A. J. Keys

Intracellular site of sucrose synthesis in leaves

Abstract Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g . UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.

Oxidation and phosphorylation associated with the conversion of glycine to serine

Abstract Particles separated from extracts of tobacco leaves by differential centrifugation catalysed the conversion of two molecules of glycine to one molecule of serine and one of carbon dioxide. The catalytic system was inhibited by inhibitors of the mitochondrial electron transport system. The reaction was slow in the absence of oxygen but attained a maximum rate at 3% by vol. of oxygen in the atmosphere. Approximately one atom of oxygen was taken up for each molecule of serine formed. Added ADP increased the rate of reaction and was converted to ATP. Some ATP was formed and oxygen consumed in the absence of glycine but both processes proceeded faster when glycine was added. Isonicotinyl hydrazide, an inhibitor of serine synthesis, decreased phosphorylation more in the presence of glycine than in its absence. Trans-aconitate, a competitive inhibitor of aconitase, did not inhibit the phosphorylation caused by adding glycine but strongly inhibited phosphorylation occuring in the absence of glycine. Phosphorylation accompanying the conversion of glycine to serine probably involves the electron transport chain of mitochondria and associated phosphorylation sites. The conversion of glycine to serine is probably responsible for the evolution of carbon dioxide in photorespiration, so our results suggest that photorespiration causes ATP to be synthesized in mitochondria during photosynthesis.

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity.

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulansSynechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the β/α barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in Vmax but did not alter the enzymes relative specificity towards either of its gaseous substrates, CO2 and O2. However replacement of alanine 340 with glutamate decreased the enzymes specificity for CO2 but had no significant effect on either the Km for ribulose-1,5-bisphosphate or CO2 or on Vmax. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.

Activation of ribulose 1,5-bisphosphate carboxylase by Ca2+

Purified RuBP carboxylase requires activation by reaction with CO2 and a divalent metal ion. Mg2+ is the most effective metal ion, and is probably involved in activation in vivo. Ca2+ is reported not to be an activator. Several oxyanions, including phosphate esters, are effectors of activation of RuBP carboxylase by Co2 and Mg2+. It is now shown that Ca2+ is an effective activator of RuBP carboxylase and that PO4 3−, FBP, NADPH and 6‐phosphogluconate are effectors of this activation. The ratio of oxygenase to carboxylase activity of enzyme activated with Ca2+ is similar to that for the enzyme activated with Mg2+.

Purified Ribulose-P2 carboxylase from wheat with high specific activity and with fast activation.

Isolation of Ribulose-P2 carboxylase/oxygenase and its purification to homogeneity often results in losses of enzyme activity. Attempts were made to decrease these losses by adding fructose-P2, SO ] , Pi or PPi to the buffers used during early stages of the extraction and purification of the enzyme. Yield and final specific activity were not improved, but fructose-P2 and PPi became bound to the enzyme and modified its activation characteristics. The enzyme prepared with fructose-P2 showed a much decreased rate of deactivation when the activating cofactors Mg 2÷ or CO2 were reduced in concentration. Specific activities of above 1.9 pmol CO2 min -1 mg -1 protein were found at intermediate stages in the purifications. High specific activities from all preparations require extraction from preilluminated young leaves of plants given excess N. Decrease in specific activity in the final stages of purification was not generally seen if Pi was added during activation of fractions with Mg a ÷ and CO2 prior to assay. Differences in tertiary and quaternary structure of the various preparations were examined using polyacrylamide gel electrophoresis and by a preliminary study of the growth and properties of crystals. Similar crystals were obtained from them all but gel electrophoresis showed that preparations made in the presence of fructose-Pz contained a greater proportion of aggregated states of ribulose-P2 carboxylase/oxygenase subunits giving protein of M.Wt. greater than 5 50,000 Daltons.

The enzymatic determination of bicarbonate and CO2 in reagents and buffer solutions.

A method for the determination of bicarbonate in buffer solutions between pH 7.5 and 8.75 and in stock solutions of NaHCO3 is described. The HCO-3 is reacted with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase (EC 4.1.1.31) and the oxaloacetate formed reduced to malate by NADH in the reaction catalyzed by malate dehydrogenase (EC 1.1.1.37). The extent of oxidation of NADH is measured spectrophotometrically. Experiments using standard solutions show that 1 mol of NADH is oxidized per mol of HCO-3 added. The method was used to establish the precautions needed to prepare buffer solutions containing less than 1% of the bicarbonate which would be present in the same buffers in equilibrium with air.

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding.

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the Km for ribulose-1,5-bisphosphate from 82 to 190 μM but its replacement with glutamine, which has polar properties, had no appreciable effect.

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.

Separation of the branch chain hexose, hamamelose, by high-performance liquid chromatography, and amperometric detection of hamamelose and related compounds

Abstract The identification and purification of the branch chain hexose hamamelose (2-hydroxymethyl- d -ribose) from plant leaves is described. The procedure includes metabolite extraction in acid and a series of isocratic HPLC separations. Amperometric detection of hamamelose is shown to be quantitative, having a detection limit below 1 nmol. Similar detection of the related compound, 2-carboxy- d -arabinitol, and its monophosphate ester, yields unusually small peaks.

Photosynthesis, Photorespiration and Enzyme Levels in Barley Wheat and Maize Grown on Nitrate and Ammonia

The effect of nitrogen nutrition on photosynthesis and photorespiration in C3 and C4 plants has been Investigated by several research groups. Fair et al. (1974) reported that barley plants grown on ammonia had higher levels of glycollate oxidase and RuBP carboxylase and an increased CO2 compensation point compared to nitrate grown material. However, although differences in the energetics of nitrogen utilization are cited as a reason for these differences in photorespiration no clear mechanism for the alteration of photorespiration by the nitrogen regime was advanced. The compensation point in maize and sugar cane has also been reported to be increased by ammonium (Grossman, Cresswell, 1973) a result confirmed in maize by Yamada, Ikeda (1980). Similar findings were recently reported for bean leaves (Marques et al. Erismann,1983). In a re-examination of earlier reports using barley wheat and maize plants grown on nitrate or ammonia we have been unable to confirm the conclusion that nitrogen nutrition causes any changes in photorespiratory CO2 loss.