Grahame J. Kelly

Light induced activation of fructose-1,6-bisphosphatase in isolated intact chloroplasts

Abstract Chloroplasts isolated from spinach leaves previously held in darkness contained no fructosebisphosphatase activity measured at pH 7.5 or 7.9, although high activity at pH 8.8 was observed. Following illumination of these chloroplasts for 7 min, enzyme activity at pH 7.9 was clearly detected, and after 24 min illumination was equal to 36% of the pH 8.8 activity; at this time activity at pH 7.5 also became apparent. The activity at pH 8.8 was not affected by illumination. Similar activation of fructosebisphosphatase in isolated pea chloroplasts was recorded.

Partial Purification and Properties of Soluble Ascorbate Peroxidases From Pea Leaves

One nonenzymic and two enzymic forms of ascorbate peroxidase were found in pea leaves, and designated A, B and C. Form A was due to a low molecular weight, heat-stable component, and could be separated from the enzymic forms by gel filtration. Forms B and C were soluble proteins with an apparent molecular weight of 57,000. These two forms could be separated by cation-exchange chromatography on CM-Sephadex C-50. This technique was incorporated into a procedure for their partial purification. Several properties of B and C were found to be similar: they were active over a wide pH range (5 to 8), they displayed very high affinities for H(2)O(2) (Km<5 μM), and Km values for ascorbate (6.5 mM and 2.9 mM, respectively) were comparable to physiological concentrations of this substrate. These properties are considered conducive to the proposed physiological role of ascorbate peroxidase, viz prevention of H(2)O(2) accumulation.

Effects of a diet of a nitrogen-limited alga (Tetraselmis suecica) on growth, survival and biochemical composition of tiger prawn (Penaeus semisulcatus) larvae.

Abstract The prasinophyte Tetraselmis suecica was grown with high (control) and low nitrogen (1760 and 176 μM nitrate) in continuous culture and fed to protozoea 1 Penaeus semisulcatus larvae, the first feeding stage of this prawn. This alga grown in the higher nitrogen medium was the better diet for the larvae: they developed faster and were heavier when fed the cells from this medium. In contrast to development, larval survival was not affected by the algal diet. The biochemical composition (protein, carbohydrate, lipid and individual fatty acids) of the algae and the larvae fed those algae were also measured. Carbohydrate increased three-fold in the lower nitrogen algae, while protein and lipid were reduced slightly compared to the control. There was a lower protein:energy ratio (0.1 to 0.2) in the lower nitrogen diets than in the control diets (ratio 0.3 to 0.4). Similarly, the n −3: n −6 ratio of fatty acids in the lower nitrogen diets (1.5) was half of that in the control diets (3.0). Although no differences were detected in the proportions of the gross biochemical components of the larvae fed the two diets, there were differences in the proportions of fatty acids. The essential fatty acid 18:3 n −3 was 1.6 times higher in the larvae fed the control diet, and this coincided with faster development to protozoea 2. The differences in the rate of larval development may be due to the higher protein:energy ratio of the control diets as well as to the different fatty acids and their ratios.

Enzyme Activities and Products of CO2 Fixation in Various Photosynthetic Organs of Wheat and Oat

Summary Enzyme activities associated with the Calvin cycle of photosynthesis and with C 4 metabolism, and rates and products of CO 2 fixation were examined with leaves, peduncles, glumes, lemmas, paleae and pericarps of oat and two varieties of wheat («Svenno» from Sweden and «Lerma Rojo» from Mexico). All tissues achieved net CO 2 fixation through the Calvin cycle. Some capacity for C 4 metabolism was detected in the first leaf and, more noticeably, in the tissues surrounding the grain; the latter were also capable of relativelyhigh CO 2 assimilation by phosphoenolpyruvate carboxylase in darkness. Ribulose 1,5-di-phosphate carboxylase activity was greater in leaves, but phosphoenolpyruvate carboxylase activity tended to predominate in the photosynthetic organs of the wheat ear and oat panicle. The wheat pericarp contained pyruvate, Pi dikinase and a relatively high phosphoenolpyruvate carboxylase activity. Glumes, lemmas, paleae and the pericarp seem adapted for catching CO 2 respired by the developing grain in addition to contributing to net CO 2 fixation.

[62] Fructose-bisphosphatase from spinach leaf chloroplast and cytoplasm

Publisher Summary Photosynthetic cells require fructose-bisphosphatase (D-fructose-l,6-bisphosphate l-phosphohydrolase) both in the chloroplast and in the cytoplasm. This chapter describes an assay method and the purification procedure for the enzyme fructose-bisphosphatase from spinach leaf chloroplast and cytoplasm. Fructose-bisphosphatase may be assayed either colorimetrically by estimating the released P i , or spectrophotometrically by coupling the production of fructose 6-phosphate to the reduction of NADP + using the enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Purification of chloroplast fructose-bisphosphatase involves preparation of spinach ( Spinacia oleracea ) leaves, addition of solid ammonium sulfate, fractionation again by addition of solid ammonium sulfate, diethylaminoethyl (DEAE)-Sephadex A-50 chromatography, and Sephadex G-200 chromatography. The first four steps of the purification procedures are common to both enzymes until chromatography on DEAE-Sephadex A-50 that separates the chloroplast enzyme from the cytoplasmic enzyme; thereafter, the procedures differ. The properties of the leaf cytoplasmic enzyme are more or less comparable to those of mammalian fructose-bisphosphatases but the chloroplast enzyme is unique in being insensitive to adenosine monophosphate (AMP) and highly sensitive to the redox state of its environment.

Hexokinases of spinach leaves

Abstract Two soluble hexokinases and a particulate hexokinase have been separated and partially purified from spinach leaves. One of the soluble hexokinases showed a high affinity for glucose ( K m = 63 μM) which was far greater than that for fructose ( K m = 9.1 mM). However, with saturating fructose the activity was twice that with saturating glucose. The particulate hexokinase showed kinetic properties similar to those of this soluble hexokinase. The second soluble hexokinase was distinct in that it was much more active with fructose than with glucose at all concentrations tested, although the K m values for these hexoses (210 μM and 71 μM respectively) were similar. The activity of this hexokinase was stimulated by the monovalent cations K + and NH 4 + .

Pyruvate orthophosphate dikinase of immature wheat grains

Abstract Pyruvate orthophosphate dikinase has been partially purified from green immature wheat grains. The enzyme was cold labile, but could be reactivated by several minutes incubation at 22°C. For phosphoenolpyruvate synthesis, enzyme activity was completely dependent on all three substrates; the apparent Michaelis constants for ATP, orthophosphate, and pyruvate were 36, 430 and 25 μM respectively. Oxalate was a strong competitive inhibitor with respect to pyruvate. The metabolic significance of pyruvate orthophosphate dikinase activity in the developing grain is considered.

Oat leaf phosphoglucose isomerase: competitive inhibition by erythrose-4-phosphate.

Phosphoglucose isomerase was partially purified from oat leaves and shown to be strongly inhibited by erythrose-4-P. Estimated Ki values were between 0.4 and 4.0 μM. The inhibition was of the competitive type with respect to either of the substrates glucose-6-P and fructose-6-P. Several other plant phosphoglucose isomerases were found to be similarly sensitive to erythrose-4-P.

Photosynthesis: Carbon Metabolism Twenty Years of Following Carbon Cycles in Photosynthetic Cells

We do not know whether or not the gentleman described above completed his planned 16 years of research on cucumber photosynthesis. If he did, he still would not have matched our efforts for Progress in Botany, in which our reviews of photosynthetic carbon metabolism have now reached their 20th year. We began at a time when the Calvin cycle was well established (although sufficient activities of all of its enzymes were not), and the processes of C4 photosynthesis and photorespiration had been only recently discovered. During the ensuing 20 years, many details concerning the operation and regulation of these carbon cycles have been revealed. Seven stand out: (1) the demonstration of adequate activities for all Calvin cycle enzymes; (2) the discovery of the chloroplast envelope’s phosphate translocator, (3) the CO2-plus-Mg2+ activation of ribulose-1,5-bisphosphate (RuBP) carboxylase and, more recently, this enzyme’s regulation by the protein “rubisco activase” and a seemingly endless array of novel sugar phosphates; (4) the demonstration of the light-mediated activation of Calvin cycle enzymes, mediated by the small proteins ferredoxin and thioredoxin; (5) the discoveries that the enzymes phosphoenolpyruvate (PEP) carboxylase, pyruvate Pi dikinase, and sucrose-P synthase are regulated by light-mediated phosphorylation/dephosphorylation; (6) the discoveries of the enzyme PPi-phosphofructokinase (PPi-PFK) and the regulatory compound fructose-2,6-P2 in the cytosol of photosynthetic cells — their roles in regulating sucrose metabolism are still being elucidated; (7) the discovery (reported in this review) that the chloroplast envelope’s adenylate translocator accommodates the uptake of ADP-glucose.

Photosynthesis. Carbon Metabolism: The Carbon Metabolisms of Unstressed and Stressed Plants

Following on from the pioneer experiments of investigators such as Joseph Priestly and Jan Ingenhousz, Nicolas de Saussure must have felt close to a full explanation of the growth of plants when he concluded that plant dry matter originated predominantly from water and air. Indeed, if we ignore biochemical details, we must conclude that he was doing well for “plants growing in an [adequately watered and] fertile soil”. Most subsequent studies have addressed the question of how an actively growing plant converts water and air (and sunshine) into its dry matter, i.e. how good plants perform photosynthesis. But what about poor plants? There is no doubt that other plant scientists have pondered upon, and studied, the responses of plants to adverse conditions, but it is doubtful whether the plant-growth scientists realised the extent to which adversity existed in even “normal” growing conditions, and the degree to which photosynthesis has had to adjust, de Saussure could not have imagined how insidiously stresses influence photosynthetic cells, although (ironically) he is credited with the discovery of crassulacean acid metabolism (see Walker 1992), probably the best-known plant adaptation to water deficit.