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Biomedical subjects

A J Keys

Publications and source records attributed to A J Keys.

At least 19 recordsLinked to original sources
PubMed2005

Adaptation of photosynthesis in marama bean Tylosema esculentum (Burchell A. Schreiber) to a high temperature, high radiation, drought-prone environment.

Marama bean, Tylosema esculentum, is a tuberous legume native to the Kalahari region of Southern Africa where it grows under high temperatures (typical daily max 37 degrees C during growing season) and radiation (frequently in excess of 2000 micromol m(-2) s(-1)) in sandy soils with low rainfall. These conditions might be expected to select for increased water-use efficiency of photosynthesis. However, marama was found to give similar leaf photosynthetic rates to other C3 plants for a given internal leaf CO2 concentration and Rubisco content. Under conditions of increasing drought, no increase in water-use efficiency of photosynthesis was observed, but stomata closed early and preceded any change in leaf water potential. The possibility of subtle adaptations of photosynthetic characteristics to its natural environment were investigated at the level of Rubisco kinetics. The specificity factor of marama Rubisco was slightly lower than that of wheat, but the apparent Km for CO2 in air (Km') was about 20% lower than that of wheat. This is consistent with better adaptation for efficient photosynthesis at high temperatures in marama compared to wheat, although the net benefit is predicted to be very small (<0.5% at 35 degrees C). The sequence of marama rbcL gene shows 27 deduced amino acid residue differences from that for wheat, and the possibility that one or more of these cause the difference in Rubisco Km' is discussed.

Adaptation, Physiological↗
PubMed2003

Manipulation of Rubisco: the amount, activity, function and regulation.

Genetic modification to increase the specificity of Rubisco for CO(2) relative to O(2) and to increase the catalytic rate of Rubisco in crop plants would have great agronomic importance. The availability of three-dimensional structures of Rubisco at atomic resolution and the characterization of site-directed mutants have greatly enhanced the understanding of the catalytic mechanism of Rubisco. Considerable progress has been made in identifying natural variation in the catalytic properties of Rubisco from different species and in developing the tools for introducing both novel and foreign Rubisco genes into plants. The additional complexities of assembling copies of the two distinct polypeptide subunits of Rubisco into a functional holoenzyme in vivo (requiring sufficient expression, post-translational modification, interaction with chaperonins, and interaction with Rubisco activase) remain a major challenge. The consequences of changing the amount of Rubisco present in leaves have been investigated by the use of antisense constructs. The manipulation of genes encoding Rubisco activase has provided a means to investigate the regulation of Rubisco activity.

Carbon Dioxide↗
PubMed1999

The localisation of 2-carboxy-D-arabinitol 1-phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L.

A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CAIP), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.

Binding Sites↗
PubMed1996

Conversion of D-hamamelose into 2-carboxy-D-arabinitol and 2-carboxy-D-arabinitol 1-phosphate in leaves of Phaseolus vulgaris L.

[1-14C]Hamamelose (2-hydroxymethyl-D-ribose) was synthesized by reaction of ribulose 5-phosphate with potassium [14C]cyanide, catalytic hydrogenation of the resulting cyanohydrin, and dephosphorylation of the product. Its identity was established by a chromatographic comparison with hamamelose isolated from the bark of witch hazel (Hamamelis virginiana L.). Following vacuum infiltration of the [1-14C]hamamelose into leaf discs from Phaseolus vulgaris L., 14C-labeled 2carboxy-D-arabinitol (CA) and 2-carboxy-D-arabinitol 1-phosphate (CA1P) were formed, in the dark. Conversion of hamamelose to both CA and CA1P in the leaf discs was inhibited by dithiothreitol and sodium fluoride, although at high concentrations of these inhibitors conversion into CA was still evident when conversion into CA1P was totally inhibited. Wheat (Triticum aestivum L.) leaves converted hamamelose into CA without formation of CA1P. Leaves from P. vulgaris contained 68 nmol.g-1 fresh weight of hamamelose in the light and 35 nmol.g-1 fresh weight in the dark. A pathway for the biosynthesis of CA1P from Calvin cycle intermediates is proposed which includes the sequence: hamamelose --> CA --> CA1P.

Anions↗
PubMed1996

2-Carboxyarabinitol 1-phosphate (CA1P) formation through a phosphate exchange reaction catalysed by the CA1P phosphatase from French bean (Phaseolus vulgaris L.).

Transfer of the phosphate group of 2-carboxy-D-arabinitol 1-phosphate (CA1P) to 14C-labelled 2-carboxy-D-arabinitol (CA) was catalysed by extracts from leaves of Phaseolus vulgaris. This phosphotransferase activity co-purified with CA1P phosphatase, described previously. This activity was increased, up to 16-fold, by addition of bicarbonate ions at pH 9-10, suggesting rate enhancement by enzyme carbamylation. A V(max) of 1.5 mumol/min per mg of protein and a K(m) (for CA) of 1.8 mM were estimated for the exchange reaction, with the purified phosphatase. 2-Carboxy-D-arabinitol 1,5-bisphosphate and 2-carboxy-D-ribitol 1,5-bisphosphate, but not D-ribulose 1,5-bisphosphate, could replace CA1P as phosphate donor to [14C]CA.

Cell Extracts↗
PubMed1994

Incorporation of carbon from photosynthetic products into 2-carboxyarabinitol-1-phosphate and 2-carboxyarabinitol.

The synthesis of 2-carboxy-D-arabinitol-1-phosphate (CA1P), the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was studied in leaves of the French bean Phaseolus vulgaris, L. Leaves were supplied with air containing 14CO2 in the light then the plants were transferred to normal air in the light or in the dark. Leaf samples were frozen in liquid nitrogen, ground to a powder and extracted with acid. Lipids, pigments and cations were removed from the extract and CA1P and 2-carboxy-D-arabinitol (CA) recovered by anion exchange chromatography. The CA1P was further purified by its specific binding to purified ribulose-1,5-bisphosphate carboxylase/oxygenase. CA and CA1P were identified by chromatographic properties and n.m.r. spectra. When plants were kept for 15 h in darkness after exposure to 14CO2, up to 2.2% and 5.5% of the radioactivity in the extracts was present in CA1P and CA, respectively. The most radioactivity appeared in these compounds when photosynthesis from 14CO2 took place at low photosynthetic photon flux density (PPFD). Under such conditions, radioactivity was detected in CA1P after only 10 min. During subsequent exposure to normal air (12CO2) at low PPFD the amount of radioactivity in CA1P remained almost constant for 6 h; in darkness the rate of incorporation of radioactivity into CA1P reached a maximum after 2 h and the radioactivity was still increasing 6 h later. At low PPFD, the amount of CA1P in the leaves reached a maximum after 2 h. In darkness, the amount of CA1P began to increase rapidly after a lag of almost 1 h, well ahead of the increase in radioactivity in CA1P.

Carbon Dioxide↗
PubMed1992

Purification and properties of a phosphatase in French bean (Phaseolus vulgaris L.) leaves that hydrolyses 2'-carboxy-D-arabinitol 1-phosphate.

An enzyme that releases P(i) from 2-carboxy-D-arabinitol 1-phosphate, a naturally occurring tightly binding inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), was purified from leaves of French bean seedlings. It was a monomeric protein of M(r) about 56,000. Catalytic activity was stimulated by increased concentrations of inorganic salts to a maximum at an ionic strength above 0.2. NADPH and D-fructose 1,6-bisphosphate increased the activity of the enzyme in both the presence and absence of 0.2 M-KCl. The pure enzyme did not require dithiothreitol for activity. The pH optimum was 7, the Km for 2-carboxy-D-arabinitol 1-phosphate was 0.43 mM and the specific activity 6.8 mumol/min per mg of protein. The enzyme had little or no activity against phosphate ester intermediates of photosynthetic metabolism and glycolysis but hydrolysed the 1,5-bisphosphates of 2'-carboxy-D-ribitol and 2'-carboxy-D-arabinitol more rapidly than 2'-carboxy-D-arabinitol 1-phosphate.

Dithiothreitol↗
PubMed1990

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.

Catalysis↗
PubMed1988

Regulation of ribulose-1,5-bisphosphate carboxylase activity by the activase system in lysed spinach chloroplasts.

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO(2) and Mg(2+) by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of DeltapH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between DeltapH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO(2) and Mg(2+).

Journal Article↗
PubMed1987

The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes.

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

Chimera↗
PubMed1986

Species variation in the predawn inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (</=0.75, while only Capsicum frutescens, Cucumis sativa, Glycine max, Nicotiana tabacum, Vigna unguiculata, and 3 Solanum species showed a pa/ma ratio </=0.5. Phaseolus vulgaris consistently showed a pa/ma ratio of </=0.1. Activities and pa/ma ratios of the same species grown in the United States and the United Kingdom were very similar. Gel filtration of extracts before assay had no effect on the observed activities and the pa/ma ratios. These data are consistent with the hypothesis that in a number of species the enzyme is partially inhibited following the night period by the presence of a tight-binding inhibitor.

Journal Article↗
PubMed1984

Inactive forms of wheat ribulose bisphosphate carboxylase. Conversion from the slowly activating into the rapidly activating form.

Wheat ribulose bisphosphate carboxylase can be converted from the slowly activating into the rapidly activating form by heat or effectors in the absence of CO2 and Mg2+. This conversion process had the same energy of activation of 95.6kJ/mol both in the presence and in the absence of effectors, whereas the free-energy change value ranged from +2.5kJ/mol to -3.4kJ/mol depending on the end product involved. Far-u.v. c.d. spectra measured before and after conversion indicated that ribulose bisphosphate carboxylase is an alpha/beta-class protein and that no significant changes in gross conformation occur. Signals in the near-u.v. region suggested that the main change during conversion is re-orientation of aromatic side chains, probably near the active site; a possible site for effector binding is discussed.

Binding Sites↗
PubMed1984

Biochemistry of photosynthesis in species of triticum of differing ploidy.

Illuminated flag leaves of Triticum monococcum(2X), T. urartu(2X), T. dicoccum(4X), T. dicoccoides(4X), and T. aestivum(6X) were exposed to (14)CO(2) for 10 seconds and subsequently allowed to continue photosynthesis in the ambient air for periods of up to 2 minutes. The relative distribution of (14)C among water-soluble products in the leaves was similar for each species at each sampling time. After the 10-second pulse of (14)CO(2), radioactivity was mainly in phosphate esters with less than 5% in C(4) acids. Subsequently, radioactivity increased in sucrose, glycine, and serine at the expense of that in phosphate esters. By 2 minutes, between 18% and 29% of the (14)C was in glycine plus serine. The results suggest rapid photorespiration in all species and an absence of C(4) photosynthesis.d-Ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) was partly purified from seedling leaves of each of the five Triticum species. Each preparation was assayed for simultaneous carboxylase and oxygenase activities in 2.1 millimolar NaHCO(3) and 265 micromolar O(2) at pH 8.2 and 25 degrees C. The mean ratio of carboxylase to oxygenase activities was 6.11 +/- 0.16 (standard error); differences between values for different species were not statistically significant. The results do not explain the faster rates of photosynthesis per unit leaf area reported for diploid and tetraploid species of Triticum compared to the hexaploid.

Journal Article↗
PubMed1984

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation.

The effects of methionine sulfoximine and ammonium chloride on [(14)C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U(14)C]glutamate in the light and in the absence of inhibitor or NH(4)Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO(2) became slightly radioactive. Ammonium chloride (10 mm) increased formation of [(14)C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and (14)CO(2). Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH(3) to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the (14)C from [U-(14)C]glutamate to be incorporated into malate and succinate, whereas most of the (14)C from [1-(14)C]glutamate was metabolized to CO(2) and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through "dark" fixation of CO(2) released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-(14)C] glutamate without inhibitor or NH(4)Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and (14)CO(2). Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.

Journal Article↗
PubMed1983

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.

Bicarbonates↗
PubMed1983

Temperature dependence of the enzymic carboxylation and oxygenation of ribulose 1,5-bisphosphate in relation to effects of temperature on photosynthesis.

Carboxylase and oxygenase activities of ribulose bisphosphate carboxylase purified from wheat were measured over the temperature range 5 to 35 degrees C either at constant O(2) and CO(2) concentrations or where the O(2) and CO(2) simulated the concentrations in water equilibrated at each temperature with the same gaseous phase. At constant CO(2) (14 micromolar) and O(2) (0.34 millimolar), the oxygenase to carboxylase ratio remained constant at 0.21 between 5 and 25 degrees C but increased to 0.26 at 35 degrees C. At O(2) and CO(2) concentrations near those expected in water equilibrated with air (21% [v/v] O(2)) containing 300 mul/l CO(2) at the various temperatures, the ratio of oxygenase to carboxylase activity increased 2.2-fold between 15 and 35 degrees C. At CO(2) and O(2) concentrations expected in water in equilibrium with subatmospheric concentrations of CO(2) in air (21% [v/v] O(2), 210 mul/l CO(2)), the oxygenase to carboxylase ratio increased from 0.25 at 10 degrees C to 0.56 at 35 degrees C. Between 20 and 30 degrees C, the apparent Q(10) value for the oxygenase reaction was 1.78 and that for the carboxylase was 1.26. Hence, the different responses of photosynthesis and photorespiration to temperature are due more to changes in the relative solubilities of CO(2) and O(2) (the solubility ratio) than to changes in kinetic parameters of the reactions catalyzed by ribulose bisphosphate carboxylase.

Journal Article↗
PubMed1978

Intramolecular labelling of sucrose made by leaves from [14C)carbon dioxide or [3-14C]serine.

Pea leaves were illuminated in air containing 150 or 1000p.p.m. of 14CO2 for various times. Alternatively, segments of wheat leaves were supplied with [3-14C]serine for 40 min in the light in air with 145, 326 or 944p.p.m. of 12CO2. Sucrose was extracted from the leaf material, hydrolysed with invertase, and 14C in the pairs of carbon atoms C-3+C-4, C-2+C-5 and C-1+C-6 in the glucose moiety was measured. The results obtained after metabolism of 14CO2 were consistent with the operation of the photosynthetic carbon-reduction cycle; the effects of CO2 concentration on distribution of 14C in the carbon chain of glucose after metabolism of [3-14C]serine is more easily explained by metabolism through the glycollate pathway than by the carbon-reduction cycle.

Carbon Dioxide↗