A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base-catalyzed methanolysis of platelet-activating factor (AGEPC) at 60 degrees C

A novel reaction was explored in which synthetic platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), upon treatment with 1 N NaOH in methanol at 60 degrees C for 20 min, sequentially released the acetyl group, then the choline moiety with concomitant formation of the monomethyl ester of 1-O-alkyl-glycero-phosphoric acid. A mechanism is proposed in which a transient cyclic phosphate intermediate is formed and then attacked by a CH3O moiety to yield a mixture of the sn-2 and sn-3 methyl esters. Proof of structure of the monomethyl ester derivative was achieved through the use of thin-layer chromatography, aluminum oxide chromatography, and examination of the trimethylsilyl derivative of the monomethyl ester by gas-liquid chromatography-mass spectrometry. Replacement of the acyl group on the 2 position with an ethyl or methyl residue completely prevented any attack by 1 N NaOH in methanol at 60 degrees C. Sphingomyelin was not attacked and only acetate removal was noted with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine under similar conditions. The significance of these findings as they relate to the influence of substituents on the chemical and biological reactivity of AGEPC is discussed.     

Ozone increases root respiration but decreases leaf CO2 assimilation in cotton and melon

It is well established that exposure of plant foliage to tropospheric ozone (O3) inhibits photosynthetic gas exchange in leaves and the translocation of current photosynthate to sink tissues. It is less clear what impact O3-reduced source strength has on the physiological responses of sink tissue such as fine roots. The responses were investigated of carbon acquisition in leaves and carbon utilization in the respiration of fine roots, following chronic (weeks) and acute (hours) exposures to O3 in open top chambers. Previous reports indicate increased, decreased, and unchanged rates of root respiration following exposure to O3. A decline in source activity is confirmed, but an increase in sink respiration is reported in fine roots of Pima cotton (cv. S-6) and muskmelon (cv. Ambrosia hybrid). Leaf source strength and root sink activity changed in opposing directions, thus there was no positive correlation that might indicate direct substrate control of root function. Additional linkages between shoot and root following exposure to O3 may be involved.     

Gas chromatography mass spectrometry studies on biologically important 8-aminoquinoline derivatives

Several substituted 8-aminoquinolines related to known antimalarial drugs have been studied by gas chromatography mass spectrometry. 5,6-Dihydroxy-8-aminoquinoline, a possible metabolite of Primaquine, can be detected by single ion monitoring after conversion to a trimethylsilyl ether derivative. The mass spectra obtained in this study indicate that there are certain ions which are characteristic of the trimethylsilyl ethers of hydroxylated 8-aminoquinolines and 5,6-dimethoxy-8-aminoquinolines. These compounds should thus be amenable to analysis if they were produced during in vivo metabolism studies. Using selected ion monitoring the derivatized compounds can be detected at submicrogram levels.     

Metabolism of leukotriene B4 in isolated rat hepatocytes. Involvement of 2,4-dienoyl-coenzyme A reductase in leukotriene B4 metabolism

Radiolabeled leukotriene (LT) B4 was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed, three of which were previously identified as 20-hydroxy-, 20-carboxy-, and 18-carboxy-19,20-dinor-LTB4. A less lipophilic major metabolite (designated HIV) was purified by two reverse phase high performance liquid chromatography separations and was found to exhibit maximal UV absorbance at 269 nm with shoulders at 260 and 280 indicating the presence of a conjugated triene chromophore. Negative ion electron capture gas chromatography/mass spectrometry analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivative of HIV, and positive ion electron ionization mass spectra of the methyl ester trimethylsilyl derivative were consistent with a structure of this metabolite being 16-carboxy-14,15-dihydro-17,18,19,20-tetranor-LTB3. The appearance of this metabolite supports the concept of further beta-oxidation of LTB4 to the carbon 16 which requires the action of 2,4-dienoyl-CoA reductase to remove the 14,15-double bond located two carbon atoms removed from the CoA thioester moiety. One minor metabolite was analyzed by negative ion continuous flow fast atom bombardment mass spectrometry which revealed an ion at m/z 444 which by high resolution mass spectrometry was shown to contain both nitrogen and sulfur. Tandem mass spectrometry suggested the presence of SO3- as well as other fragments corresponding to the amino acid taurine. Incubation of isolated rat hepatocytes with [14C]taurine as well as [3H]LTB4 revealed the incorporation of both radioactive isotopes into this metabolite. The data supported the identification of this metabolite as tauro-18-carboxy-19,20-dinor-LTB4. Amino acid conjugation of leukotrienes has not been previously reported and suggests that such intermediates might participate in enterohepatic circulation of LTB4 metabolites in the intact animal and thus serve as an alternative metabolic route for LTB4 elimination.     

Use of an affinity chromatography method for isolating monospecific Salmonella antibodies

Obtaining antibodies to individual components of Salmonella antigenic complex is highly important for investigations aimed at the study of the antigenic structure of bacteria, their serological identification and the development of diagnostic preparations. The method of obtaining antibodies by the oxidation of Salmonella antigens with sodium periodate and creating immunosorbents based on these antibodies with subsequent affinity chromatography has been developed. Monospecific antibodies thus obtained (O2, O4, O9) have been studied and used as monospecific preparations in the agglutination test, the immunofluorescence test and the immunosorbent assay. The development of methods for stabilizing these preparations, thus ensuring their wide practical use, may be of interest.     

水分对土壤呼吸的影响及机理

土壤呼吸是陆地碳循环的重要环节,在全球变化的背景下,研究水分对土壤呼吸的影响,能为探索陆地生态系统在碳循环方面的源—汇功能和揭示碳的失汇之迷提供有力的证据。综述了水分对土壤呼吸的影响及其机理。土壤呼吸是一个复杂的生态学过程,大气降水对土壤呼吸的影响结果是因时、因地而异,在湿润的生态系统或者干湿交替的生态系统中比较湿润的季节．降水事件对土壤呼吸可能会产生比较明显的抑制现象;而在干旱的生态系统或有干湿交替季节的生态系统中比较干旱的季节里,降水事件可能会强烈地激发土壤呼吸。其对土壤呼吸的影响机理包括水分对土壤孔隙中CO2替代、对CO2扩散的阻滞、对微生物活动的刺激和对微生物生物量的影响等。由于实验方法和标准的不一致以及影响土壤呼吸的因素的多样性。水分量的变化对土壤呼吸的影响很难以一个统一的方程来描述,总的来说,最优的水分状况通常是接近最大田间持水力,当土壤处于过于或过湿状态时,土壤呼吸会受到抑制。水分量的变化对土壤呼吸的影响机制在于可溶性有机质、土壤的通透性、微生物与植物根系生命活动等都随土壤水分状况不同而发生相应的改变。关于水分与土壤呼吸的关系研究今后应该主要集中在：(1)水分对根系呼吸和土壤微生物呼吸分别产生的影响;(2)全球变化后水分格局的变化对全球陆地生态系统土壤呼吸格局的潜在影响;(3)人类活动通过直接或间接改变水分状况而对土壤释放CO2的贡献率。     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

Respiratory carbon metabolism in the high mountain plant species Ranunculus glacialis

Very little is known about the primary carbon metabolism of the high mountain plant Ranunculus glacialis. It is a species with C3 photosynthesis, but with exceptionally high malate content in its leaves, the biological significance of which remains unclear. 13C/12C-isotope ratio mass spectrometry (IRMS) and 13C-nuclear magnetic resonance (NMR) labelling were used to study the carbon metabolism of R. glacialis, paying special attention to respiration. Although leaf dark respiration was high, the temperature response had a Q10 of 2, and the respiratory quotient (CO2 produced divided by O2 consumed) was nearly 1, indicating that the respiratory pool is comprised of carbohydrates. Malate, which may be a large carbon substrate, was not respired. However, when CO2 fixed by photosynthesis was labelled, little labelling of the CO2 subsequently respired in the dark was detected, indicating that: (i) most of the carbon recently assimilated during photosynthesis is not respired in the dark; and (ii) the carbon used for respiration originates from (unlabelled) reserves. This is the first demonstration of such a low metabolic coupling of assimilated and respired carbon in leaves. The biological significance of the uncoupling between assimilation and respiration is discussed.     

2,3-Erythro-dihydroxyhexacosanoic acid and homologs: isolation from yeast cerebrin phosphate and determination of their structures

Homologs of methyl esters of very polar fatty acids were obtained by methanolysis of cerebrin phosphate isolated from baker's yeast. The major ester component was isolated by preparative gas-liquid chromatography and was found to be 2,3-dihydroxyhexacosanoic acid as deduced from the mass spectra of its trimethylsilyl ether and isopropylidene derivative, reaction with periodate, and comparison of its chromatographic behavior with that of synthetic erythro- and threo-dihydroxy acids. Its infrared spectrum supported the above conclusions. From their retention times by gas-liquid chromatography, the homologs were found to be saturated, unbranched 2,3-dihydroxy fatty acids with 24-27 carbon atoms. The synthesis of the new fatty acids, erythro- and threo-2,3-dihydroxyhexacosanoic acids, is also reported. A method for separating trans-2-hexacosenoic acid, a key intermediate of the above synthesis, and its isomer, trans-3-hexacosenoic acid, both formed by dehydrobromination of 2-bromohexacosanoic acid, is also described.     

Hydrogen shuttles in gills of water versus air breathing osteoglossids.

1. Using subcellular preparations of gills from Arapaima, an obligate air breather, and aruana, a related osteoglossid that is an obligate water breather, a comparison was made of the relative roles of the malate-aspartate cycle and the alpha-glycerophosphate (alpha-GP) cycle in transferring reducing equivalents from the cytosol to the mitochondria. 2. In aruana gill preparations, the alpha-GP cycle could be most clearly demonstrated by reconstructing it with purified isolated mitochondria, using the oxidation rate of exogenous NADH as a measure of the cycling activity. 3. Subcellular preparations of Arapaima gill, in contrast to the aruana gill, were not responsive to exogenous alpha-glycerophosphate, but a glutamate-malate stimulated O2 uptake was sensitive to aminooxyacetate, an aminotransferase inhibitor, a result that would be expected if the respiration were based on malate-aspartate cycling. 4. It was concluded that, compared to the alpha-glycerophosphate cycle, the malate-aspartate cycle was relatively more active in Arapaima gill than in aruana gill, and possible implications were discussed.     

Magnitude of ouabain-sensitive respiration of lamb hepatocytes (Ovis aries)

In lamb hepatocyte preparations with viabilities greater than 90%, ouabain-sensitive respiration accounted for approximately 50% of the total cellular O2 consumption. Lamb hepatocyte preparations with viability of less than 50% exhibited lower (P less than 0.05) total and ouabain-sensitive respiration. The decrease in ouabain-sensitive respiration in these preparations entirely accounted for the drop in total respiration.     

Crystallization and positional specificity of hydroperoxidation of Fusarium lipoxygenase.

A lipoxygenase obtained from the fungus Fusarium oxysporum was purified and crystallized. Using the purified enzyme, the positional specificity of linoleate peroxidation was studied. Linoleate hydroperoxides were converted into the corresponding trimethylsilyl derivative by reduction, catalytic hydrogenation and treatment with hexamethyldisilazane/trimethylchlorosilane/pyridine and then analyzed by combined gas-liquid chromatography-mass spectrometry. Fusarium lipoxygenase was found to produce 9- or 13-hydroperoxy-octadecadienoates from linoleate. The ratio of 9- to 13-hydroperoxides produced by the enzyme was also determined by high performance liquid chromatography of their methyl esters. When the enzymic reaction proceeded at pH 9.0 and 12.0, the ratio of 9- to 13-hydroperoxide isomers was 70 : 30 and 56 : 44, respectively. With the use of the heavy isotope of oxygen (18O2), atoms of oxygen introduced into hydroperoxides were found to be derived from the gaseous phase and not from the aqueous phase.     

Soil respiration, root biomass, and root turnover following long-term exposure of northern forests to elevated atmospheric CO2 and tropospheric O3

The Rhinelander free-air CO(2) enrichment (FACE) experiment is designed to understand ecosystem response to elevated atmospheric carbon dioxide (+CO(2)) and elevated tropospheric ozone (+O(3)). The objectives of this study were: to understand how soil respiration responded to the experimental treatments; to determine whether fine-root biomass was correlated to rates of soil respiration; and to measure rates of fine-root turnover in aspen (Populus tremuloides) forests and determine whether root turnover might be driving patterns in soil respiration. Soil respiration was measured, root biomass was determined, and estimates of root production, mortality and biomass turnover were made. Soil respiration was greatest in the +CO(2) and +CO(2) +O(3) treatments across all three plant communities. Soil respiration was correlated with increases in fine-root biomass. In the aspen community, annual fine-root production and mortality (g m(-2)) were positively affected by +O(3). After 10 yr of exposure, +CO(2) +O(3)-induced increases in belowground carbon allocation suggest that the positive effects of elevated CO(2) on belowground net primary productivity (NPP) may not be offset by negative effects of O(3). For the aspen community, fine-root biomass is actually stimulated by +O(3), and especially +CO(2) +O(3).     

Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass. Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration. Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration. During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier. As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased. Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2. Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2. Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1. We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates.     

Permafrost collapse alters soil carbon stocks,respiration, CH4, and N2O in upland tundra

Release of greenhouse gases from thawing permafrost is potentially the largest terrestrial feedback to climate change and one of the most likely to occur; however, estimates of its strength vary by a factor of thirty. Some of this uncertainty stems from abrupt thaw processes known as thermokarst (permafrost collapse due to ground ice melt), which alter controls on carbon and nitrogen cycling and expose organic matter from meters below the surface. Thermokarst may affect 20–50% of tundra uplands by the end of the century; however, little is known about the effect of different thermokarst morphologies on carbon and nitrogen release. We measured soil organic matter displacement, ecosystem respiration, and soil gas concentrations at 26 upland thermokarst features on the North Slope of Alaska. Features included the three most common upland thermokarst morphologies: active‐layer detachment slides, thermo‐erosion gullies, and retrogressive thaw slumps. We found that thermokarst morphology interacted with landscape parameters to determine both the initial displacement of organic matter and subsequent carbon and nitrogen cycling. The large proportion of ecosystem carbon exported off‐site by slumps and slides resulted in decreased ecosystem respiration postfailure, while gullies removed a smaller portion of ecosystem carbon but strongly increased respiration and N₂O concentration. Elevated N₂O in gully soils persisted through most of the growing season, indicating sustained nitrification and denitrification in disturbed soils, representing a potential noncarbon permafrost climate feedback. While upland thermokarst formation did not substantially alter redox conditions within features, it redistributed organic matter into both oxic and anoxic environments. Across morphologies, residual organic matter cover, and predisturbance respiration explained 83% of the variation in respiration response. Consistent differences between upland thermokarst types may contribute to the incorporation of this nonlinear process into projections of carbon and nitrogen release from degrading permafrost.     

The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559.

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.     

Glutaraldehyde fixation chemistry: oxygen-consuming reactions

In tissue fixed with glutaraldehyde, dissolved O2 is rapidly consumed by two processes: residual respiration and glutaraldehyde-induced chemical uptake. The nature of the chemistry which consumes O2 during tissue fixation was investigated by studying model reactions of glutaraldehyde with amines and with homogenized tissue suspensions. The addition of glutaraldehyde to solutions of most primary amines and ammonia stimulated rapid O2 consumption. The reaction of glutaraldehyde with primary amines (e.g., 25 mM ethanolamine, glycine, or methylamine) consumed 50% of the dissolved O2 in 15 to 20 s at 37 degrees C. The initial rate of O2 uptake followed second-order kinetics with respect to the primary amine concentration. The total amount of O2 consumed was sufficient to account for the stoichiometric conversion of the primary amines to pyridines. These data are consistent with the synthesis of pyridine derivatives from glutaraldehyde-amine precursors in which the last step is an irreversible oxidation of dihydropyridines to pyridines. The addition of glutaraldehyde to homogenized muscle suspensions, in which respiration was chemically inhibited, significantly increased the rate of O2 uptake. Thus, in tissue O2 is rapidly depleted both by respiration and the chemical demands of the glutaraldehyde-amine reactions during the cross-linking process. Since these experiments were done under conditions commonly used for tissue fixation, hypoxia should be assumed to exist in biological preparations fixed with glutaraldehyde.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

The 13C/12C isotopic signal of day-respired CO2 in variegated leaves of Pelargonium × hortorum

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.     

Identification of citrate as the albumin-bound inhibitor of the ferroxidase activity of ceruloplasmin

The influence of several commercial albumin preparations on the ferroxidase activity of ceruloplasmin (ferroxidase I, ferrous: O₂ oxidoreductase EC 1.16.3.1) at pH 6.0 was determined using ferric-transferrin formation. The ability of several albumin preparations to inhibit the ferroxidase activity of ceruloplasmin differs more than three hundredfold. It appears to depend on the method of isolation of albumin rather than the source of albumin, suggesting the existence of an inhibitor bound to albumin. The inhibitor was isolated after chromatography of an albumin preparation on Sephadex G-200. It was identified as citrate by thin layer chromatography and by comparison of the spectrum of the sulfide-pentabromoacetone derivative. Albumin preparations, even with bound citrate, do not exert a significant inhibitory effect at pH 7.4.