Spinach Ribulose Diphosphate Carboxylase. I. Purification and Properties of the Enzyme *

A bstract. The D-glyceraldehyde 3-P dehydrogenases of spinaoh leaf, pea seed, and pea shoot were purified. The NADP 'and NAD-linked enzymes of either spinach leaves and pea shoots could not be separated. Changes in the ratio of NADP-to NAD-linked activity oif the spinach leaf and pea shoot enzymes were observed during both purification and stonage of crude extracts. The spinach leaf, pea shoot, and pea seed enzymes differ electrophoretically from each other and from the rabbit muscle enzyme.The The occurrence and distribution of the NAD and NADP-linked glyceraldehyde 3-P dehydrogenases inl higher planits has been investigated in many laboratories. In higher plants the NADP-linked activity was found to occur only in the photosynthetic tissue (10,15). On the other hand, the NAD-linked activity was present in non-chlorophyllous as wvell as chlorophyll containing tissues (12,15

“…E., Eversoni R. G. and \W. Cockburn, unl)ubli shed ol)seriations and n)-glvceric acid 3t-P has b)een sliown to iihibit ribulose I-diP carbl)oxvlase ( 30 …”

Section: Discussionmentioning

confidence: 99%

d-Glyceraldehyde 3-Phosphate Dehydrogenases of Higher Plants

Schulman¹,

Gibbs²

1968

“…Spinach leaf RuBP carboxylase is a large protein having a mol wt of 557,000 and is composed of eight large subunits, mol wt 55,800, and eight small subunits, mol wt 12,000 (21,22). RuBP carboxylases have been isolated from procaryotic systems which vary in mol wt, structural composition, and regulatory properties.…”

mentioning

confidence: 99%

Carbon Isotope Fractionation by Ribulose-1,5-Bisophosphate Carboxylase from Various Organisms

Estep¹,

Tabita²,

Parker³

et al. 1978

Carbon Isotope fractionaton by structurally and catalytically distinct rbulose-1,5-blspbosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under itrogen. The Spinach leaf RuBP carboxylase is a large protein having a mol wt of 557,000 and is composed of eight large subunits, mol wt 55,800, and eight small subunits, mol wt 12,000 (21, 22). RuBP carboxylases have been isolated from procaryotic systems which vary in mol wt, structural composition, and regulatory properties. For example, when isolated and purified from the purple, nonsulfur, bacterium Rhodospirillum rubrum, the carboxylase has a mol wt of 114,000, is composed of only two large subunits, and is not regulated by the effector 6-P-gluconate (12, 27). Furthermore, two structurally and catalytically distinct RuBP carboxylases have been isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides (9). Peak I carboxylase is structurally and catalytically similar to the enzyme from higher plants. Peak II carboxylase has a mol wt of 360,000 and is composed of only six large subunits. It is also apparent that a diversity of structure may exist in the blue-green algae (28,29), similar to the bacteria (9, 27); i.e. Hydrogenomonas eutropha, a chemosynthetic bacterium, has a high mol wt carboxylase similar in structure to that of the spinach enzyme (12).In this paper we have examined carbon isotope fractionation by RuBP carboxylases from spinach, R rubrum, R sphaeroides, Agmenellum quadruplicatum, and H. eutropha, for several reasons. First, we have produced a reproducible fractionation value, A dissolved C02, by carefully controlling reaction conditions and purification procedures of both the enzyme and the product phosphoglyceric acid. Second, certain reaction conditions were purposely modified to test their effects on the fractionation: NaHCO concentration, 02 level, temperature, buffer, substrate, and metal cofactors. In addition, several purification states ofeach enzyme were used in order to determine the effect on the A dissolved CO2. Moreover, in an attempt to see if enzymes of different mol wt, purity, quaternary structure, and catalytic prop-

“…More recently Paulsen and Lane (13) described a preparation of RuDP' carboxylase that was homogeneous by ultracentrifugation criteria. The majority of studies of the enzyme, including those on control of photosynthetic CO. fixation, have used similar preparations (1-3, 11-15, 17, 20).…”

mentioning

confidence: 99%

Ribulose Diphosphate Carboxylase from Freshly Ruptured Spinach Chloroplasts Having an in Vivo Km[CO₂]

Bahr

Jensen²

1974

110

The properties of a form of ribulose diphosphate carboxylase having a high affinity for CO2 have been studied. Its apparent Km(HCOsa) of 0.5 to 0.8 mM (pH 7.8) METHODSChloroplasts were prepared by the method of Jensen and Bassham as modified by Jensen (8) from 5-to 6-week old spinach leaves (Spinacia oleracea L., var. Viroflay) grown in an environmental chamber. The chloroplasts were about 70% class I by phase contrast microscopy (16) and capable of lightdriven fixation at rates up to 100 jumoles (mg Chl hr)-1.Light-driven fixation was measured by adding chloroplasts to isotonic medium containing 0.33 M sorbitol, 50 mm HEPES, pH 7.8 with NaOH, 2 mm EDTA, 1 mm MnCl2, 1 mm MgCl2, 0.5 mM K,HPO,, 2 mm sodium isoascorbate, 5 mm sodium pyrophosphate, and NaH'4CO, (5.5 juc/pmole) as indicated.Light intensity was 2000 ft-c, temperature, 25 C.RuDP carboxylase activity was measured in the dark upon lysis of intact chloroplasts in hypotonic medium containing HEPES buffer, pH 7.8 with NaOH, 5 mM DTE, 25 mM MgCl2, RuDP or RSP plus ATP, and NaH"CO, (5.5 ,uc/Mmole) at 25 C. The concentrations of the components varied with the experiment and are indicated in the appropriate figure legends. Within the range used, there was no effect of chloroplast concentration on the Km or stability properties of the carboxylase. DTE, required for maximal activity of phosphoribulokinase, had no effect on the activity of RuDP carboxylase. It was included routinely in these experiments to permit direct comparison between RuDP and RSP plus ATP as substrates. In both light and dark fixation experiments the media were bubbled with CO2-free N2 in 2-ml serum-capped plastic beakers prior to addition of NaH"4CO3. Aliquots (25-100 td1)were removed at specified intervals, added to two volumes of formic acid, and dried on a planchet; and the acid-stable "4C was determined by a planchet counter. Chlorophyll was assayed by the method of Vernon (19).RuDP (Sigma Chemical Co.) was assayed by allowing complete conversion to 14C-glycerate-3-P with excess NaH"CO3 and spinach chloroplasts in hypotonic buffer. Identical results were obtained using purified RuDP carboxylase in place of spinach chloroplasts. Cooper et al. (3) have shown that the true substrate for RuDP carboxylase is C02, not HCO-. In this paper we use "HCO3-" to refer to the total carbonate species added to the reaction vessel, of which HCO-is the predominant form at pH 7.8. The concentration of CO2 has been calculated on the assumption of equilibrium between CO2 and HCO,-, and using the known liquid and gas volumes of the reaction vessels and the constants given by Umbreit et al. (18).