The function of the small subunits of ribulose bisphosphate carboxylase-oxygenase

When ribulose bisphosphate carboxylase-oxygenase from Synechococcus (strain RRIMP N1) was precipitated under mildly acidic conditions, most of its small subunits remained in solution. The precipitated enzyme readily redissolved at neutral pH and remained as an octamer of large subunits with some small subunits still attached. Optimum pH for this separation was 5.3 and disulfide-reducing reagents were not necessary. The fraction of small subunits removed by a single precipitation increased with increasing NaCl concentration. Catalytic activity of small subunit-depleted enzyme was linearly proportional to the fraction of small subunits remaining, while the carboxylase:oxygenase activity ratio and the affinity for CO2 remained constant. When isolated small subunits were added back to depleted enzyme preparations at neutral pH, reassociation occurred with return of catalytic activity. Under the usual assay conditions at pH 7.7, the binding constant of the small subunits was estimated to be about 10(-9) M. The small subunits also bound avidly to surfaces. However, loss of small subunits from the enzyme during the course of purification was minimal. The results are consistent with a simple model in which only those large subunits which have a small subunit bound to them are catalytically competent. Thus, an essential, even if indirect, role for the small subunits in catalysis is indicated.     

Structural and regulatory studies on the ribulose bisphosphate carboxylase-oxygenase of Anabaena cylindrica

Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase fromthe cyanobacterium Anabaena cylindrica has been purified tohomogeneity by the criterion of polyacrylamide gel electrophoresisand shown to consist of two types of subunits of molecular weights51K (large) and 12K (small). The enzyme is of the higher planttype and probably consists of 8 large plus small subunits. Isoelectricfocusing of the S-carboxymethylated protein in 8 M urea revealeda profile of consisting of 3 major polypeptides plus 1 minorpolypeptide. Some characteristics of the carboxylase and oxygenasereactions were studied using simultaneous measurements of bothactivities. Pyridoxal 5'-phosphate inhibited both activitiesequally. Neither the carboxylase nor oxygenase reaction wasaffected by glutamate (5 mM), although 6-phosphogluconate andfructose 1,6-bisphosphate inhibited both reactions. RuBP oxygenasewas more sensitive to 6-phosphogluconate (0.5 and 1.0 mM) thanRuBP carboxylase. Marked changes in the oxygenase to carboxylaseactivity ratio of the purified enzyme were effected by homologousantiserum (which preferentially inhibited carboxylation). ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received May 22, 1980; )     

Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase)

This historical minireview deals with events leading to the eventual discovery of Rubisco (ribulose bisphosphate carboxylase-oxygenase). This abundant leaf protein is not only responsible for the net fixation of CO₂ in all plants, but also causes the loss of carbon through photorespiration. The latter is a special ‘problem’ of the so-called ‘C₃’ plants. The protein was first called ‘Fraction 1 protein’ before it was recognized to be the same as Rubisco. Instead of reinventing words, text as needed has been freely used from three earlier publications (Wildman and Kwanyuen 1978; Wildman 1992, 1998) This revised version was published online in June 2006 with corrections to the Cover Date.     

Conditions leading to precipitation of ribulose bisphosphate carboxylase-oxygenase differ from those leading to enzyme activation

The relation between conditions leading to precipitation and/or activation of Ru-P₂ carboxylase have been explored in order to test the hypothesis that conformational changes leading to precipitation might be identical to those which are presumed to lead to enzyme activation. From the results of kinetic and solubility studies, we conclude that this hypothesis is not valid, since changes in solubility of Ru-P₂ carboxylase occur ten times as fast as changes in enzyme kinetics.Abbreviations Ru-P₂ ribulose 1,5-bisphosphate - PVP polyvinylpyrrolidone - DTE dithioerythritol - Bicine N,N-bishydroxy-2-ethylglycine     

Conditions leading to precipitation of ribulose bisphosphate carboxylase-oxygenase differ from those leading to enzyme activation

The relation between conditions leading to precipitation and/or activation of Ru-P₂ carboxylase have been explored in order to test the hypothesis that conformational changes leading to precipitation might be identical to those which are presumed to lead to enzyme activation. From the results of kinetic and solubility studies, we conclude that this hypothesis is not valid, since changes in solubility of Ru-P₂ carboxylase occur ten times as fast as changes in enzyme kinetics.     

Proteinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris

Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.     

Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.     

Heterogeneity of alkaline small subunits of ribulose 1,5 bisphosphate carboxylase-oxygenase from Medicago sativa and M. falcata

The isolated leaf proteins of lucerne (Medicago sativa L. and M. falcata L.) were fractionated by Sepharose 6B column chromatography. Analysis of fractionated proteins indicated that the 2nd peak component was almost entirely ribulose 1,5 bisphosphate carboxylase-oxygenase (Rubisco) which represented 57% of the total recovered protein.Rubisco yielded one large subunit (LSU) and one small subunit (SSU) polypeptide after SDS gel electrophoresis.Isoelectric focusing of the SSU of Rubisco from genotypes of M. sativa cv. Hunter River (HR), Hairy Peruvian (HP) and of M. falcata (MF) showed two SSU components for HR and HP, and three components for MF. Most components of genotypes were located in the alkaline region of the gel. While the pIs of the SSU components of HR and HP were identical they differed from those of the SSU of MF thus demonstrating heterogeneity for SSU in Medicago.It is suggested that the alkaline nature of SSU may have some adaptive physiological significance.Abbreviations Rubisco ribulose bisphosphate 1,5-carboxylase-oxygenase - LSU large subunit - SSU small subunit - HR Hunter River - HP Hairy Peruvian - MF Medicago falcata - SDS Sodium dodecyl sulphate - TCA trichloracetic acid     

Seasonal variation in ribulose bisphosphate carboxylase activity in Pinus silvestris

Ribulose bisphosphate carboxylase activity was examined in Pinus silvestris L. during successive seasons. The enzyme activities were studied both in seedlings, kept under controlled conditions in a climate chamber, and in needles from a 15-year-old tree in a natural stand. The enzyme activities were analysed in cell-free extracts prepared with Tween 80 as protective agent. The carboxylase activity fluctuated periodically both in the seedlings and in the natural stand. In the seedlings, the weight-related activity in the older needles increased 50–100% (in the cotyledons c. 200%) in the beginning of the “summer”. It decreased as the new shoot developed. The specific activity increased c. 100%. With chlorophyll as base, the activity usually decreased during “summer”. In the developing current needles the carboxylase activity increased when expressed on a weight or on a protein basis. The decrease in weight-related carboxylase activity in the older needles was preceded by, or simultaneous with, loss of total protein. It is suggested that protein, including the carboxylase, is utilized as nitrogen reserve for the new shoot. During hardening by combined photoperiod and thermoperiod, the carboxylase activity decreased when expressed relative to dry weight and protein. Calculated on a chlorophyll basis, the activity was rather constant. In the natural stand the activity in the one- and two-year-old needles increased during spring and summer and decreased during autumn and winter. Even at severe winter stress substantial carboxylase activity remained in the needles. The activity of the enzyme in vivo is discussed with respect to electron transport and net photosynthesis.     

Independent regulation of synthesis of form I and form II ribulose bisphosphate carboxylase-oxygenase in Rhodopseudomonas sphaeroides.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) activity was greatly enhanced when Rhodopseudomonas sphaeroides was grown in a mineral salts medium supplied with 1.5% CO2 in hydrogen. Analysis of cell extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that cells growing on 1.5% CO2 in H2 specifically accumulated RuBPC-O polypeptides. Quantitative immunological determinations revealed that accumulation of form I and form II RuBPC-O closely correlates with the increase of specific activity. However, the two enzymes appeared to be derepressed at different levels. Upon transfer from heterotrophic to autotrophic (1.5% CO2) growth conditions, the intracellular form I RuBPC-O concentration was augmented 17-fold, whereas the form II RuBPC-O content increased only fourfold. As a result, the form I-form II ratio changed from 0.5 to about 2.0. Since this change in the RuBPC-O ratio occurred in the early stage of growth, it suggests that form I RuBPC-O is required for growth under drastic CO2 limitation. The difference in the extent of derepression of form I and form II RuBPC-O also indicates that the synthesis of each enzyme is regulated somewhat independently of the other.     

Purification and properties of glutamate dehydrogenase in Scots pine (Pinus sylvestris) needles

NADH-dependent glutamate dehydrogenase (GDH. EC 1. 4. 1.2) was isolated from the needles of Scots pine (Pinus sylverstris L.) grown on a rural and on a heavily polluted industrial area, and it was purified about 500 fold. The purification procedure included salt I'ractionation, ion exchange and affinity chromatography. Miehaelis constants for 2-oxoglularale (1.7 mM). for ammonium sultate (19 mM ) and for NADH (42.5 resp. 53 μM) the pH optimum (8.5) the requirements for Ca²⁺ ions, the temperature dependence ofl the enzyme activity (incubation from 0 to 82°C). and the relation between forest region and electrophoretie isoenzyme pattern were determined. The possible role of GDH in the adaptation of plants to ammonia assimilation (detoxification) under stress conditions, particularly with respect to air pollution, is discussed.     

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.     

Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles

Four-year-old seedlings of Scots pine ( Pinus sylvestris L.) were exposed to filtered air (FA), and to FA supplemented with NH₃ (60 and 240 μg m⁻³) in controlled-environment chambers for 14 weeks. Exposure to the higher NH₃ concentration resulted in an increased activity of glutamine synthetase (GS, EC 6.3.1.2), and an increase in the concentrations of soluble proteins, total nitrogen, free amino acids and leaf pigments in the needles. The GS activity (μmol g⁻¹ fresh weight h⁻¹) in the needle extract increased to levels 69% higher than in FA and the soluble protein concentration to levels 22% higher. Total nitrogen concentration in the needles was 42% higher than in FA, while the free amino acid concentration was 300% higher, which was caused by an increase in arginine, glutamate, aspartate and glutamine. Chlorophyll a , chlorophyll b and carotenoid concentrations were 29, 38 and 11% higher, respectively. Neither the glutamate dehydrogenase (GDH, EC 1.4.1.2) activity nor the concentrations of free NH₄⁺ and glucose in the needles were affected by exposure to NH₃. After NH₃ fumigation at 240 μg m⁻³ the starch concentration decreased by 39% relative to the FA. The results indicate that the metabolism of Scots pine acclimates to concentrations of NH₃ which are 3 to 10 times higher than the average concentration in areas with intensive stock farming. The possible mechanisms underlying acclimation to NH₃ are discussed.     

Pinus halepensis,Pinus pinaster,Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers,before and after Inoculation with the Pinewood Nematode Bursaphelenchus xylophilus

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time‐course study of the essential oils (EOs) of 2‐year‐old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.     

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4°C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl₂, 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58°C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25°C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.