Bicarbonate Fixation Research Articles

Intraplastidic Bicarbonate Concentration and Fixation during Plastid Development

The amount of bicarbonate fixation by etioplasts and 1–24 h etio-chloroplasts, isolated from unilluminated and partially illuminated dark-grown Avena sativa laminae, has been measured as total fixation in association with the levels of activity of some of the enzymes involved in the fixation of CO 2 . The bicarbonate permeability of the plastid envelopes during the same stages of development has also been investigated. The results show: a) dark fixation of bicarbonate remains constant throughout, whereas light-dependent fixation of bicarbonate exhibits a lag-phase up to two hours during plastid development and increases markedly afterwards; b) changes in the levels of activity of ribulose-1.5-diphosphate carboxylase show a similar lag-phase, whilst ribulokinase and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase show an immediate increase in levels of activity and there is no light-dependent increase in the levels of activity of phospho-enolpyruvate carboxylase; c) the intraplastidic concentration of bicarbonate in etioplasts and 1–2 h etio-chloroplasts is similar to that of the surrounding medium whereas etio-chloroplasts isolated from laminae that have been illuminated for 4 h and longer show an increasing accumulation of bicarbonate in the intraplastidic space. These results are discussed in relation to the possible appearance of a «CO 2 -pump» in the later stages of plastid development.

Partial Purification and Properties of Ornithine Transcarbamoylase from Nostoc muscorum Kützing

Ornithine transcarbamoylase (carbamoyl phosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) has been partially purified from the blue-green alga Nostoc muscorum Kützing, an organism in which the enzyme seems to be involved in a bicarbonate-fixing pathway leading to citrulline. Pertinent to possible regulation of this pathway, the enzyme shows hyperbolic substrate kinetics, has a molecular weight estimated at 75,000 daltons, and its catalytic capability is little influenced by a selection of metabolites that might conceivably act as regulators in vivo. Thus it seems unlikely that this enzyme is the control point for bicarbonate fixation. In terms of energy of activation (12.3 kcal/mole), size and Km for carbamoylphosphate, the Nostoc enzyme resembled preparations from liver and higher plants more than preparations from Streptococcus and Mycoplasma. The enzymes from Streptococcus and Mycoplasma are probably specialized for citrulline breakdown rather than citrulline synthesis. The Km for ornithine was 2.5 mm at a saturating concentration of carbamoylphosphate and the Km for carbamoylphosphate was 0.7 mm at an ornithine concentration of 2 mm. Ornithine was inhibitory at concentrations greater than 2 mm. Phosphate was a competitive inhibitor with respect to carbamoylphosphate. The pH optimum for citrulline synthesis was 9.5.

Secretion analysis: secretin testing in pancreatic cancer.

Modern testing of pancreatic function and the diagnosis of pancreatic disease is facilitated by the addition of augmented stimulus to the standard stimulus used in secretin testing. The combined standard-augmented secretin test protocol enables not only diagnosis using the classical patterns of secretion (qualitative and quantitative defects), but also permits delineation of secretory capacity. In the abnormal patient, the augmented test increases the defect in the characteristic diagnostic parameter. Thus, there is fixation of flow in pancreatic cancer and fixation of bicarbonate in pancreatic inflammation.

Experimental studies on the effects of copper on a marine food chain

Effects of copper sulphate on a marine food chain were investigated in large tanks at Loch Ewe on the west coast of Scotland. The food chain consisted of phytoplankton, the bivalve Tellina tenuis da Costa, and O-group plaice (Pleuronectes platessa L). During initial months after settlement, T. tenuis siphons are an important food for juvenile plaice, which feed by grazing the siphon tips; these subsequently regenerate. Copper dose rates of 10, 30 and 100 μg Cu/1 were investigated; the distribution of added copper was monitored and its metabolic effects were determined. Copper levels in water, sand, in algae on tank walls, in T. tenuis shell and flesh, and in plaice muscle and viscera were measured by atomic absorption spectrophotometry. Copper accumulated in sand, T. tenuis flesh and shell, and plaice viscera. Accumulations were dose-dependent and in no case was a plateau concentration reached. Most of the added dose was taken up by the sand; accumulations in T. tenuis shell were small — less than twice background in all cases. High levels were found in T. tenuis flesh, where concentrations after 100 days were 270, 470 and 1100 μg Cu/g dry flesh for dose concentrations of 10, 30 and 100 μg Cu/1, respectively. The mean control concentration was less than 50 μg Cu/g dry flesh. There was no accumulation of copper in plaice muscle. Visceral accumulations after 100 days were 71, 147 and 567 μg Cu/g dry flesh for dose concentrations of 10, 30 and 100 μg Cu/1, respectively. The mean control level was 30 μg Cu/g dry flesh. The effect of copper on phytoplankton metabolism was investigated by measuring plant pigment levels and rates of primary photosynthetic fixation of C14-labelled bicarbonate. All dose concentrations reduced both the standing crop and the rate of photosynthesis per unit of chlorophyll a. The effects of copper on growth and condition of T. tenuis and P. platessa were investigated. All dose concentrations adversely affected T. tenuis condition (dry flesh weight for standard individual). The effect was most marked during deposition of winter reserves. In the absence of plaice predation, there was a decrease in the mean siphon weight for T. tenuis exposed to 30 and 100 μg Cu/1. For plaice, all dose concentrations of copper resulted in reduced growth, but there was no significant change in condition or biochemical composition. The ash weight of fish exposed to copper was significantly higher than for controls.

Studies on the control of gluconeogenesis in sheep: effect of glucose infusion.

1. Effect of glucose infusions on the rate of gluconeogenesis, as indicated by changes in the irreversible loss of plasma glucose, synthesis of glucose from ruminal propionate or fixation of blood bicarbonate into glucose, has been examined in sheep.Sheep, feeding at hourly intervals, received intravenous infusions of [U-14C]glucose, NaH14CO3, or [6-3H]glucose, infused simultaneously with an intraruminal infusion of [2-14C]propionate. Glucose (4–144 mg/min) was infused intravenously at a single rate or at two different successive rates of equal duration. The glucose infusions were over a 6 h period and followed estimates of pre-infusion kinetic measurements.2. The infusion of glucose suppressed endogenous glucose production in direct proportion to that infused for sheep given diets of lucrene or wheat. The maximum rate of suppression recorded was equivalent to about 50–60% of the endogenous production rate of glucose.Transfer rates of carbon-14 from blood bicarbonate or ruminal propionate to plasma glucose were reduced during glucose loadings. The results indicate that administration of glucose was more effective in suppressing the synthesis of glucose from substrates other than ruminal propionate.3. The percentage of glucose carbon derived from blood bicarbonate was lower in sheep given wheat than in sheep given lucerne. It was suggested that this difference may have been due to absorption of glucose from the alimentary canal of sheep on the former ration. Approximately 8% of the wheat starch may have escaped fermentation and been absorbed as glucose in these animals.

Studies on the control of gluconeogenesis in sheep: effect of propionate, casein and butyrate infusions

1. Short-term effects of infusions of propionate, amino acids and butyrate on gluconeogenesis, as indicated by changes in the irreversible loss of plasma glucose, synthesis of glucose from ruminal propionate or fixation of blood bicarbonate into glucose have been examined in sheep given their daily ration in twenty-four equal portions at hourly intervals.Sheep received intravenous infusions of [6-3H]glucose usually, in combination with [U-14C]glucose or NaH14CO3or with intraruminal infusions of [2-14C]propionate. Substrates were infused over a 3–7 h period and followed estimates of pre-infusion kinetic measurements.2. It was demonstrated that intraruminal and intramesenteric vein infusions of sodium propionate and intra-abomasal infusions of casein hydrolysate stimulated gluconeogenesis. Glucose synthesis showed a linear response to the infusion of these substrates, which varied from 0·35–6·35 mmol propionate/min and 50–160 mg casein/min.3. The increment in the measured production rate of propionate in the rumen was consistently less than the rate of addition of propionate to the rumen.4. Intramesenteric vein infusions of sodium butyrate at successive rates of 0·25 and 0·50 mmol/min produced only an initial transient increase in plasma glucose production. Since the rate of glucose synthesis from ruminal propionate was not altered, it was suggested that butyrate initiated glycogen mobilization.

Indoleacetic acid stimulation of phosphorylation and bicarbonate fixation by chloroplast preparations in light

The rate of phosphorylation in illuminated chloroplast preparations from spinach, Swiss chard, or Acetabularia mediterranea was increased by physiological concentrations of indoleacetic acid (IAA), optimum effect being obtained at 10−7 M. Treated chloroplasts gave an increased P/2e ratio, suggesting a greater degree of coupling between electron transport and ATP synthesis. Five minutes after incubation the effect of the hormone was already noticeable. During prolonged illumination the effect of IAA was greatly magnified, raising the phosphorylation rate more than threefold over the control rate. Stimulation of 14C-bicarbonate fixation was also observed in the presence of the same levels of IAA.

Bicarbonate Fixation by Cell-free Extracts and by Mycelium of Neocosmospora vasinfecta

Summary: The most active carboxylating enzyme in cell-free extracts of Neocosmospora vasinfecta was identified as pyruvate carboxylase (EC 6.4.1.1) on the basis of its substrate specificity, biotin dependence and cofactor requirements. Following 48 h. growth on acetate as sole carbon source, the specific activity of the extracted enzyme was 1·4 to 3 times greater than when the organism was maintained on glucose. Bicarbonate fixation in vivo was considerably reduced in the acetategrown mycelium, but was enhanced when ethyl pyruvate was supplied. The mycelial pyruvic acid content was almost an order of magnitude lower in acetategrown mycelium than in glucose-grown. These results indicate that bicarbonate fixation in acetate-grown mycelium may be limited by the availability of pyruvic acid.

Bicarbonate Fixation and Malate Compartmentation in Relation to Salt-induced Stoichiometric Synthesis of Organic Acid

The relationship of malate synthesis to K(+) absorption from solutions of K(2)SO(4) and KHCO(3) was compared in nonvacuolate barley (Hordeum vulgare) root tips and whole excised roots. The comparison has permitted separation of the process which evokes organic acid synthesis from that which leads to stoichiometry between net acid equivalents formed and excess K(+) absorbed from K(2)SO(4), on the one hand, and total K(+) absorbed from KHCO(3), on the other. Both in tips and in roots K(+) uptake from 20 mN salt solution exceeds malate synthesis in the first hour. In vacuolate roots the expected stoichiometry is achieved with time. When root tips are transferred to dilute CaSO(4), malate is rapidly metabolized, and K(+) is lost to the solution. By contrast, in excised whole roots the malate level remains unchanged, the salt-induced organic acid presumably being retained in the vacuole. In excised roots malonate leads to a marked drop in malate levels in untreated roots as well as in roots which have experienced salt-induced net malate synthesis. In consequence, it is contended that malonate makes available normally sequestered vacuolar malate.The general hypothesis is offered that the bicarbonate level of the cytoplasm controls organic acid synthesis by phosphoenolpyruvate carboxylase, and that the cytoplasmic bicarbonate level is raised either by exchange of cytoplasmic H(+) for external cation, or by bicarbonate absorption directly. Stoichiometry, in turn, is achieved by the accumulation in the vacuole of the double salt of malate.

ROOT GROWTH AND DARK FIXATION OF CARBON DIOXIDE IN CALCICOLES AND CALCIFUGES

SummaryThe inhibition of root growth by bicarbonate ions has been further investigated by an examination of the process of dark fixation of carbon dioxide by Arrhenatherum elatius and Deschampsia flexuosa. Excised roots incorporated [14C]bicarbonate mostly into malate in 4 hours. Intact plants grown on solutions containing 10 m‐equiv./1 bicarbonate for 24 hours showed large increases in the total acid content of the roots. Malate was increased in both species and citrate also in Arrhenatherum elatius. The increases in total acidity were similar and could be explained solely by the dark fixation of bicarbonate. Seedlings grown on solutions of sodium hydrogen malate showed similar inhibitions to those observed on sodium bicarbonate solutions. Root growth of Deschampsia flexuosa was inhibited throughout a range of malate concentrations (1–20 m‐equiv./l) whereas Arrhenatherum elatius was stimulated at low malate concentrations and only slightly inhibited at 20 m‐equiv/1. The inhibiting action of bicarbonate on the root growth of Deschampsia flexuosa is associated with the increase in malate in the roots by dark fixation. The results are discussed in terms of the organic acid content and metabolism of calcicole and calcifuge species.

MICROBIAL GROWTH ON C1 COMPOUNDS. SYNTHESIS OF CELL CONSTITUENTS BY METHANE- AND METHANOL-GROWN PSEUDOMONAS METHANICA.

1. A study has been made of the incorporation of carbon from [(14)C]methane, [(14)C]methanol and [(14)C]bicarbonate by cultures of Pseudomonas methanica growing on methane, and [(14)C]methanol by cultures of the same organism growing on methanol. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compound for periods up to 3min., has been analysed by chromatography and radioautography. 3. Over 90% of the radioactivity fixed from [(14)C]methane or [(14)C]methanol at the earliest times of sampling appeared in phosphorylated compounds. Glucose phosphate and fructose phosphate together constituted the largest part of the radioactive phosphates (70-90%); phosphoglycerate was a relatively minor component (2-17%). Other compounds becoming labelled during the incubation included glycine, serine, glutamate, aspartate, malate, citrate and alanine. 4. The first stable products of [(14)C]bicarbonate fixation were malate and aspartate (containing between them over 90% of the total radioactivity fixed at the earliest times of sampling). 5. The percentage of the total radioactivity fixed that was contained in each of the radioactive compounds has been plotted against time. The slopes of the curves obtained show that hexose phosphates are primary stable products of [(14)C]methane and [(14)C]methanol incorporation and that aspartate and malate are primary stable products of [(14)C]bicarbonate incorporation. 6. No carboxydismutase activity has been found in cell-free extracts of the organism. This fact, together with the other findings, shows that an autotrophic metabolism involving the ribulose diphosphate cycle of carbon dioxide fixation cannot be operating.