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Lawrence Berkeley National Laboratory Recent Work Lawrence Berkeley National Laboratory Recent Work Title CARBON METABOLISM OF CHLOROPLASTS IN THE DARK: OXIDATIVE PENTOSE PHOSPHATE CYCLE VERSUS GLY-COLYTIC PATHWAY Permalink https://escholarship.org/uc/item/14j5m5n9 Author Kaiser, W. M. Publication Date 1978-07-01 eScholarship.org Powered by the California Digital Library University of California Submitted to PLANTA LBL-8104 Preprint e. .-1- CARBON METABOLISM OF CHLOROPLASTS IN THE DARK: OXIDATIVE PENTOSE PHOSPHATE CYCLE VERSUS GLYCOLYTIC PATHWAY r-.ECEIVED ~AWRENCE W. M. Kaiser and J. A. Bassham BERKIil::Y LABORATORY AUG 30 1978 LIBRARY AND July 1978 ,.... r.CUMENTS SECTJON Prepared for the U. S. Department of Energy under Contract W-7405-ENG-48 TWO-WEEK LOAN COpy This is a Library Circulating Copy _ which may be borr"wed for two weeks. For a personal retention copy, call Tech. Info. Diuision, Ext. 6782 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor the Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the . United States Government or any agency thereof, or the Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or the Regents of the University of California. CARBON METABOLISM OF CHLOROPLASTS IN THE DARK: OXIDATIVE PENTOSE PHOSPHATE. CYCLE VERSUS GLYCOLYTIC'PATHWAY ~. \ . w. M. Kaiser* and.J. A. Bassham Laboratory of C~em1cal Biodynamics Lawrence Berkeley Laboratory University of California Berkeley, CA 94720 *Present Address: Institut fuer Botanik und Pharmazeutische Biologie mit Botanischem Garten der Universitaet, Lehrstuhl Botanik I, D-87 Wuerzburg, Mittlerer Dallenbergweg 64, F.R.G. Mailing Address: W. M. Kaiser, Institut fuer Botanik und Pharmazeutische Bio)ogie mit Botanischem Garten der Universitaet, Lehrstuhl .Botanik I, 0-87 Wuerzburg, Mittlerer Oallenbergweg 64 1 ABSTRACT The conversion of U-labelled l4C-glucose-6-phoiphate into other products by a soluble fraction of lysed chloroplasts has been studied. It was found· that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this ~raction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequ~nce including a phospho-' fructokinase step. The conversion of glucose-6-phosphate via the oxidativ~ pentose phosphate cycle stopped with the formation of pentose monophosphat~s. This was found not to be due to a lack in transaldolase (or transketolase) activity, but rather to the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied. Key words: Carbon metabolism - chloropl~sts - darkness. \ 2 . ABBREVIATIONS ~DHAP = dihydroxyac~tone phosphate; GAP = 3-phosphog1ycera1dehyde; PGA = 3-phosphog1ycerate;. HMP = hexose monophosphate~ including F6P = fructose-6-phosphate G6P =, gl ucose-6-phosphate GIP = gl ucose-1- phosphate; 6-PGL = 6-phosphog1uconat~; PMP = pento~e monophosphates, including R5P = ribose-5-phosphate, Ru5P = ribu1ose-5-phosphate, X5P = xy1u1ose-5-phosphate; E4P = erythrose-4-phosphate; S7P = sedoheptu1ose-7-phosphate; FBP = fructose-1 ,6-bispho~phate; SBP = sedoheptulose-l,,7-bisphosphate; RuBP = ribulose-1,5-bisphosphate Footnote (po 11): . l)If Mg++ was completely che1ated with EDTA, formation of bisphosphates and DHAP was strongly inhibited (data not shown). 3 lNTRODUCTI ON Chloroplastic starch can be degraded by a hydrolytic pathway into maltose and glucose or, via phosphorylase, into G1P (Heldt et al., 1977; Peavey et al., 1977;.Steupp et al., 1976): Since fixed carbon can pas~ through the ch1orop1astenve16pe ori1y in the form of triose"phosphate or . PGA (for a review see Heldt, 1976), the primary products of starch degrada­ tion have to be converted into these compounds in order to be exported into the cytoplasm. Theoretically, the conversion of G1P into triose phosphates could occur either vja an oxidative pentose phosphate cycle or via a glyco- lytic pathway mediated by phosphofructokinase. The occurance of some reactions of oxidative pentose phosphate cycle in chloroplasts is well (:!stab1ished (Krause and Bassham, 1969; Lendzian and Ziegler, 1970; Lendzian 'and Bassham, 1975). The recent purification and characterization of a ch1orop1astic phosphofructokinase (E.C. 2.7.1.11) gives strong evidence for a glycolytic pathway in chloroplasts (Kelly and Latzko, 1977, a,b). It is still an open question to what extent each process really contributes to a'conversion of G1P into triose phosphate an~ other compounds in the dark metabolism of chloroplasts. Some recent data on starch degradation in chloroplasts have been obtained with intact spinach chloroplasts which. were preloaded with 14C-1abe11ed starch (Heldt et al., 1977; Peavey et a1., 1977; Steupp et a1., 1976). There are some disadvantages in this· method: the specif1c activity of labelled starch is not exactly known; due to the necessary preincubation and washing procedures, the chloroplasts have already been isolated for a long time when the actual measurement of dark metabolism begins; starch degradation has to be measured at Pi-levels which are far above any Pi-level optimal for 4 photosynthesis; finally, and most important for regulatory studies, significant features of the stroma composition, such as pH, ionic strength and metabolite levels can be manipulated only to a very limited extent due to the imperme- , ' . ability of the chloroplast envelope. For the studies reported here we therefore used the soluble fraction of lysed chloroplasts, -in which the catabolism of U-1abe11ed 14C_G6P was followed under conditions allowing a separation of the reactions of oxidative pentose phosphate cycle and of a glycolytic pathway. MATERIALS AND METHODS Chemicals' and Enzymes. U-1~bel1ed 14C_G6P was prepared from 14C-g1ucose with lyophilized hexokinase' and stoichiometric amounts of ATP, lYOphilized transa1do1ase (E.C. 2.2.1.1) and transketolase (E.C. 2.2.1.1.) were obtained from Sigma, Stock # 6008 and T-6133, resp. Plant Material. Spinach was grown in vermiculite fertilized with Hoag1ands solution, under artificial light (3000 ft-c) with an 8 h light period- and a 16 h dark period at a temperature of 13°C. Chloroplasts were isolated from young leaves according to Jensen and Bassham (1966). Preparation of stroma enzymes. A suspension of freshly isolated intact chloroplasts containing about 2 mg chlorophyll was centrifuged for 1 min at 1000 g (4°C), -and the pellet was resuspended in 1.2 m1 of a lysing solution containing 0.025 M HEPES-NaOH pH 7.6, 2mt~ MgC1 and 1 mM EOTA, if not mentioned 2 otherwise. After 10 min at O°C, the suspension was centrifuged for 15 min at 27,000 g (4°C). The resulting clear supernatant contained the soluble components from the chloroplasts and had an average protein content of about 5 mg protein in 1.2 m1. This solution was usually made CO2-free by intermittently flushing with N2 on a Vortex mixer for about 3 min (O°C). The pellet was 3 l.NTRODUCTI ON Chloroplastic starch can be degraded by a hydrolytic pathway into maltose and glucose or, via phosphorylase, into G1P (Heldt et al., 1977; Peavey et al.~ 1977;.Steupp et al., 1976). Since fixed carbon can pass . , through the chloroplast. envelope only in the form of triose phosphate or PGA (for a review see Heldt, 1976), the primary products of starch degrada- tion have to be converted into these compounds in order to be exported into the cytoplasm. Theoretically, the conversion of G1P into triose phosphates could occur either via an oxidative pentose phosphate cycle or via a glyco- lytic pathway mediated by phosphofructokinase. The· occurance of some reactions of oxidative pentose. phosphate cycle in chloroplasts is well established (Krause and Bassham, 1969; Lendzian and Ziegler, 1970; Lendzian and Bassham, 1975). The recent purification and characterization of a chloroplastic phosphofructokinase (E.C. 2.7.1.11) gives strong evidence for a glycolytic pathway in chloroplasts (Kelly and Latzko, 1977, a,b). It is still an open question to what extent each process . really contributes to a'conversion of G1P into triose pnosphate and other compounds in the dark metabolism of chloroplasts. - Some recent data on starch degradation in chloroplasts have been obtained with intact spinach chloroplasts which were preloaded with 14C-labelled starch (Heldt et al q 1977; Peavey et al., 1977; Steupp et al., 1976). There are some disadvantages in this method: the specifi~ activity of labelled starch is not exactly known; due to·the necessary preincubation and washing procedures, the chloroplasts have already been isolated for a long time when the actual meas~rement of dark metabolism begins; starch degradation has to be measured at Pi-levels which are far above any Pi-level optimal for 4 photosynthesis; finally, and most important for regulatory studies, significant features of the stroma composition, such as pH, ionic strength and metabolite levels can be manipulated only to a very limited extent due to the imperme­ ability of the chloroplast envelope. For the studies reported here we therefore used the soluble fraction of lysed chloroplasts,fn which the catabolism of U-l~belled 14C_G6P was followed under conditions allowing a separation of the reactions of oxidative pentose phosphate cycle and of a glycolytic pathway.
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