Thiol-dependent regulation of glycerate metabolism in leaf extracts : the role of glycerate kinase in c(4) plants

We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C₄ plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C₃ species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C₄ species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C₄ plants is postulated.     

Dihydroxyacetone phosphate reductase in plants

Two forms of dihydroxyacetone phosphate reductase are present in spinach, soybean, pea, and mesophyll cells of corn leaves. An improved homogenizing medium was developed to measure this activity. The enzyme was detectable only after dialysis of the 35 to 70% saturated (NH₄)₂SO₄ fraction and the two forms were separated by chromatography on either DEAE cellulose or Sephacryl S-200. About 80% of the reductase was one form in the chloroplast and the rest was a second form in the cytosol as determined by chromatography and by fractionation of subcellular organelles. The amount of activity detectable in the chloroplast fraction was 10.7 micromoles of dihydroxyacetone phosphate reductase per hour per milligram chlorophyll from spinach leaves and 4.9 from pea leaves. The chloroplast form eluted first from DEAE cellulose and, being smaller, it eluted second from Sephacryl S-200. Activity of the chloroplast form was stimulated 3- to 5-fold by the addition of 1 millimolar dithiothreitol or 50 microgram reduced Escherichia coli thioredoxin or 4 micrograms spinach thioredoxin to the assay mixture. This stimulation was not observed with monothiols. Activity of the cytosolic form was not affected by either reduced thioredoxin or dithiothreitol.     

Handbook of Biosolar Resources : Vol. 1, parts 1 and 2, ‘Basic Principles’ Edited by A. Mitsui and C.C. Black (Editor-in-Chief,O.R. Zaborsky) CRC Press; Boca Raton,USA, 1982 643 pages. $95.00 each part; approx. £113 for both parts

Glycerate kinase from spinach leaves was purified to near homogeneity using PEG/MgCl₂ fractionation, ion exchange, molecular sieving and affinity chromatography. The purified enzyme is a monomer of M_r 40 000, shows a pI-value of 4.8 and a broad pH optimum of 6.5–8.5 and is specific for D-isomer of glycerate. The high activity of crude enzyme (≈ 150 μmol. h^?1.mg chl^?1) indicates that glycerate kinase does not limit the oxidative photosynthetic carbon cycle.     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves

A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg²⁺ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl₂ and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl₂, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K_m (NADH) was 74 micromolar and K_m (NADPH) was 81 micromolar. Apparent V_max was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide.     

Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH₄)₂SO₄ was stable when frozen as small beads in liquid nitrogen and stored at −80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO₂ and Mg²⁺ activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana

Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f₁ and f₂) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C₃ and C₄ plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C₃, C₄, or CAM photosynthesis.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

Association of a thioredoxin-like protein with chloroplaast coupling factor (CF1).

The δ subunit isolated from chloroplast coupling factor (CF₁) preparations partially replaced thioredoxin in the dithiothreitol-linked activation of chloroplast fructose 1,6-bisphosphatase. The δ subunit fraction also stimulated the dithiothreitol-dependent ATPase of heated CF₁ in a manner analogous to that obseryed with each of the three thioredoxins isolated from spinach leaves (thioredoxins f, m, and c). The δ subunit used in most of these experiments was obtained from CF₁ that had been isolated by a newly devised procedure based on acid precipitation.     

Evidence for a light dependent increase of phosphoglucomutase activity in isolated, intact spinach chloroplasts

Phosphoglucomutase (PGM) activity was measured in spinach (Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glc 1,6-P₂) in the assay. Initial PGM activity increased 2- to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Initial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, dl-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3-phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glc 1,6-P₂ was maximal between pH 7.5 and 8.5. Stromal concentrations of Glc 1,6-P₂ were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc 1,6-P₂ concentration.     

Possible control of maize leaf sucrose-phosphate synthase activity by light modulation

Sucrose phosphate synthase (SPS) activity was measured in extracts of maize (Zea mays L.) and soybean (Glycine max L. [Merr.]) leaves over a single day/night cycle. There was a 2- to 3-fold postillumination increase in extractable enzyme activity in maize leaves, whereas the activity of soybean SPS was only about 30% higher in extracts prepared from light- compared to dark-adapted leaves. Alterations in extractable maize leaf SPS activity correlated with light/dark transitions suggesting that the enzyme may be light modulated. Diurnal variations of extractable maize leaf SPS activity were also observed in a greenhouse experiment. A transition from high (light) to low (dark) extractable SPS activity occurred near the light compensation point for photosynthesis (about 20 micromole photons per square meter per second). Further increases in irradiance did not increase extractable SPS activity. Substrate affinities for uridine 5′-diphosphoglucose (Michaelis constant = 3.5 and 5.1 millimolar) and fructose-6 phosphate (half maximal concentration = 1.0 and 2.5 millimolar) were lower for partially purified SPS obtained from light compared to dark acclimated maize leaves. Light-induced changes in extractable SPS activity were stable for at least one column chromatography step. The above results indicate that light-induced changes in SPS activity may be important in controlling the photosynthetic production of sucrose.     

Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize

Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C₃ plants (wheat, pea, green bean, plum, and castor bean), in some C₃ leaves (tobacco, spinach, sunflower, and wheat), and in C₄ (maize) kernels. The enzyme in the C₃ plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C₃ seeds and leaves is much less than in C₄ leaves. PPDK is present in kernels of the C₄ plant, Zea mays in amounts comparable to those in C₄ leaves.

Regulatory properties of the enzyme from C₃ tissues (wheat) are similar to those of the enzyme from C₄ leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).

Following incorporation of ¹⁴CO₂ by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development.

     

Shikimate kinase from spinach chloroplasts : purification, characterization, and regulatory function in aromatic amino Acid biosynthesis

Shikimate kinase was purified to near homogenity from spinach Spinacia oleracea L. chloroplasts and found to consist of a single 31 kilodalton polypeptide. The purified enzyme was unstable, but could be stabilized by a variety of added proteins, including oxidized and reduced thioredoxins. Whereas the isolated enzyme was stimulated by mono- and dithiol reagents, the enzyme in intact chloroplasts was unaffected by added thiols and showed only minor response to dark/light transitions. These results indicate that the previously reported stimulation of shikimate kinase activity by reduced thioredoxins is due to enzyme stabilization rather than to activation. In the current study, the purified enzyme was inhibited by added ADP and showed a strong response to energy charge. When intact chloroplasts were incubated in the dark in presence of shikimate, phosphoenolpyruvate and a source of ATP (dihydroxyacetone phosphate or ATP itself under appropriate conditions), aromatic amino acids were formed: phenylalanine and tyrosine. The data indicate that energy charge plays a role in regulating shikimate kinase, thereby controlling the shikimate pathway. An unidentified enzyme of the latter part of the pathway, leading from shikimate-3-phosphate to phenylalanine, appears to be activated by light.     

Photoactivation and Regulation of Spinach Leaf Fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase, in isolated intact chloroplast from spinach leaves, is photoactivated by ferredoxin/thioredoxin system. The mechanism involved is conversion of enzyme disulfide to sulfhydryl groups as the photoactivation is inhibited by sulfhydryl group modifying agents which are able to penetrate the chloroplast envelope. Reduction of ferredoxin on the reducing side of photosystem I is found to be a key event and active electron flow to ferredoxin must be maintained for keeping the enzyme in activated state. DCMU - a classical electron transport chain inhibitor and other exogenously added electron acceptors, which intercept electrons on or before ferredoxin cause deactivation of fructose 1,6-bisphosphatase in light. The rate of deactivation, in dark, is also enhanced by exogenously added electron acceptors and sulfhydryl group modifying agents. The mechanism of regulation of fructose 1,6-bisphosphatase is discussed.     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C₃ (spinach) and C₄ (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000-dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.

Kalanchoë FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoë ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.

Kalanchoë FBPase could be partially activated by Mg²⁺ at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca²⁺, especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoë SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg²⁺ concentration. However, like FBPase, increased Mg²⁺ concentrations enhanced the thioredoxin-linked activation of this enzyme.

In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoë leaves which required reduced thioredoxin m for activity.

These results indicate that Kalanchoë FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C₃ and C₄ plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the functional segregation of glucan synthesis and degradation.

     

Enzyme Regulation in C(4) Photosynthesis : PURIFICATION AND PROPERTIES OF THIOREDOXIN-LINKED NADP-MALATE DEHYDROGENASE FROM CORN LEAVES

NADP-malate dehydrogenase, a light-modulated enzyme of C₄ photosynthesis, was purified to homogeneity from leaves of corn. The pure enzyme was activated by thioredoxin m that was reduced either photochemically (with ferredoxin and ferredoxin-thioredoxin reductase) or chemically (with dithiothreitol). Unactivated corn leaf NADP-malate dehydrogenase had a molecular weight of 50,000 to 60,000 and was chromophorefree. The enzyme appeared to have a high content of serine and glycine and to contain both S—S and SH groups. Consequently, NADP-malate dehydrogenase seems to be capable of undergoing reversible oxidation/reduction during its photoregulation.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.