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Ultracytochemical localisation of adenosine triphosphatase activity on the plasmalemma of Chara corallina
Micron,
Vol.13, No.3, pp.309-3;0, Printed in Great Britain
Ultracytochemical Chara corallina
]982.
0047-7206/82/030309-02503.00/0 Pergamon Press Ltd.
localisation of adenosine triphosphatase activity on the plasmalemma of
G.D. Price and M.I. Whitecross Botany Department, Australian National University, Canberra, A.C.T.
2600
A proton-pumping adenosine triphosphatase H+ATP'ase) on the plasmalermma of cells of Chara corallina has been proposed as being responsible for the electrogenic component of the membrane potential (~m)I'2'3" The proton motive force (PMF) across the membrane, generated by the H + ATP'ase, is seen as driving such ion transport systems as a H+/CI - symport 4'$" Although a salt-stimulated ATP'ase has been isolated from homogenates of Chara 6 , no cytochemical localisation of ATP'ase activity on the plasmalemma has been reported. Chara cells possess large numbers of elaborate, 3-dimensional, branched-tubular invaginations of the plasmalemma - the plasmalemmasomes (PLS - see Fig. i). Electron microscope studies in this laboratory and elsewhere have shown that the density of PLS is greater in the acid bands of Chara than in the alkaline bands 7'8+ Chloride uptake is also greater in the acid bands 7 . These observations, together with the evident increase in membrane surface area due to PLS, suggest that PLS may be involved in H + efflux in the acid bands. Any demonstration of ATP'ase activity at the membrane level, especially associated with the tubules of PLS would be consistent with PLS having such a function. ATP'ase cytochemistry by electron microscopy was performed by a lead phosphate precipitation technique with suitable controls (after Browning et al.9). Mature internodal cells of C.corallina were fixed (60 or 90 min) in 5% glutaraldehyde in 0.08M PIPES-NaOH (+40 mM NaCl + 1 mM CaCI2) pH 8 on ice, prior to incubation, after which tissue was processed in a standard way (i.e. osmication ÷ ethanol dehydration ÷ Spurr's resin). Unstained or lightly stained (uranium) sections were examined in a JEOL i00 C TEM. ATP'ase reaction product was localised at discrete sites on the inside of the plasmalemma (Fig. 2) and on the inside of the tubules of PLS (Fig. 3), when cells were incubated in complete medium. Minor amounts of reaction product were also evident on the chloroplast envelope (Fig. 2), the tonoplast (Fig. 4), and occasionally in the ground cytoplasm. A shorter fixation period prior to incubation resulted in enhanced product deposition but a significant reduction in ultrastructural preservation (cf. Figs. 2,3). Controls employing absence of substrate or addition of NaF (Fig. 5) demonstrated no activity. Substitution of 8-glycerophosphate for ATP gave no deposition on the plasmalemma but small amounts were discernible in the cytoplasm. This study has therefore succeeded in providing further evidence consistent with the presence of H+ATP'ases on the plasmalemma of Chara. Demonstrated ATP'ase activity associated with PLS tubules also supports the notion that the PLS enhance H + efflux locally, thereby creating H + gradients capable of energising transport of other ions. Further studies will need to demonstrate conclusively the H + pumping nature of the plasmalemma ATP'ase. Further consideration must also be given to the implications of ATP'ase activity on the tonoplast and the chloroplast envelope. I.
Shimmen, T., Tazawa, M.
(1977).
J.Memb.Biol.
(1979).
37:167-192
2.
Kiefer, D.W., Spanswick, R.M.
3.
Smith, P.T., Walker, N.A.
(1981).
J.Memb. Biol. 60: 223-236
Plant Physiol. 6 4 : 1 6 5 - 6 8
4.
Smith, F.A., Walker, N.A.
(1976).
J.Exp.Bot. 309
27:451-59
310
G.D.
5.
Sanders, D.
(1980).
6.
Atkinson, M.R., Polya, G.
7.
Price, G.D.
(1981)
Price and M. I. Whitecross
J.Memb. Biol. 53: 129-41 (1967)
M.Sc. Thesis
Aust. J.Biol.Sci.
20: 1069-86
(A.N.U.)
8.
Franceschi, V.R., Lucas, W.J.
9.
Browning, A.J., Hall, J.L., Baker, D.A.
(1980)
Protoplasma 1 0 4 : 2 5 3 - 2 7 1 (1980)
Protoplasma 1 0 4 : 5 5 - 6 5
k
T
®
O*S ~ m
,
Fig. i.
Ultrastructural view of PLS M = mitochondrion.
conventional,
Fig. 2.
Complete medium (unstained; 60 min. fixation). Deposits plasmalemma (PL) and chloroplast (CHL) envelope.
Fig. 3.
Complete medium
Fig. 4.
Complete medium (unstained; 60 min fixation). (T). NUC = nucleus.
Fig. 5.
Complete medium + i0 mM NaF
(uranium stained;
2 hour fixation;
90 min fixation).
(unstained).
lead & uranium stained).
(arrows) evident on the
Deposits in the PLS tubules.
Deposits evident on the tonoplast
No reaction product.
Acknowledgements The authors wish to thank M. Fenning for preparation of EM prints; of the A.N.U. TEM Unit for technical support.
G. Weston and the staff
Vol.13, No.3, pp.309-3;0, Printed in Great Britain
Ultracytochemical Chara corallina
]982.
0047-7206/82/030309-02503.00/0 Pergamon Press Ltd.
localisation of adenosine triphosphatase activity on the plasmalemma of
G.D. Price and M.I. Whitecross Botany Department, Australian National University, Canberra, A.C.T.
2600
A proton-pumping adenosine triphosphatase H+ATP'ase) on the plasmalermma of cells of Chara corallina has been proposed as being responsible for the electrogenic component of the membrane potential (~m)I'2'3" The proton motive force (PMF) across the membrane, generated by the H + ATP'ase, is seen as driving such ion transport systems as a H+/CI - symport 4'$" Although a salt-stimulated ATP'ase has been isolated from homogenates of Chara 6 , no cytochemical localisation of ATP'ase activity on the plasmalemma has been reported. Chara cells possess large numbers of elaborate, 3-dimensional, branched-tubular invaginations of the plasmalemma - the plasmalemmasomes (PLS - see Fig. i). Electron microscope studies in this laboratory and elsewhere have shown that the density of PLS is greater in the acid bands of Chara than in the alkaline bands 7'8+ Chloride uptake is also greater in the acid bands 7 . These observations, together with the evident increase in membrane surface area due to PLS, suggest that PLS may be involved in H + efflux in the acid bands. Any demonstration of ATP'ase activity at the membrane level, especially associated with the tubules of PLS would be consistent with PLS having such a function. ATP'ase cytochemistry by electron microscopy was performed by a lead phosphate precipitation technique with suitable controls (after Browning et al.9). Mature internodal cells of C.corallina were fixed (60 or 90 min) in 5% glutaraldehyde in 0.08M PIPES-NaOH (+40 mM NaCl + 1 mM CaCI2) pH 8 on ice, prior to incubation, after which tissue was processed in a standard way (i.e. osmication ÷ ethanol dehydration ÷ Spurr's resin). Unstained or lightly stained (uranium) sections were examined in a JEOL i00 C TEM. ATP'ase reaction product was localised at discrete sites on the inside of the plasmalemma (Fig. 2) and on the inside of the tubules of PLS (Fig. 3), when cells were incubated in complete medium. Minor amounts of reaction product were also evident on the chloroplast envelope (Fig. 2), the tonoplast (Fig. 4), and occasionally in the ground cytoplasm. A shorter fixation period prior to incubation resulted in enhanced product deposition but a significant reduction in ultrastructural preservation (cf. Figs. 2,3). Controls employing absence of substrate or addition of NaF (Fig. 5) demonstrated no activity. Substitution of 8-glycerophosphate for ATP gave no deposition on the plasmalemma but small amounts were discernible in the cytoplasm. This study has therefore succeeded in providing further evidence consistent with the presence of H+ATP'ases on the plasmalemma of Chara. Demonstrated ATP'ase activity associated with PLS tubules also supports the notion that the PLS enhance H + efflux locally, thereby creating H + gradients capable of energising transport of other ions. Further studies will need to demonstrate conclusively the H + pumping nature of the plasmalemma ATP'ase. Further consideration must also be given to the implications of ATP'ase activity on the tonoplast and the chloroplast envelope. I.
Shimmen, T., Tazawa, M.
(1977).
J.Memb.Biol.
(1979).
37:167-192
2.
Kiefer, D.W., Spanswick, R.M.
3.
Smith, P.T., Walker, N.A.
(1981).
J.Memb. Biol. 60: 223-236
Plant Physiol. 6 4 : 1 6 5 - 6 8
4.
Smith, F.A., Walker, N.A.
(1976).
J.Exp.Bot. 309
27:451-59
310
G.D.
5.
Sanders, D.
(1980).
6.
Atkinson, M.R., Polya, G.
7.
Price, G.D.
(1981)
Price and M. I. Whitecross
J.Memb. Biol. 53: 129-41 (1967)
M.Sc. Thesis
Aust. J.Biol.Sci.
20: 1069-86
(A.N.U.)
8.
Franceschi, V.R., Lucas, W.J.
9.
Browning, A.J., Hall, J.L., Baker, D.A.
(1980)
Protoplasma 1 0 4 : 2 5 3 - 2 7 1 (1980)
Protoplasma 1 0 4 : 5 5 - 6 5
k
T
®
O*S ~ m
,
Fig. i.
Ultrastructural view of PLS M = mitochondrion.
conventional,
Fig. 2.
Complete medium (unstained; 60 min. fixation). Deposits plasmalemma (PL) and chloroplast (CHL) envelope.
Fig. 3.
Complete medium
Fig. 4.
Complete medium (unstained; 60 min fixation). (T). NUC = nucleus.
Fig. 5.
Complete medium + i0 mM NaF
(uranium stained;
2 hour fixation;
90 min fixation).
(unstained).
lead & uranium stained).
(arrows) evident on the
Deposits in the PLS tubules.
Deposits evident on the tonoplast
No reaction product.
Acknowledgements The authors wish to thank M. Fenning for preparation of EM prints; of the A.N.U. TEM Unit for technical support.
G. Weston and the staff