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Photoregulation of K+-ATPase in vitro by Red and Far Red Light in Extracts from Cucumber Hypocotyls
Original Paper Photoregulation of K+ -ATPase in vitro by Red and Far Red Light in Extracts from Cucumber Hypocotyls:~ BRIAN THOMAS and SUSAN E. TULL DivisiDn 'Of Physiology and Chemistry, Glasshouse Crops Research Institute, Worthing Road, Littlehampton, West Sussex, BN16 3PU, U. K. Received October 30, 1980 . Accepted December 15, 1980
Summary Particulate material from cucumber hypocotyls was purified by differential centrifugation and agarose gel chromatography. The fraction was heterogeneous in nature and contained bDth phytochrome and ATPase activity. The latter consisted of at least tWD different components, a non-specific acid phosphatase and a potassium stimulated ATPase (K+-ATPase). The K+-ATPase appeared to be strongly inhibited by Ca+2 and was subject to photoregulation by red and far red light (R and F) in vitro. F caused an increase in Km for ATP and this was reversible by subsequent R. ND significant effect on Vmax was caused by the light treatments. Photoregulation of ATPase in vitro was only seen if R was given in vivo prior to extraction. The effect of R in vivo was reversible by F indicating that this also was a phytochrome response. The acid phDsphatase activity was not subject to photoregulation but its presence prevented further characterisation of the K+ -ATPase and its photoregulation. The results are interpreted as an indirect effect of phytochrome on the K+-ATPase possibly by affecting membrane permeability and substrate availability.
Key words: Cucumis sativus L., ATPase, phytochrome, membranes.
Introduction Phytochr'Ome-mediated reSP'Onses t'O light are 'Often manifested as effects 'On the m'Ovement 'Of i'Ons (e.g. Satter and Galston, 1971; Br'Ownlee and Kendrick, 1979; Hale and R'Oux, 1980) alth'Ough the mechanism inv'Olved is n'Ot kn'Own. The active m'Ovement 'Of i'Ons in higher plants appears to be driven by A TPases, s'Ome 'Of which ". Part of this work was funded by ARC and carried out in the BDtany Department, University of Reading, Whiteknights, Reading, RG6 2AS. Abbreviations: BSA - bDvine serum albumin; EDTA - ethylene diamine-tetraacetic acid; ATP - adenosine 5'-triphosphate; ATPase - ATP phosphohydrolase (E.C. 3.6.1.3); R - red light; F - far red light; Pr - red absorbing form of phytochrome; Pfr - far red absorbing form of phytochrome; MOPS - morpholinopropanesulphonic acid; E.R. - endoplasmic reticulum.
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are stimulated by cations. There is considerable evidence for example that K+-ATPase associated with the plasma membrane drives active monovalent caltion transport (Leonard and Hotchkiss, 1976; Balke and Hodges, 1979) either directly or by the electrical component of the proton motive force (Dupont and Leonard, 1980). The importance of ion or proton-pumping ATPases has prompted the suggestion that their regulation offers a possible mode of action for both growth promoters and inhibitors (Hager et aI., 1971; Kasamo, 1979; Kasamo and Yamaki, 1976). The observations that ATPase in a particulate fraction from mung bean hypocotyls is subject to photoregulation in vitro by red light (R) and far red light (F) (Jose and Schafer, 1979) extends this possibility to include phytochrome. Unfortunately the ATPase in the mung bean was only partially characterised (Jose, 1977 b) and its role in cell function is uncertain. As part of an investigation into the photoresponses of commercially important glasshouse crops we observed that a membrane fraction from cucumber hypocotyl hooks prepared as described for the mung fraction by Jose and Schafer (1979) contained K+-ATPase. We report here on some properties of the enzyme including its photoregulation in vitro which differs from that reported for mung ATPase. Materials and Methods Plant Material Cucumber seeds (Cucumis sativus L., cv. Long Green Ridge, purchased from Suttons Seeds, Charvil, U.K.) were germinated on moist cellulose wadding in closed plastic boxes for 72 h at 25°C in darkness. Sections of about 10 mm immediately below the cotyledons and including the hook region were excised from the cucumber hypocotyls. Harvests were carried out in laboratory light except for experiments involving phytochrome measurements or photoregulation of enzyme activity where a dim green safelight was used. In these experiments harvested tissue was kept in the dark or treated with red or far red light as required prior to homogenisation, and subsequent purification steps and enzyme assays were also performed in darkness. Preparation of particulate fraction The particulate ATPase was prepared as described by Jose and Schafer (1979) using the techniques of differential centrifugation with the fraction pelletable between 10,000 g and 45,000 g (45 KP) being further purified by gel chromatography on Sepharose CL-2B. This step removes phytochrome non-specifically associated with ribonucleo-protein (Jose 1977 a) and produces a membrane fraction eluting at the void volume (Vo) containing phytochrome (data not shown), ATPase and acid phosphatase (Figure 1). The phytochrome in such preparations is tightly associated with the membrane rather than an electrostatically bound loosely held peripheral protein (Jose, 1977 a). The bulk of the ATPase co-elutes with the peak at Vo; the remainder eluting as a broad band behind this peak. The fractions comprising the peak at Vo were pooled and concentrated by centrifuging at 45,,000 g for 15 minutes. The pellet (Pa), resuspended in 3 ml 25 mM MOPS pH 7.0, 1 mM EDTA and 14 mM mercaptoethanol, was used in the subsequent assays. Assays ATPase actlvlty was determined by measuring the release of inorganic phosphate from ATP. Assay mixtures contained 45 mM Tris maleate pH 6.5, 25 mM MgCI 2, 5 mM
Z. Pflanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
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Fig. 1: Chromatography of membrane preparation on Sepharose CL-2B showing distribuAcid phosphatase 0-0, and protein . -•. Column dimensions tion of ATPase 150 X 26 mm, flow rate 1 ml min-t, 2.5 ml fractions. Fractions indicated as Vo were pooled and centrifuged to obtain P a.
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Tris-ATP except where indicated in the text. Released inorganic phosphate was determined using Fiske and Subbarow reducer (Sigma Chemical Co.) at a wavelength of 660 nm. Acid phosphatase was determined by measuring p-nitrophenol released from p-nitrophenyl phosphate. Assay mixtures contained 45 mM Tris maleate pH 6.0, 23 mM MgCI 2, 5 mM p-nitrophenyl phosphate. The reaction was stopped and colour developed with 2 vols of 5 % Na2COa and the absorbance was measured at 405 nm. Protein was determined by the method of Lowry et al. (1951) using crystalline BSA as standard. Light Sources Red and far red with incident photon flux area densities of 10,umol m-2s-1 and 5,umol m- 2s- 1 were produced by slide projectors fitted with interference filters (Schott and Gen. Mainz . .l.max 660 nm 1/2 bw 20 nm - Red; },max 730 nm '/2 bw 20 nm - Far Red). Replication Each experiment was repeated a mmlmum of 3 times with similar results obtained on each occasion. The data presented are in each case from one experiment and each value is the mean from 3 or 4 replicates. Standard errors were, in all experiments, <: 4 % of the mean values.
Results
Enzyme properties - pH The activity of the ATPase over the pH range 5.5 to 8.5 was determined. Activity was high at all pH values tested with an optimum at approximately pH 7.0 in the presence of 50 mM Mg+2. Addition of 25mM K+ led to increased activity between pH 5.5 and 7.0 as compared to the activity in the presence of Mg+ 2 alone. K+ stimulation Z. PJlanzenphysiol. Ed. 102. S. 283-292. 1981.
286
BRIAN THOMAS and SUSAN E. TULL
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PJlanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
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showed an optimum between pH 5.7 and 6.1 and was not observed> pH 7.5. From these observations it was decided to perform subsequent assays at pH 6.5. Cation effects
The ATPase activity m P s was greatly affected by inorganic cations. Mg+ 2 increased ATPase activity with maximum effect (usually >100 Ofo increase) at about 20 mM (Figure 2). K+ further stimulated ATPase activity commonly by 25 to 30 Ofo as compared with Mg+ 2 -ATPase values with an optimum K+ concentration of 25 mM at pH 6.5 (Figure 3). Ca+ 2, in contrast, strongly inhibuted Mg+ 2-ATPase activity (Figure 4) with 50 Ofo inhibition at 500 ,uM. No effect of Ca+ 2 or K+ was observed in the absence of Mg+2. Neither the inorganic anions Cl-, S04- 2 and NO s- nor the organic cation Tris had any effect on ATPase aotivity. In the presence of the mild non-ionic detergent Triton-X 100 at 0.01 % the total activity in the sample increased and was redistributed almost equally between soluble and insoluble fractions (Table 1). The detergent in the soluble fraction was therefore able to substitute for the membrane lipids present in the particulate preparation. Mg+ 2 and K+ stimulation and Ca+ 2 inhibition were retained in the solubilised fraction with the relative effectiveness of Mg+ 2 and Ca+ 2 being greater in detergent treated samples compared to controls. Response to light
Initial attempts to observe photoregulation used P s obtained from tissues given 2 mins R in vivo. In these experiments R ,and F given in vitro prior to the assay had only a small effect on ATPase activity at a substrate concentration of 5 mM. Further Z. Pflanzenphysiol. Bd. 102. S. 283-292. 1981.
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BRIAN THOMAS and SUSAN E . TULL
Table 1: Solubilization of ATPase by Triton-X 100. Untreated p )
Triton-XI 00 treated soluble':' a b
insoluble':' b
a
b
100
100
78
113
80
204
+ 20 mM Mg+2 167
167
232
337
238
608
212
255
371
311
795
117
96
139
89
227
Control
+ 20 mM Mg+2 + 25 mMK + 212 + 20mMMg+ 1 + 5 mM Ca+2 117
a
Table shows the effect of treating p) with 0.01 % Triton-Xl 00 on ATPase activity. a relative ATPase activity (Ilmoles PO.' ml p).1 . h· I). b = relative specific activity (f.lmoles PO • . mg protein-I. h- I). All values relative to untreated p) control which was 241lmoles PO.' mg protein-I. h- I = 100 %.
=
':. Soluble and insoluble are supernatant and resuspended pellet from 45,000 g for 15 min spin after treatment with Triton-XIOO for 30 min.
analysis of the response was carried out by determining the effect of light on ATPase activity over a range of substrate concentrations. Figure 5 shows results from two such experiments as Lineweaver-Burke plots. In one experiment -the tissue was given 2 minutes R prior to extraction and in the other this treatment was omitJted. In samples given R in vivo, F consistently increased the Km value for ATP whereas a subsequent red reversed the effect although the amount of reversal achieved was inconsistent. The results here show a partial reversal which was a typical result but full reversal has occasionally been observed. Despite the use of standardised methods and conditions Km values varied between preparations but were always in the range 0.3-1 mM for samples given R in vivo only. These values are consistent with those described for corn root plasma membrane K+ -ATPase (Dupont and Leonard, 1980). Following treatment with F and R in vitro, changes in Vmax were small. The plots therefore do not resemble non-competitive or allosteric effect but best describe a competitive inhibition of ATPase caused by F. Although the precise values of the kinetic constants vary from preparation to preparation the same pattern of response to Rand F given in vitro was consistently obtained. If no light treatment was given in vivo prior to homogenisation, subsequent in vitro light treatments were ineffective (Figure 5). Similarly Rand F given in vitro had no effect if R given in vivo was followed immediately by F (data not shown). It appears, therefore, that the potential for photoregulation in vitro is under phytochrome control. Reversibility of the in vitro light treatments can be demonstrated for one cycle of photoconversions. The number of assays to determine each Km value accurately has limited the number of reversals examined both on account of the quantity of experimental material av.ailable and also ·the problems of adequate replication. Z. P/lanzenphysiol. Ed. 102. S. 283-292. 1981.
Light and ATPase activity
289
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Fig. 5: Double reciprocal plots of ATPase activity versus ATP concentration after various light treatments. a = plants given no light treatments in vivo. b = plants given 2 min R in vivo prior to extraction. Light treatments in vitro prior to assay were: No light treat1 min F followed by 1 min R 0-0. All lines fitted by linement ___ , 1 min F ar regression analysis. r >0.95 in all cases.
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Acid phosphatase activity When p-nitrophenyl phosphate was incubated with the P 3 fraction, considerable non-specific (acid) phosphatase aotivity was obtained. The distribution of nitrophenyl phosphatase on Sepharose CL-2B also shows a large peak co-eluting with Vo and a later broad band of soluble activity (Figure 1). This enzyme activity was high in the absence of Mg+ 2 and its activity was increased only slightly «10 Ofo) in the presence of 25 mM MgCI 2 • Mg+ 2 could be replaced by Ca+ 2 but in the presence of Mg+2 neither Ca+ 2 nor K+ affected nitrophenyl phosphatase activity. Nitrophenyl phosphatase was completely insensitive to light treatments in vivo or in vitro. Attempts to separate the nitrophenyl phosphatase from the ATPase activ~ty using ionic and detergent treatments were unsuccessful.
Discussion The phytochrome-containing particulate fraction used in this paper was prepared in such a way as to avoid contamination by electrostatically bound Pfr which when Z. Pjlanzenphysiol. Bd. 102. S. 283-292. 1981.
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BRIAN THOMAS
and
SUSAN
E.
TULL
adsorbed to ribonucleo-protein represents a major artifact in the preparation of pelletable phytochrome in cucurbrts (Quail, 1975). The use of gel chromatography results in a membrane fraction containing tightly bound phytochrome representing a true in vivo association (Pratt, 1978; Jose, 1977 a) although the nature of the membrane fraotion is not known. Enzymes present in the fraction are K+-ATPase which has on occasion been used as a plasma membrane marker (Nagahashi et aI., 1978) but multiple location of this enzyme in the cell has also been suggested (Hendriks, 1977). Acid phosphatase, also present, has been found to be associated both with Golgi membranes (Pierce and Hendrix, 1979) and E. R. (Pyliotis et aI., 1979), and NADH-cytochrome-C reductase, a marker for E.R., co-elutes with Vo in our extracts (unpublished data). The preparation is therefore almost certainly heterogeneous in nature. Investigaotions of such crude active preparations represent a necessary intermediary step in the prepamtion of homogeneous extracts for detailed investigation. With the exception of light mediatted gibberellin release from etioplasts (Hilton and Smith, 1980), demonstrations of in vitro responses have been limited to heterogeneous prepara.rions (Penel and Greppin, 1979; Jose and Schafer, 1979; Gaunt and Plumpton, 1978) and any interpretation of the results should be made with caution. The photoregulation of ATPase in a cell-free system has previously been demonstrated only in extracts from mung bean, (Jose, 1977 b; Jose and Schafer, 1979). In this paper we show such a regulation in extracts from cucumber seedlings although the details are somewhat different. In our hands photoregulation in cucumber is shown as a change in ATPase Km values following irradiation in vitro providing that the tissue had been treated with R in vivo. No effect of light in vitro was observed if the light treatment was omitted or if R was immediaotely followed by F in vivo. The system therefore involves two separate effects of light; the first light treatment potentiating the system for the in vitro treatment. This is similar to the situation described for the mung bean (Jose, 1977 b). Reversibility of R by a subsequent F in vivo and F by a subsequent R in vitro indicates that both light effects are mediated by phytochrome. A major problem in the interpretation of the in vitro light response is the presence of a high level of acid phosphatase activity in our preparations. In its presence it is not possible to characterize fully the K+-ATPase activity to establish whether it is equivalent to the K+-ATPase thought to be involved in ion transport (Dupont and Leonard, 1980). The properties of the acid phosphatase are, however, clearly different from the K+-ATPase. It is neither Ca+ 2 inhibited or K+ stimulated and it is not subject to photoregulation. It may contribute to the high level of activity in the absence of Mg+ 2 and help explain why the Km of ATPase varies between preparations. On the other hand it cannot explain the effects of F and R on the ATPase Km values nor the difference between the cucumber and mung bean extracts, especially as the latter also contains acid phosphatase activity (unpublished observations). Z. PJlanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
291
The similarity between the effect of light and thaJt predicted for a competitive inhibitor suggeSlts that ph}'1tochrome may act by affecting the availability of substrate rMher than by a direct effect on the enzyme. Such an effect might be obtained if, for example, a significant proportion of the A TP-binding sites of ,t he ATPase were orientated inwards in membrane vesicles in the preparation. The effect of phytochrome could then be due to changes in permeability of the vesicle membranes. In this case an increase in Km following F would mean that removing Pfr decreases permeability and hence accessibility of substrate. This explanation would be consistent with the suggestion made by several workers thaJt phytochrome acts by controlling membrane permeability (see Marme, 1977; Brownlee et al., 1979). Another possibility is that phytochrome could control the localised concentration of free K+ or Ca+ 2 carried through in the membrane preparation procedure. Dreyer and Weisenseel (1979) have postulated that the regulation of the level of cytosolic Ca+ 2 is the prime function of phytochrome with Ca+2 acting as a secondary messenger. In our system regulation of ATPase in this way would be due to inhibition by Ca+ 2, an unusual but not unique property of the enzyme (Hendriks, 1977). The mechanism by which ATPase activity is inhibited is unknown and it could be argued that it is the result of a non-specific effect of Ca+2 on the membrane lipids. This seems unlikely, however, as Ca+ 2 is equally as effeotive in inhibiting ATPase activity solubilised by Triton-X 100 as it is in inhibiting the particulate enzyme. This suggests that the effect of Ca+ 2 is actually on the enzyme itself and may thus be of some biological significance. It is not known whether a specific calcium binding protein such as calmodulin (Stevens et al., 1976) is involved in this Ca+ 2 regulation but by analogy with animal systems the distribution of such proteins might be important if Ca+ 2 is indeed the secondary messenger for phytochrome control. The difference between the type of in vitro photomodulation of ATPase activity in extracts of cucumbers and mung beans argues against a direct regulation of the enzyme by phytochrome as we would expect that the primary response representing a particular mode of action of phytochrome should be the same irrespective of source. The response must, however, be at least an early secondary event, the characterization of which would help us to understand the mode of action of phytochrome. Further investigation to this end is cominuing in this laboratory. Acknowledgements The authors would like to thank Dr. D. Vince-Prue for her interest and advice and Dr. B. Jordan for helpful discussion. Special thanks to Dr. G. Hobson for critical reading of the manuscript.
References BALKE, N. E. and T. K. HODGES: Plant Physio!. 63, 48-52 (1979). BROWNLEE, C. and R. E. KENDRICK: Plant Physio!. 64, 206-210 (1979). BROWNLEE, C. N., N. ROTH-BEJERANO, and R. E. KENDRICK: Sci. Prog. Oxf. 66, 217-229
(1979).
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Pjlanzenphysiol. Bd. 102.
s. 283-292.
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BRIAN THOMAS and SUSAN E. TULL
DREYER, E. M. and M. H. WEISENSEEL: Planta, 146,31-39 (1979). DUPONT, F. M. and R. T. LEONARD: Plant Physio!., 65,931-938 (1980). GAUNT, ]. K. and E. S. PLUMPTON: Biochern. Soc. Trans., 6, 143 (1978). HAGER, A., H. MENZEL, and A. KRAUSS: Planta, 100, 47-75 (1971). HALE, C. C. and s. ]. Roux: Plant Physio!., 65, 658-662 (1980). HENDRIKS, T.: Plant Sci. Lett. 9, 351-363 (1977). HILTON,]. R. and H. SMITH: Planta, 148, 312-318 (1980). JOSE, A. M.: Planta 134, 287-293 (1977 a). - Planta, 137, 203-206 (1977 b). JOSE, A. M. and E. SCHAFER, Planta, 146, 75-81 (1979). KASAMO, K.: Plant & Cell Physio!., 20, 293-300 (1979). KASAMO, K. and T. YAMAKI: Plant & Cell Physio!., 17, 149-164 (1976). LEONARD, R. T. and C. W. HOTCHKISS: Plant Physio!., 58,331-335 (1976). LOWRY, O. H., N. ]. ROSEBROUGH, A. L. FARR, and R. ]. RANDALL: J. Biochern. 193, 265-275 (1951). MARME, D.: Ann. Rev. Plant Physio!., 28,173-198 (1977). NAGAHASHI, G., R. T. LEONARD, and W. T. THOMSON: Plant Physio!', 61, 993-999 (1978). PENEL, C. and H. GREPPIN: Physio!. Plant., 46, 208-210 (1979). PIERCE, W. S. and D. L. HENDRIX: Planta, 146, 161-169 (1979). PRATT, L. H.: Photochern. Photobio!' 27, 81-105 (1978). PYLIOTIS, N. A., A. E. ASHFORD, M. I. WHITE CROSS, and]. V. JACOBSEN: Planta, 147, 134-140 (1979). QUAIL, P. H.: Planta, 123, 223-234 (1975). SATTER, R. L. and A. W. GALSTON: Plant Physio!', 48,740-746 (1971). STEVENS, F. c., M. WALSH, H. C. Ho, T. S. TEO, and]. H. WANG: J. BiD!. Chern. 251, 4495-4500 (1976).
z.
Pjlanzenphysiol. Bd. 102. S. 283-292. 1981.
Summary Particulate material from cucumber hypocotyls was purified by differential centrifugation and agarose gel chromatography. The fraction was heterogeneous in nature and contained bDth phytochrome and ATPase activity. The latter consisted of at least tWD different components, a non-specific acid phosphatase and a potassium stimulated ATPase (K+-ATPase). The K+-ATPase appeared to be strongly inhibited by Ca+2 and was subject to photoregulation by red and far red light (R and F) in vitro. F caused an increase in Km for ATP and this was reversible by subsequent R. ND significant effect on Vmax was caused by the light treatments. Photoregulation of ATPase in vitro was only seen if R was given in vivo prior to extraction. The effect of R in vivo was reversible by F indicating that this also was a phytochrome response. The acid phDsphatase activity was not subject to photoregulation but its presence prevented further characterisation of the K+ -ATPase and its photoregulation. The results are interpreted as an indirect effect of phytochrome on the K+-ATPase possibly by affecting membrane permeability and substrate availability.
Key words: Cucumis sativus L., ATPase, phytochrome, membranes.
Introduction Phytochr'Ome-mediated reSP'Onses t'O light are 'Often manifested as effects 'On the m'Ovement 'Of i'Ons (e.g. Satter and Galston, 1971; Br'Ownlee and Kendrick, 1979; Hale and R'Oux, 1980) alth'Ough the mechanism inv'Olved is n'Ot kn'Own. The active m'Ovement 'Of i'Ons in higher plants appears to be driven by A TPases, s'Ome 'Of which ". Part of this work was funded by ARC and carried out in the BDtany Department, University of Reading, Whiteknights, Reading, RG6 2AS. Abbreviations: BSA - bDvine serum albumin; EDTA - ethylene diamine-tetraacetic acid; ATP - adenosine 5'-triphosphate; ATPase - ATP phosphohydrolase (E.C. 3.6.1.3); R - red light; F - far red light; Pr - red absorbing form of phytochrome; Pfr - far red absorbing form of phytochrome; MOPS - morpholinopropanesulphonic acid; E.R. - endoplasmic reticulum.
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BRIAN THOMAS and SUSAN E. TULL
are stimulated by cations. There is considerable evidence for example that K+-ATPase associated with the plasma membrane drives active monovalent caltion transport (Leonard and Hotchkiss, 1976; Balke and Hodges, 1979) either directly or by the electrical component of the proton motive force (Dupont and Leonard, 1980). The importance of ion or proton-pumping ATPases has prompted the suggestion that their regulation offers a possible mode of action for both growth promoters and inhibitors (Hager et aI., 1971; Kasamo, 1979; Kasamo and Yamaki, 1976). The observations that ATPase in a particulate fraction from mung bean hypocotyls is subject to photoregulation in vitro by red light (R) and far red light (F) (Jose and Schafer, 1979) extends this possibility to include phytochrome. Unfortunately the ATPase in the mung bean was only partially characterised (Jose, 1977 b) and its role in cell function is uncertain. As part of an investigation into the photoresponses of commercially important glasshouse crops we observed that a membrane fraction from cucumber hypocotyl hooks prepared as described for the mung fraction by Jose and Schafer (1979) contained K+-ATPase. We report here on some properties of the enzyme including its photoregulation in vitro which differs from that reported for mung ATPase. Materials and Methods Plant Material Cucumber seeds (Cucumis sativus L., cv. Long Green Ridge, purchased from Suttons Seeds, Charvil, U.K.) were germinated on moist cellulose wadding in closed plastic boxes for 72 h at 25°C in darkness. Sections of about 10 mm immediately below the cotyledons and including the hook region were excised from the cucumber hypocotyls. Harvests were carried out in laboratory light except for experiments involving phytochrome measurements or photoregulation of enzyme activity where a dim green safelight was used. In these experiments harvested tissue was kept in the dark or treated with red or far red light as required prior to homogenisation, and subsequent purification steps and enzyme assays were also performed in darkness. Preparation of particulate fraction The particulate ATPase was prepared as described by Jose and Schafer (1979) using the techniques of differential centrifugation with the fraction pelletable between 10,000 g and 45,000 g (45 KP) being further purified by gel chromatography on Sepharose CL-2B. This step removes phytochrome non-specifically associated with ribonucleo-protein (Jose 1977 a) and produces a membrane fraction eluting at the void volume (Vo) containing phytochrome (data not shown), ATPase and acid phosphatase (Figure 1). The phytochrome in such preparations is tightly associated with the membrane rather than an electrostatically bound loosely held peripheral protein (Jose, 1977 a). The bulk of the ATPase co-elutes with the peak at Vo; the remainder eluting as a broad band behind this peak. The fractions comprising the peak at Vo were pooled and concentrated by centrifuging at 45,,000 g for 15 minutes. The pellet (Pa), resuspended in 3 ml 25 mM MOPS pH 7.0, 1 mM EDTA and 14 mM mercaptoethanol, was used in the subsequent assays. Assays ATPase actlvlty was determined by measuring the release of inorganic phosphate from ATP. Assay mixtures contained 45 mM Tris maleate pH 6.5, 25 mM MgCI 2, 5 mM
Z. Pflanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
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Fig. 1: Chromatography of membrane preparation on Sepharose CL-2B showing distribuAcid phosphatase 0-0, and protein . -•. Column dimensions tion of ATPase 150 X 26 mm, flow rate 1 ml min-t, 2.5 ml fractions. Fractions indicated as Vo were pooled and centrifuged to obtain P a.
.-e,
Tris-ATP except where indicated in the text. Released inorganic phosphate was determined using Fiske and Subbarow reducer (Sigma Chemical Co.) at a wavelength of 660 nm. Acid phosphatase was determined by measuring p-nitrophenol released from p-nitrophenyl phosphate. Assay mixtures contained 45 mM Tris maleate pH 6.0, 23 mM MgCI 2, 5 mM p-nitrophenyl phosphate. The reaction was stopped and colour developed with 2 vols of 5 % Na2COa and the absorbance was measured at 405 nm. Protein was determined by the method of Lowry et al. (1951) using crystalline BSA as standard. Light Sources Red and far red with incident photon flux area densities of 10,umol m-2s-1 and 5,umol m- 2s- 1 were produced by slide projectors fitted with interference filters (Schott and Gen. Mainz . .l.max 660 nm 1/2 bw 20 nm - Red; },max 730 nm '/2 bw 20 nm - Far Red). Replication Each experiment was repeated a mmlmum of 3 times with similar results obtained on each occasion. The data presented are in each case from one experiment and each value is the mean from 3 or 4 replicates. Standard errors were, in all experiments, <: 4 % of the mean values.
Results
Enzyme properties - pH The activity of the ATPase over the pH range 5.5 to 8.5 was determined. Activity was high at all pH values tested with an optimum at approximately pH 7.0 in the presence of 50 mM Mg+2. Addition of 25mM K+ led to increased activity between pH 5.5 and 7.0 as compared to the activity in the presence of Mg+ 2 alone. K+ stimulation Z. PJlanzenphysiol. Ed. 102. S. 283-292. 1981.
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BRIAN THOMAS and SUSAN E. TULL
250
/
---- 200
0/0---.
°
150
100 •
---==== .,
o
5
,
10
,
20
15
,
25
[MgC121 / mM
Fig. 2: Effect of Mg+2 on ATPase activity at pH 6.5. Activity is expressed relative to ATPase activity in the absence of Mg+2 which in this experiment is 35 ,umoles· mg protein-I . h- I = 100 0/0.
130
/o~ 0/0
----120 ~
;:
g QI
,;::
Si
QI
a:
110
.
I
I°
/
.
0/
°
100 ~.------------~,------------~---------~ o 10 20 30 [KCL] / mM
Fig. 3: Effect of K+ on ATPase activity at pH 6.5. Activity is expressed relative to ATPase activity in the presence of 25 mM MgC12 and the absence of KCl which in this experiment is 17 ,umoles . mg protein-I. h- I = 1000/0.
z.
PJlanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
100
\
.
\
£>
u "
'>"
50
.~. ~
0"
287
o
05
10
--
.
.- .
'"
15
20
25
rCa (N03 )2] / mM
Fig. 4: Inhibition of ATPase activity by Ca+ 2 pH 6.5. Activity is expressed relative to A TPase activity in the presence of 20 mM MgCI2 and the absence of Ca(NOsh which in this experiment was 69 ,umoles' mg protein-I. h- 1 = 100 0/0.
showed an optimum between pH 5.7 and 6.1 and was not observed> pH 7.5. From these observations it was decided to perform subsequent assays at pH 6.5. Cation effects
The ATPase activity m P s was greatly affected by inorganic cations. Mg+ 2 increased ATPase activity with maximum effect (usually >100 Ofo increase) at about 20 mM (Figure 2). K+ further stimulated ATPase activity commonly by 25 to 30 Ofo as compared with Mg+ 2 -ATPase values with an optimum K+ concentration of 25 mM at pH 6.5 (Figure 3). Ca+ 2, in contrast, strongly inhibuted Mg+ 2-ATPase activity (Figure 4) with 50 Ofo inhibition at 500 ,uM. No effect of Ca+ 2 or K+ was observed in the absence of Mg+2. Neither the inorganic anions Cl-, S04- 2 and NO s- nor the organic cation Tris had any effect on ATPase aotivity. In the presence of the mild non-ionic detergent Triton-X 100 at 0.01 % the total activity in the sample increased and was redistributed almost equally between soluble and insoluble fractions (Table 1). The detergent in the soluble fraction was therefore able to substitute for the membrane lipids present in the particulate preparation. Mg+ 2 and K+ stimulation and Ca+ 2 inhibition were retained in the solubilised fraction with the relative effectiveness of Mg+ 2 and Ca+ 2 being greater in detergent treated samples compared to controls. Response to light
Initial attempts to observe photoregulation used P s obtained from tissues given 2 mins R in vivo. In these experiments R ,and F given in vitro prior to the assay had only a small effect on ATPase activity at a substrate concentration of 5 mM. Further Z. Pflanzenphysiol. Bd. 102. S. 283-292. 1981.
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BRIAN THOMAS and SUSAN E . TULL
Table 1: Solubilization of ATPase by Triton-X 100. Untreated p )
Triton-XI 00 treated soluble':' a b
insoluble':' b
a
b
100
100
78
113
80
204
+ 20 mM Mg+2 167
167
232
337
238
608
212
255
371
311
795
117
96
139
89
227
Control
+ 20 mM Mg+2 + 25 mMK + 212 + 20mMMg+ 1 + 5 mM Ca+2 117
a
Table shows the effect of treating p) with 0.01 % Triton-Xl 00 on ATPase activity. a relative ATPase activity (Ilmoles PO.' ml p).1 . h· I). b = relative specific activity (f.lmoles PO • . mg protein-I. h- I). All values relative to untreated p) control which was 241lmoles PO.' mg protein-I. h- I = 100 %.
=
':. Soluble and insoluble are supernatant and resuspended pellet from 45,000 g for 15 min spin after treatment with Triton-XIOO for 30 min.
analysis of the response was carried out by determining the effect of light on ATPase activity over a range of substrate concentrations. Figure 5 shows results from two such experiments as Lineweaver-Burke plots. In one experiment -the tissue was given 2 minutes R prior to extraction and in the other this treatment was omitJted. In samples given R in vivo, F consistently increased the Km value for ATP whereas a subsequent red reversed the effect although the amount of reversal achieved was inconsistent. The results here show a partial reversal which was a typical result but full reversal has occasionally been observed. Despite the use of standardised methods and conditions Km values varied between preparations but were always in the range 0.3-1 mM for samples given R in vivo only. These values are consistent with those described for corn root plasma membrane K+ -ATPase (Dupont and Leonard, 1980). Following treatment with F and R in vitro, changes in Vmax were small. The plots therefore do not resemble non-competitive or allosteric effect but best describe a competitive inhibition of ATPase caused by F. Although the precise values of the kinetic constants vary from preparation to preparation the same pattern of response to Rand F given in vitro was consistently obtained. If no light treatment was given in vivo prior to homogenisation, subsequent in vitro light treatments were ineffective (Figure 5). Similarly Rand F given in vitro had no effect if R given in vivo was followed immediately by F (data not shown). It appears, therefore, that the potential for photoregulation in vitro is under phytochrome control. Reversibility of the in vitro light treatments can be demonstrated for one cycle of photoconversions. The number of assays to determine each Km value accurately has limited the number of reversals examined both on account of the quantity of experimental material av.ailable and also ·the problems of adequate replication. Z. P/lanzenphysiol. Ed. 102. S. 283-292. 1981.
Light and ATPase activity
289
12 In vitro Km(mM)
•0
0·35 0.33 0·37
of F-R
6
,. E c:
0
-3
ci 0
-1
In vitro Km(mM)
.0
of cF-R
0
20
3
5
3
5
7
b
0·68 1· 72 0·90
10
-3
-1
o [ATP]-l /mM
Fig. 5: Double reciprocal plots of ATPase activity versus ATP concentration after various light treatments. a = plants given no light treatments in vivo. b = plants given 2 min R in vivo prior to extraction. Light treatments in vitro prior to assay were: No light treat1 min F followed by 1 min R 0-0. All lines fitted by linement ___ , 1 min F ar regression analysis. r >0.95 in all cases.
e-e,
Acid phosphatase activity When p-nitrophenyl phosphate was incubated with the P 3 fraction, considerable non-specific (acid) phosphatase aotivity was obtained. The distribution of nitrophenyl phosphatase on Sepharose CL-2B also shows a large peak co-eluting with Vo and a later broad band of soluble activity (Figure 1). This enzyme activity was high in the absence of Mg+ 2 and its activity was increased only slightly «10 Ofo) in the presence of 25 mM MgCI 2 • Mg+ 2 could be replaced by Ca+ 2 but in the presence of Mg+2 neither Ca+ 2 nor K+ affected nitrophenyl phosphatase activity. Nitrophenyl phosphatase was completely insensitive to light treatments in vivo or in vitro. Attempts to separate the nitrophenyl phosphatase from the ATPase activ~ty using ionic and detergent treatments were unsuccessful.
Discussion The phytochrome-containing particulate fraction used in this paper was prepared in such a way as to avoid contamination by electrostatically bound Pfr which when Z. Pjlanzenphysiol. Bd. 102. S. 283-292. 1981.
290
BRIAN THOMAS
and
SUSAN
E.
TULL
adsorbed to ribonucleo-protein represents a major artifact in the preparation of pelletable phytochrome in cucurbrts (Quail, 1975). The use of gel chromatography results in a membrane fraction containing tightly bound phytochrome representing a true in vivo association (Pratt, 1978; Jose, 1977 a) although the nature of the membrane fraotion is not known. Enzymes present in the fraction are K+-ATPase which has on occasion been used as a plasma membrane marker (Nagahashi et aI., 1978) but multiple location of this enzyme in the cell has also been suggested (Hendriks, 1977). Acid phosphatase, also present, has been found to be associated both with Golgi membranes (Pierce and Hendrix, 1979) and E. R. (Pyliotis et aI., 1979), and NADH-cytochrome-C reductase, a marker for E.R., co-elutes with Vo in our extracts (unpublished data). The preparation is therefore almost certainly heterogeneous in nature. Investigaotions of such crude active preparations represent a necessary intermediary step in the prepamtion of homogeneous extracts for detailed investigation. With the exception of light mediatted gibberellin release from etioplasts (Hilton and Smith, 1980), demonstrations of in vitro responses have been limited to heterogeneous prepara.rions (Penel and Greppin, 1979; Jose and Schafer, 1979; Gaunt and Plumpton, 1978) and any interpretation of the results should be made with caution. The photoregulation of ATPase in a cell-free system has previously been demonstrated only in extracts from mung bean, (Jose, 1977 b; Jose and Schafer, 1979). In this paper we show such a regulation in extracts from cucumber seedlings although the details are somewhat different. In our hands photoregulation in cucumber is shown as a change in ATPase Km values following irradiation in vitro providing that the tissue had been treated with R in vivo. No effect of light in vitro was observed if the light treatment was omitted or if R was immediaotely followed by F in vivo. The system therefore involves two separate effects of light; the first light treatment potentiating the system for the in vitro treatment. This is similar to the situation described for the mung bean (Jose, 1977 b). Reversibility of R by a subsequent F in vivo and F by a subsequent R in vitro indicates that both light effects are mediated by phytochrome. A major problem in the interpretation of the in vitro light response is the presence of a high level of acid phosphatase activity in our preparations. In its presence it is not possible to characterize fully the K+-ATPase activity to establish whether it is equivalent to the K+-ATPase thought to be involved in ion transport (Dupont and Leonard, 1980). The properties of the acid phosphatase are, however, clearly different from the K+-ATPase. It is neither Ca+ 2 inhibited or K+ stimulated and it is not subject to photoregulation. It may contribute to the high level of activity in the absence of Mg+ 2 and help explain why the Km of ATPase varies between preparations. On the other hand it cannot explain the effects of F and R on the ATPase Km values nor the difference between the cucumber and mung bean extracts, especially as the latter also contains acid phosphatase activity (unpublished observations). Z. PJlanzenphysiol. Bd. 102. S. 283-292. 1981.
Light and ATPase activity
291
The similarity between the effect of light and thaJt predicted for a competitive inhibitor suggeSlts that ph}'1tochrome may act by affecting the availability of substrate rMher than by a direct effect on the enzyme. Such an effect might be obtained if, for example, a significant proportion of the A TP-binding sites of ,t he ATPase were orientated inwards in membrane vesicles in the preparation. The effect of phytochrome could then be due to changes in permeability of the vesicle membranes. In this case an increase in Km following F would mean that removing Pfr decreases permeability and hence accessibility of substrate. This explanation would be consistent with the suggestion made by several workers thaJt phytochrome acts by controlling membrane permeability (see Marme, 1977; Brownlee et al., 1979). Another possibility is that phytochrome could control the localised concentration of free K+ or Ca+ 2 carried through in the membrane preparation procedure. Dreyer and Weisenseel (1979) have postulated that the regulation of the level of cytosolic Ca+ 2 is the prime function of phytochrome with Ca+2 acting as a secondary messenger. In our system regulation of ATPase in this way would be due to inhibition by Ca+ 2, an unusual but not unique property of the enzyme (Hendriks, 1977). The mechanism by which ATPase activity is inhibited is unknown and it could be argued that it is the result of a non-specific effect of Ca+2 on the membrane lipids. This seems unlikely, however, as Ca+ 2 is equally as effeotive in inhibiting ATPase activity solubilised by Triton-X 100 as it is in inhibiting the particulate enzyme. This suggests that the effect of Ca+ 2 is actually on the enzyme itself and may thus be of some biological significance. It is not known whether a specific calcium binding protein such as calmodulin (Stevens et al., 1976) is involved in this Ca+ 2 regulation but by analogy with animal systems the distribution of such proteins might be important if Ca+ 2 is indeed the secondary messenger for phytochrome control. The difference between the type of in vitro photomodulation of ATPase activity in extracts of cucumbers and mung beans argues against a direct regulation of the enzyme by phytochrome as we would expect that the primary response representing a particular mode of action of phytochrome should be the same irrespective of source. The response must, however, be at least an early secondary event, the characterization of which would help us to understand the mode of action of phytochrome. Further investigation to this end is cominuing in this laboratory. Acknowledgements The authors would like to thank Dr. D. Vince-Prue for her interest and advice and Dr. B. Jordan for helpful discussion. Special thanks to Dr. G. Hobson for critical reading of the manuscript.
References BALKE, N. E. and T. K. HODGES: Plant Physio!. 63, 48-52 (1979). BROWNLEE, C. and R. E. KENDRICK: Plant Physio!. 64, 206-210 (1979). BROWNLEE, C. N., N. ROTH-BEJERANO, and R. E. KENDRICK: Sci. Prog. Oxf. 66, 217-229
(1979).
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s. 283-292.
1981.
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DREYER, E. M. and M. H. WEISENSEEL: Planta, 146,31-39 (1979). DUPONT, F. M. and R. T. LEONARD: Plant Physio!., 65,931-938 (1980). GAUNT, ]. K. and E. S. PLUMPTON: Biochern. Soc. Trans., 6, 143 (1978). HAGER, A., H. MENZEL, and A. KRAUSS: Planta, 100, 47-75 (1971). HALE, C. C. and s. ]. Roux: Plant Physio!., 65, 658-662 (1980). HENDRIKS, T.: Plant Sci. Lett. 9, 351-363 (1977). HILTON,]. R. and H. SMITH: Planta, 148, 312-318 (1980). JOSE, A. M.: Planta 134, 287-293 (1977 a). - Planta, 137, 203-206 (1977 b). JOSE, A. M. and E. SCHAFER, Planta, 146, 75-81 (1979). KASAMO, K.: Plant & Cell Physio!., 20, 293-300 (1979). KASAMO, K. and T. YAMAKI: Plant & Cell Physio!., 17, 149-164 (1976). LEONARD, R. T. and C. W. HOTCHKISS: Plant Physio!., 58,331-335 (1976). LOWRY, O. H., N. ]. ROSEBROUGH, A. L. FARR, and R. ]. RANDALL: J. Biochern. 193, 265-275 (1951). MARME, D.: Ann. Rev. Plant Physio!., 28,173-198 (1977). NAGAHASHI, G., R. T. LEONARD, and W. T. THOMSON: Plant Physio!', 61, 993-999 (1978). PENEL, C. and H. GREPPIN: Physio!. Plant., 46, 208-210 (1979). PIERCE, W. S. and D. L. HENDRIX: Planta, 146, 161-169 (1979). PRATT, L. H.: Photochern. Photobio!' 27, 81-105 (1978). PYLIOTIS, N. A., A. E. ASHFORD, M. I. WHITE CROSS, and]. V. JACOBSEN: Planta, 147, 134-140 (1979). QUAIL, P. H.: Planta, 123, 223-234 (1975). SATTER, R. L. and A. W. GALSTON: Plant Physio!', 48,740-746 (1971). STEVENS, F. c., M. WALSH, H. C. Ho, T. S. TEO, and]. H. WANG: J. BiD!. Chern. 251, 4495-4500 (1976).
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