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Light-Hduced Breakdown of NADPH-Protochlorophyllide Oxidoreductase in Vitro' Received for Publication October 13, 1982 and in Revised Form January 14, 1983

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Light-Hduced Breakdown of NADPH-Protochlorophyllide Oxidoreductase in Vitro' Received for Publication October 13, 1982 and in Revised Form January 14, 1983 Plant Physiol. (1983) 72, 229-236 0032-0889/83/72/0229/08/$00.50/0 Light-hduced Breakdown of NADPH-Protochlorophyllide Oxidoreductase In Vitro' Received for publication October 13, 1982 and in revised form January 14, 1983 STEVE A. KAY AND W. TREVOR GRIFFITHS Department ofBiochemistry, The Medical School, University ofBristol, Bristol BS8 ITD, England ABSTRACT d at 22°C or 33°C in the dark, exactly as reported earlier (2). Preparation of Etioplasts and Etioplast Membrane Fractions. Light-induced loss of the enzyme protochlorophylide reductase (EC Shoots of oats and rye were homogenized in an Atomix blender 1.6.99.1.), already described as a characteristic of whole plants, has now and etioplasts isolated by differential centrifugation of the ho- been demonstrated in vitro using etioplast membrane preparations ofAvexa mogenates using a modification ofthe previously reported method Sativa L. var Peniarth and Secal cerealk L. var RheidoL Some evidence is (10). Etioplast pellets were gently resuspended using a cotton presented, based upon temperature, pH, and inhibitor sensitivity of the wool-covered glass rod and then filtered through glass wool to process, that loss of enzyme may be the result of proteolysis. The lght- produce a homogeneous suspension. induced process can, in vitro, be largely prevented by addition of the Oat and rye etioplast membranes were prepared by lysing substrates of the reductase, protochlorophyflide and NADPH. It is con- whole, intact etioplasts in hypotonic buffer (20 mM sucrose, 20 mM cluded that Ught causes the breakdown of the reductase in vivo and in vitro Tes, 20 mM Hepes, 2 mM MgCl2, 5 mm cysteine, pH 7.5) followed by producing ligand-free enzyme as a consequence of the photoconversion by centrifugation (21). In some experiments (see text), a further reaction. purification step was carried out by centrifugation on a 20%1o to 40o (w/w) discontinuous sucrose gradient as before (21). Samples were finally resuspended in a buffer containing 0.5 M sucrose, 20 mM Tes, 20 mM Hepes, 2 mM MgCl2, 5 mm cysteine, pH 7.5. All steps were performed under a dim green safelight at 4°C. Enzyme Assay. Pchlide reductase (EC 1.6.99.1) was assayed as described by Griffiths (10). A unit of enzyme activity was taken It has previously been demonstrated (17, 18) that illumination as that causing the conversion of 1 nmol Pchlide to Chlide/min of etiolated plants leads to a dramatic decrease in the activity of under standard assay conditions (10). the plastid-localized enzyme Pchlide reductase. It has also been In Vitro Incubation Conditions. Samples of etioplasts and etio- observed that such in vivo illumination simultaneously caused plast membranes (typically, 200 ul ofmaterial containing approx- oxidation of the plastid NADPH and a decrease in Pchlide level imately 1 mg of protein) were illuminated on ice for 2 min by a as a result ofphotoconversion. Redarkening ofbriefly illuminated 50-w focussed quartz halogen lamp at a distance of5 cm, followed plants produced an exact reversal ofthe situation with re-reduction by 3 min red light (Kodak Wratten filter No. 25) of an intensity of the plastid NADP and resynthesis of Pchlide accompanying of 15 w m-2. Incubation on ice ensured that no significant rise in recovery ofPchlide reductase activity (17-19). After identification temperature occurred during the illumination. The illumination ofthe enzyme protein (1, 20), it became apparent that the observed conditions were chosen as those producing maximal photoconver- changes in enzyme activity results from a degradation and resyn- sion of Pchlide in etioplasts with the minimum of photooxidative thesis of the actual enzyme protein under conditions of illumina- damage. The plastids or plastid membranes were then incubated tion and redarkening. However, the mechanism whereby light at room temperature in darkness and aliquots (approximately 15 produced this change is unexplained. Again, the significance, if id) removed at various time intervals for either enzyme assay or any, of the accompanying changes in Pchlide and NADPH, in analysis by SDS-PAGE2. In experiments where compounds were regulation ofthe level ofPchlide reductase, also remained obscure. added to membrane preparations, additions were made 5 mi In the present paper, the light-induced loss ofPchlide reductase prior to illumination at a final concentration of 2 mm, except for has been mimicked by illumination of cell-free preparations in Pchlide and oxidized GSH which were added at 80 and 50 ,UM, vitro. Further, and more significantly, the process is shown to be respectively. In the proteolytic inhibitor studies, inhibitors were largely inhibited by addition of Pchlide and NADPH prior to 10 to at illumination. From the data, a simple model to account for the added to membrane samples min prior illumination, light-induced loss of reductase activity is proposed. An account of concentrations of 0 to 100 Ag ml-'. this work has already been presented briefly at the Nato Advanced For the determination ofthe pH profile ofreductase breakdown, on "Molecular models of samples ofetioplast membranes were suspended to a concentration Study Institution photoresponsiveness", of 5 mg protein mln' in a buffer consisting of 10 mm phthalate, 10 Pisa, Italy, September 1982. mm Mes, 10 mm Hepes, and 10 mm Tricine adjusted to the various pH values. The samples at different pH values were then illumi- MATERIALS AND METHODS nated with 2 min white light, followed by 3 min red light, to effect Plant Material. Oats (Avena sativa L. var Peniarth) were grown complete photoconversion. After incubation at room temperature for 6 or 7 d in complete darkness as previously described (9). Rye for a further 30 min in darkness, aliquots were taken for enzyme (Secale cereale L. var Rheidol) was germinated and grown for 7 assay. 'Supported by grant GR/B/71992 from the Science and Engineering 2Abbreviations: PAGE, polyacrylamide gel electrophoresis; G-6-P, glu- Research Council. cose-6-phosphate; G-6-PDH, glucose-6-phosphate dehydrogenase. 229 230 KAY AND GRIFFITHS Plant Physiol. Vol. 72, 1983 Controls appropriate to each particular experiment were carried out under the conditions specified in the text. Electrophoretic Analysis of Peptides. Aliquots ofmembranes or plastids were solubilized in a buffer containing 1% (w/v) SDS, 10% (v/v) glycerol, 62.5 mM Tris-HCl, pH 6.8. Approximately 70 ,ug of protein was loaded on each track and electrophoresed on 12% or 15% polyacrylamide gels according to Laemmli (16). Mol wt marker proteins were always run and consisted of BSA (66 kD), ovalbumin (43 kD), carbonic anhydrase (29 kD), and lyso- zyme (12 kD). Gels were fixed and then stained with Coomassie Brilliant Blue R. Protein Assay. Protein was assayed by the method of Bramhall et al. (5) using BSA as standard. Spectral Measurements. Absorbance spectra of samples sus- pended in buffer were recorded using a split-beam spectrophotom- eter as previously described (10). Chemicals. Pchlide was purified from acetone extracts of etio- lated oats as described previously (10). NADPH was produced from a regenerating system containing G-6-P (4 mM), NADP (0.5 mM), and G-6-PDH (0.1 units). The protease inhibitors were obtained from Sigma Chemical Co. Ltd. (London, U.K.). All other chemicals were A.R. grade where possible from sources already described (10). FIG. 2. Light-induced decrease in Pchlide reductase activity in etioplast RESULTS membranes. Etioplast membranes prepared by osmotic lysis of oat etio- Etioplasts prepared from 7-d-old dark-grown oats contain a plasts were incubated at a protein concentration of 7.0 mg ml-' with (@) very active Pchlide reductase with, typically, a specific activity of and without (0) prior incubation as described in Figure 1. Aliquots were about 3.2 units/mg protein. The enzyme in such preparations removed at intervals and assayed for residual reductase activity. Activities remains stable for prolonged periods if stored at -20°C in dark- are expressed as percentages of the activity of the original membranes. ness. At room temperature (22°C) in darkness, the enzyme is again relatively stable with activity readily detectable, albeit at a is present. In marked contrast, however, if the etioplasts are reduced level, even after 3 h under such conditions (Fig. 1), and illuminated prior to incubation at room temperature, then a after only 50 min as much as 90o of the original enzymic activity dramatic and rapid decrease in the activity of the enzyme occurs with approximately 40% lost within 15 min of illumination (Fig. 1), and after 2 h, only about 25% of the activity remains. This observation is very reminiscent of the behavior of the enzyme in whole leaves under similar conditions (17-19). A similar behavior was observed on illumination of isolated ~~~~~~~~~~~~ . ....... etioplast membranes. Before illumination, such membranes had a reductase specific activity of 4.13 units/mg protein. Over a 3-h incubation in darkness, this declined to about 65% of its original level. In contrast, however, illumination of the membranes prior to such an incubation in darkness led to a 50% drop in reductase activity within 20 min, and after 3 h, less than 20% of the original activity was still detectable (Fig. 2). Figure 3 shows the peptide profiles ofetioplasts (tracks I and 2) and membranes (tracks 3 and 4) sampled before (tracks 1 and 3) and 3 h after (tracks 2 and 4) illumination, respectively. The specific activity of Pchlide reductase in each sample is also in- cluded. In the etioplasts prior to illumination (track 1), the pres- ence of the reductase is quite discernable (arrowed bands of mol wt 36 and 38 kD) despite the large number of other etioplast proteins in such preparations. After illumination followed by 3 h *'.:7.MW'...Si9...........':!gincubation in darkness, however, the intensity of the reductase bands have greatly diminished (Fig.
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