The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

Abstract

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...

Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity

“…Co-purification of PRK with other enzymes of the reductive pentose phosphate cycle has been suggested to be due to the formation of multienzyme complexes [22,23]. Of these only that of Gontero et al [23] contains both G3PDH and PRK.…”

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

“…This conclusion is relevant in view of recent proposals that NAD(P)-GAPDH binds to a multimeric complex of 500 kDa encompassing ribulose-l,5-bisphosphate carboxylase/oxygenase, phosphoribulose kinase and other proteins (Gontero et al 1988;Sainis et al 1989). This putative complex is difficult to reconcile with the present observations, since the NAD(P)-GAPDH in its common regulatory form is about as large as the proposed complex, and it is much smaller in light-activated state.…”

“…It is possible that the protein is free in the stromal gel (Pupillo and Giuliani-Piccari 1973). Alternatively, NAD(P)-GAPDH has been postulated to be part of a multimeric complex with other proteins, phosphoribulose kinase in particular (Wara-Aswapati et al 1980;Gontero et al 1989;Sainis et al 1989;Clasper et al 1991), or to be bound to the chloroplast envelope (Anderson and Ben Bassat 1981;Brinkmann et al 1989). Also, the significance of the light effect is little understood.…”

The aggregation states of spinach phosphoribulokinase

“…Phosphoribulokinase extracted in the presence of low levels (or absence) of thiols has been resolved into two forms of essentially inactive PRK, which can be activated with DTT or DTT plus thioredoxin. The low-Mr form appears identical to the pure, active form of the enzyme, while the high-Mr form behaves like a molecule of more than 550 kDa (Bradbeer et al 1981;Gontero et al 1988;Sainis et al 1989;Wara-Aswapati et al 1980;Wolosiuk and Buchanan 1978).…”

“…While this explanation assumes that aggregation is an artifact of tissue processing, other models assume that the high-Mr complex represents a normal physiological state of PRK. Apparent co-purification of PRK with a number of Calvin-cycle enzymes, including phosphoriboisomerase and ribulosebisphosphate carboxylase/oxygenase (Rubisco), led to the suggestion that these enzymes form an active muttienzyme complex (Gontero et al 1988;Sainis and Harris 1986;Sainis et al 1989). However, thiol treatment of PRK is required in vitro to mimic the enzyme activation observed in chloroplasts (Laing et al 1981;Wirtz et al 1982) and this treatment leads to dissociation of PRK from the putative complex (Gontero et al 1988;Wara-Aswapati et al 1980).…”