CHANCE and HAGIHARA, 1961). The final mitochondrial peIlet was suspended in a cold solution containing 0.25 M sucrose, 5 mM Tris-chloride, and 1 mM ethyleneglycol-bis-(aminoethyl)-tetra acetate (EGTA), pH 74. 367
368
J. F. DOWELLAW.ND R. B. BEECHEY
The protein concentration was approximately 10 mg/ml. Submitochondriaf particles were prepared by subjecting a mitochondrial suspension (5 mg of protein/ ml) to the output of a 100 W MSE ultrasonicator for 2 min. The supematant, after centrifuging for 1.2 x lO”gjmin, was recentrifuged for 2 x 106g/min to yield a pellet of submitochondrial particles which we call EGTA particles. Calcium particles were prepared by the same procedure save that CaCl, was added to the mitochondrial suspension before sonication to give a buffered concentration of 10-B M free calcium ions (PORTZEHLet aE., 1964). Oxygen consumption was measured at 25°C using an oxygen electrode inserted into a 2 ml Perspex cell. RESULTS
AND DISCUSSION
We have found that for the oxidation of L-glycerol-l-phosphate by flightmuscle mitochondria, isolated from Sarcophaga barbata, Pi& brassicae, Apis me&f&a, Schistocerca gregaria, and _iMzrscadome&a, normal Michaelis-Menten kinetics are observed in the presence of lo-’ M free Ca2+. In the absence of Ca2+ allosteric kinetics are observed. (See Table 1 and Fig. 1, curves a and b.) The variation of mitochondrial respiration rates with the concentration of L-glycerol-lphosphate in the presence and absence of lo-’ M Ca2+ is essentially the same as reported for CaZZiphora zomitoria by HANSFORDand CHAPPELL (1967). The data illustrated in Fig. 2 show that Sr2+ will replace Ca2’, but the concentrations required are one hundredfold greater. Magnesium ions are ten thousandfold less effective than Ca2+; the concentration of Mg2+ could not be increased in this experiment because of the inadequate buffering capacity of the EDTA-Mg2+ system used. At higher concentrations of L-glycerol-l-phosphate, the requirement of flight-muscle mitochondria for either Ca”+, Sr2+, or Mg2+ to achieve a maximum rate of oxygen consumption disappears (Fig. 1, curve b). Calcium particles also show a marked difference in their affinity for L-glycerol-lphosphate dependent upon the presence or absence of Ca2+. In the presence of lo-‘M Ca2+ (Fig. 1, curve c) the oxidation of L-glycerol-l-phosphate follows normal Michaelis-Menten kinetics; I& = 1.8 mM & 0.28, 10 assays. However, in the absence of Ca*+ (Fig. 1, curve d), the variation of oxygen consumption with L-glycerol-l-phosphate follows a sigmoid curve. The mean L-glycerol-l-phosphate concentration giving 50 per cent of the maximum rate of oxygen consumption was 4.9 mM + 1.4, 10 assays. The pattern of response to Ca2+, Sr”+, and h’lg2+ is identical with that shown by intact mitochondria. This indicates that the allosteric rate-limiting factor is identical in both intact mitochondria and calcium particles. The absence of a similar effect of Ca2+ on the oxidation of either pyruvic acid or proline by intact flight-muscle mitochondria and on the oxidation of NADH by calcium particles limits the site’ of action of Ca2+ to the L-glycerol-l-phosphate dehydrogenase protein. The possibility of Ca a+ affecting an L-glycerol-l-phosphate porter (MITCHELL, 1966) is eliminated by the presence of allosteric kinetics in the calcium particles, where the cristal membrane is inside out (LEE and ERXSTER, 1966). The effects of Caaf on the oxidation of L-glycerol-I-phosphate by EGTA
-
50
194
7.5
194 194 194 -
144 184
2.5 5
10 15 20 30
+ lo-’ M Caa+
97 101 101 101 -
91
-
64 78
13 48 132 184 194 -
+ lo-’ M Ca’+
No Ca2+
-
59 90 93 -
42
9 26
No Ca*+-
Schistocerca gregaria
-
510 510 510 -
510
22s 410
+ lo-’ M Ca*-‘-
+ IO-’ M Ca2+ 29 50 57 58 58 58 -
No Cae +. 15 45 90 150 320 440 -
Musca domestica
27
6 11 18 36
5
2 3
No Caa+
Pieris brassicae
L-GLYCEROL-~-PHOSPHATE BY FLIGHT-MUSCLE MITOCHONDRIA
An uncoupling agent, 4,5,6,7-tetrachloro-Z-trifluoromethylbenzimidazole
Mitochondrial suspension (0.5-Z mg protein) was added to 154 mM KC1 containing 10 mM phosphate and 2 mM KGTA, pH 7.1. (1 mpM)-was also present (BEECHEY, 1966). Oxygen uptake was followed as the L-glycerol-l-phosphate concentration was increased, in the presence and absence of IO--’ M Ca2+. The respiration rates were corrected for endogenous respiration which in no’case exceeded 5 n-moles O,/min per mg protein.
-
Apis mellifera
Concentration of L-glyceroll-phosphate (mM)
OF
Mitochondrial respiration rate (n-moles O,/min per mg protein)
TABLE ~---EFFECT OF Ca’+ ON THE OXIDATION
g g g j:
E 8
k
&
s ;! i; 2
2
E E z
5
; $ z
t
E
370
J. F. DONNELLANANDR. B. BEECHEY
particles are marginal (see Fig. 1, curves e and f). The oxidation usually follows Michaelis-Menten kinetics both in the presence of lo-’ M Ca2+ (KJ1 = 4.0 mM f l-47, 15 assays) and the absence of Ca2+ (K-l1 = 5.6 mM + 1.1, 15 assays). The L-glycerol-l-phosphate dehydrogenase activity present in Triton X-l 14 solubilized extracts of both EGTA and calcium particles showed little response to the presence or absence of Ca2+ (Kx = 4.8 mM f l-35, 8 assays in the presence of lo-’ M Ca2+ and KLV = 4.9 mM? 1.5, 8 assays, in the absence of Ca2+). Solubilization by Triton X-114 in the presence and absence of Ca a+ had no effect on these results.
FIG. 1. Oxidation of L-glycerol-l -phosphate by mitochondria and either calcium or EGTA particles isolated from Sarcophuga barbatu flight-muscle. Mitochondrial or particle suspension was added to 154 mM KC1 containing 10 mM phosphate, 2 n-&l EGTA, 1 mM KCN, and 0.25 mg phenazine methosulphate, pH 7.1. Oxygen uptake was followed as the concentration of L-glycerol-l-phosphate was increased in the presence and absence of lo-’ M free Ca?+; curve a, mitochondria (0.4 mg of protein) plus Ca*+; curve b, mitochondria (O-4 mg of protein) EGTA only; curve c, calcium particles (O-53 mg of protein) plus Ca2+; curve d, calcium particles (0.53 mg of protein) EGTA only; curve e, EGTA particles (0.53 mg of protein) plus Ca2+; curve f, EGTA particles (0.53 mg of protein) EGTA only.
The simplest explanation for these observations is as follows: In intact flightmuscle mitochondria, L-glycerol-l-phosphate is acting as a homotropic effector and Cat+ as a heterotropic effector. When mitochondria are sonicated in the presence of 10m6 M Ca 2+ the dehydrogenase molecule is in the polymeric form and this conformation is retained when the cristal membrane vesiculates forming calcium particles. In this conformation the polymer is able to undergo depolymerization and polymerization in response to the absence or presence of Ca2+ or L-glycerol-l-phosphate. However, when the mitochondria are sonicated in the presence of EGTA (Ca”T concentration < lob9 RI) the L-glycerol-l-phosphate dehydrogenase molecule is monomeric. On formation of the submitochondrial
GLYCEROL-1 -PHOSPHATE OXIDATIOK BY FLIGHT-MUSCLE MITOCHONDRIA
371
FIG. 2. Effect of CaZ+, Sr*+, and Mg2+ on the oxidation of 2.5 mM L-glycerol-lphosphate by flight-muscle mitochondria isolated from Sarcophaga barbutu. Mitochondrial suspension (0.58 mg protein) was added to 154 mM KC1 containing 10 mM phosphate, 2 mM EDTA, 1 m,uhl 4,5,6,7-tetrachloro-2&fluoromethylbenzimidazole and 2.5 mM L-glycerol-l-phosphate, pH 7.1. Oxygen uptake was followed as the level of divalent metal ion was increased by addition of the appropriate volume of 200 mM CaCle (O), SrCl, (O), or MgCI, (m).
particles the new membrane monomers in the presence curves e and f in Fig. 1. Preliminary experiments and guinea-pig, show that similarly by Ca2+. Acknowledgement-We
conformation is such that the interaction between the of Ca2+ is severely restricted, hence the hyperbolic with skeletal muscle mitochondria, isolated from rat the oxidation of L-glycerol-l-phosphate is affected
would
like to thank Mr.
D. W. JENNER for skilled assistance.
REFERENCES BEECHEY R. B. (1966) The uncoupling of respiratory-chain phosphorylation by 4,5,6,7tetrachloro-2-trifluoromethylbenzimidazole. Biochem. J. 98, 284-289. CH.LUCE B. and HAGIHARA B. (1961) Direct spectroscopic measurements of interaction of components of the respiratory chain with ATP, ADP, phosphate and uncoupling agents.
Proc. 5th int. Congr. Biochem. 5, 3-33. ESTABROOK R. W. and SACKTOR B. (1958) a-Glycerophosphate oxidase of flight muscle mitochondria. J. biol. Chem. 233, 1014-1019. HANSFORD R. G. and CHAPPELL J. B. (1967) The effect of Ca2+ on the oxidation of glycerol Biochem. Biophys. Res. Comm. 27, phosphate by blowfly flight-muscle mitochondria.
686-692.
372
J. F. DONNELLANANDR. B. BJZECHEY
LEE C. P. and ERNSTER L. (1966) The energy-linked nicotinamide nucleotide transhydrogenaae reaction: its characteristics and its use as a tool for the study of oxidative phosphorylation. BBA Library 7, 218-234. MITCHELL P. (1966) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 41, 445-502. PORTZEHLI-I., CALDWELLP. C., and R&GG J. C. (1964) The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591.