When Ribulose-1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCI2 followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose-1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose-1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose-1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose-1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose-1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose-1,5-bisphosphate carboxylase peak from phosphonbulokinase peak. 'Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase; Rib-5-P, ribose-5-phosphate; Ru-5-P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; fr wt, fresh weight. 368 ters such that metabolic channeling would be facilitated (25). Given the high concentration of Rubisco it is highly unlikely that Rubisco molecules would be devoid of extensive proteinprotein interactions or would be capable of free diffusion in the stromal environment. We have recently reported (23) that Rubisco, when purified from stromal extracts of pea chloroplasts on sucrose gradients, showed Rib-5-P and Ru-5-P dependent CO2 fixation activities. The expression of these activities required in addition to Rubisco either phosphoribulokinase (kinase) (Ru-5-P dependent CO2 fixation) or kinase and phosphoriboisomerase (isomerase) (Rib-5-P dependent CO2 fixation). This Rubisco preparation showed equivalent rates of CO2 fixation using RuBP or Ru-5-P when treated with 10 mM DTT. Also, the Ru-5-P dependent activity was linear for longer time periods than the RuBP dependent activity (25). These studies have suggested some binding specificity between kinase and carboxylase and some kinetic benefit to the carboxylase reaction. The work reported here is an extension of these studies using both spinach and peas. It is an effort to further characterize the association and the kinetics of this multiple reaction sequence. MATERIALS AND METHODSChloroplasts were isolated from 6 to 8 week old spinach leaves (Spinacia oleracea) and 10 to 20 d old pea shoots (Pissum sativum) according to Cerovic et al. (4). Chloroplasts were lysed in 25 mm Bicine buffer (pH 8). The membranes were removed by centrifugation (25,000g for 15 min) and supernatants were processed on sucrose gradients (5-30%) made either in 25 mM Bicine (pH 8) containing 20 mM each of MgCl2 and bicarbonate...
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