A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.
The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase. (+info)
Arterial blood gas tensions during upper gastrointestinal endoscopy.
Arterial blood gas tensions were measured before and during upper gastrointestinal endoscopy, with (group I) and without (group 2) sedation with intravenous diazepam. There was a highly significant fall in the PaO2, which occurred in both groups and was therefore not attributable to diazepam. Measurement of FEV, and FVC before endoscopy had no predictive value for those patients whose PaO2 fell the most. (+info)
Does gill boundary layer carbonic anhydrase contribute to carbon dioxide excretion: a comparison between dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss).
In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=-0.11+/-0.01; N=22; mean +/- s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=-0.002+/-0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=-0.005+/-0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from -0.133+/-0.03 to -0.063+/-0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/-22.8 micromol l-1; acetazolamide delta [H+]=81.3+/-21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/-0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/-0.01). The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from -0.123+/-0.01 to -0.065+/-0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from -0.11+/-0.01 to -0.07+/-0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion. (+info)
Prior protein intake may affect phenylalanine kinetics measured in healthy adult volunteers consuming 1 g protein. kg-1. d-1.
Study of the amino acid metabolism of vulnerable groups, such as pregnant women, children and patients, is needed. Our existing protocol is preceded by 2 d of adaptation to a low 13C formula diet at a protein intake of 1 g. kg-1. d-1 to minimize variations in breath 13CO2 enrichment and protein metabolism. To expand on our potential study populations, a less invasive protocol needs to be developed. We have already established that a stable background 13CO2 enrichment can be achieved on the study day without prior adaptation to the low 13C formula. Therefore, this study investigates phenylalanine kinetics in response to variations in prior protein intake. Healthy adult subjects were each fed nutritionally adequate mixed diets containing 0.8, 1.4 and 2.0 g protein. kg-1. d-1 for 2 d. On d 3, subjects consumed an amino acid-based formula diet containing the equivalent of 1 g protein. kg-1. d-1 hourly for 10 h and primed hourly oral doses of L-[1-13C]phenylalanine for the final 6 h. Phenylalanine kinetics were calculated from plasma-free phenylalanine enrichment and breath 13CO2 excretion. A significant quadratic response of prior protein intake on phenylalanine flux (P = 0.012) and oxidation (P = 0.009) was identified, such that both variables were lower following adaptation to a protein intake of 1.4 g. kg-1. d-1. We conclude that variations in protein intake, between 0.8 and 2.0 g. kg-1. d-1, prior to the study day may affect amino acid kinetics and; therefore, it is prudent to continue to control protein intake prior to an amino acid kinetics study. (+info)
Role of a novel photosystem II-associated carbonic anhydrase in photosynthetic carbon assimilation in Chlamydomonas reinhardtii.
Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2-concentrating mechanism (CCM), which helps to overcome CO2 limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II-mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular alpha-CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2 for carboxylation. (+info)
Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum.
Nitrate has been shown to shunt the electron flow in Clostridium thermoaceticum from CO2 to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J. M. Frostl, C. Seifritz, and H. L. Drake, J. Bacteriol. 178:4597-4603, 1996). Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway. The CO oxidation activity in crude extracts of nitrate (30 mM)-supplemented cultures was fivefold less than that of nitrate-free cultures, while the H2 oxidation activity was six- to sevenfold lower. The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis. Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures. Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%). Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged. Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level. At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered. We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min-1 mg-1) in crude extracts of C. thermoaceticum. Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins. CO2 also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity. (+info)
Glucose kinetics during prolonged exercise in highly trained human subjects: effect of glucose ingestion.
1. The objectives of this study were (1) to investigate whether glucose ingestion during prolonged exercise reduces whole body muscle glycogen oxidation, (2) to determine the extent to which glucose disappearing from the plasma is oxidized during exercise with and without carbohydrate ingestion and (3) to obtain an estimate of gluconeogenesis. 2. After an overnight fast, six well-trained cyclists exercised on three occasions for 120 min on a bicycle ergometer at 50 % maximum velocity of O2 uptake and ingested either water (Fast), or a 4 % glucose solution (Lo-Glu) or a 22 % glucose solution (Hi-Glu) during exercise. 3. Dual tracer infusion of [U-13C]-glucose and [6,6-2H2]-glucose was given to measure the rate of appearance (Ra) of glucose, muscle glycogen oxidation, glucose carbon recycling, metabolic clearance rate (MCR) and non-oxidative disposal of glucose. 4. Glucose ingestion markedly increased total Ra especially with Hi-Glu. After 120 min Ra and rate of disappearance (Rd) of glucose were 51-52 micromol kg-1 min-1 during Fast, 73-74 micromol kg-1 min-1 during Lo-Glu and 117-119 micromol kg-1 min-1 during Hi-Glu. The percentage of Rd oxidized was between 96 and 100 % in all trials. 5. Glycogen oxidation during exercise was not reduced by glucose ingestion. The vast majority of glucose disappearing from the plasma is oxidized and MCR increased markedly with glucose ingestion. Glucose carbon recycling was minimal suggesting that gluconeogenesis in these conditions is negligible. (+info)
Differences in spontaneous breathing pattern and mechanics in patients with severe COPD recovering from acute exacerbation.
The aims of this study were to assess spontaneous breathing patterns in patients with chronic obstructive pulmonary disease (COPD) recovering from acute exacerbation and to assess the relationship between different breathing patterns and clinical and functional parameters of respiratory impairment. Thirty-four COPD patients underwent assessment of lung function tests, arterial blood gases, haemodynamics, breathing pattern (respiratory frequency (fR), tidal volume (VT), inspiratory and expiratory time (tI and tE), duty cycle (tI/ttot), VT/tI) and mechanics (oesophageal pressure (Poes), work of breathing (WOB), pressure-time product and index, and dynamic intrinsic positive end-expiratory pressure (PEEPi,dyn)). According to the presence (group 1) or absence (group 2) of Poes swings during the expiratory phase (premature inspiration), 20 (59%) patients were included in group 1 and 14 (41%) in group 2. Premature inspirations were observed 4.5+/-6.4 times x min(-1) (range 1-31), i.e. 20+/-21% (3.7-100%) of total fR calculated from VT tracings. In group 1 the coefficient of variation in VT, tE, tI/ttot, PEEPi,dyn, Poes and WOB of the eight consecutive breaths immediately preceding the premature inspiration was greater than that of eight consecutive breaths in group 2. There were no significant differences in the assessed parameters between the two groups in the overall population, whereas patients with chronic hypoxaemia in group 1 showed a more severe impairment in clinical conditions, mechanics and lung function than hypoxaemic patients in group 2. In spontaneously breathing patients with chronic obstructive pulmonary disease recovering from an acute exacerbation, detectable activity of inspiratory muscles during expiration was found in more than half of the cases. This phenomenon was not associated with any significant differences in anthropometric, demographic, physiological or clinical characteristics. (+info)