Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

Carbon metabolism and gas exchange in leaves of Zea mays L.

The aim of this work was to investigate the mechanism of formation of triose phosphates and 3-phosphoglycerate during photosynthetic induction in leaves of Zea mays. Simultaneous measurements of gas exchange, chlorophyll a fluorescence and metabolite contents of maize leaves were made. Leaves illuminated in the absence of CO₂ showed a build-up of triose phosphates during the first 2 min of illumination which was comparable to the build-up observed in the presence of CO₂. Isolated mesophyll protoplasts, which lack the Calvin cycle, also showed a build-up of triose phosphates upon illumination. Leaves contained amounts of phosphoglycerate mutase and enolase adequate to account for the formation of triose phosphates and 3-phosphoglycerate from intermediates of the C₄ cycle and their precursors.     

3-Phosphoglycerate Phosphatase in Plants: II. Distribution, Physiological Considerations, and Comparison with P-Glycolate Phosphatase

3-Phosphoglycerate phosphatase and phosphoglycolate phosphatase were found in leaves of all 52 plants examined. Activities of both phosphatases varied widely between 1 to 20 micromoles per minute per milligram chlorophyll. Plants were grouped into two categories based upon the relative ratio of activity of 3-phosphoglycerate phosphatase to phosphoglycolate phosphatase. This ratio varied between 2:1 to 4:1 in the C₄-plants except corn leaves which had a low level of 3-phosphoglycerate phosphatase. This ratio was reversed and varied between 1:2 to 1:6 in all C₃-plants except one bean variety which had large amounts of both phosphatases. By differential grinding procedures for C₄ plants a major part of the 3-phosphoglycerate phosphatase was found in the mesophyll cells and P-glycolate phosphatase in the bundle sheath cells. Phosphoglycolate phosphatase, but not 3-phosphoglycerate phosphatase, was located in chloroplasts of C₃- and C₄- plants. Formation of 3-phosphoglycerate phosphatase increased 4- to 12-fold during greening of etiolated sugarcane leaves. This cytosol phosphatase displayed a diurnal variation in sugarcane leaves by increasing 50% during late daylight hours and early evening. It is proposed that the soluble form of 3-phosphoglycerate phosphatase is necessary for carbon transport between the bundle sheath and mesophyll cells during photosynthesis by C₄-plants. In C₃- and C₄-plants this phosphatase initiates the conversion of 3-phosphoglycerate to serine which is an alternate metabolic pathway to glycolate metabolism and photorespiration.     

Carbon assimilation pathways in sulfate reducing bacteria. Formate,carbon dioxide,carbon monoxide,and acetate assimilation by Desulfovibrio baarsii

Desulfovibrio baarsii is a sulfate reducing bacterium, which can grown on formate plus sulfate as sole energy source and formate and CO₂ as sole carbon sources. It is shown by ¹⁴C labelling studies that more than 60% of the cell carbon is derived from CO₂ and the rest from formate. The cells thus grow autotrophically. Labelling studies with [¹⁴C]acetate, ¹⁴CO and [¹⁴C]formate indicate that CO₂ fixation does not proceed via the Calvin cycle. The labelling patterns of alanine, aspartate, glutamate, and glucosamine indicate that acetate (or activated acetic acid) is an early intermediate in formate and CO₂ assimilation; the methyl group of acetate is derived from formate, and the carboxyl group from CO₂ via CO; pyruvate is formed from acetyl-CoA by reductive carboxylation. The capacity to synthesize an acetate unit from two C₁-compounds obviously distinguishes D. baarsii from those Desulfovibrio species, which require acetate as a carbon source in addition to CO₂.     

Anaerobic Degradation of Uric Acid by Gut Bacteria of Termites

A study was done of anaerobic degradation of uric acid (UA) by representative strains of uricolytic bacteria isolated from guts of Reticulitermes flavipes termites. Streptococcus strain UAD-1 degraded UA incompletely, secreting a fluorescent compound into the medium, unless formate (or a formicogenic compound) was present as a cosubstrate. Formate functioned as a reductant, and its oxidation to CO₂ by formate dehydrogenase provided 2H⁺ + 2e⁻ needed to drive uricolysis to completion. Uricolysis by Streptococcus UAD-1 thus corresponded to the following equation: 1UA + 1formate → 4CO₂ + 1acetate + 4NH₃. Urea did not appear to be an intermediate in CO₂ and NH₃ formation during uricolysis by strain UAD-1. Formate dehydrogenase and uricolytic activities of strain UAD-1 were inducible by growth of cells on UA. Bacteroides termitidis strain UAD-50 degraded UA as follows: 1UA → 3.5 CO₂ + 0.75acetate + 4NH₃. Exogenous formate was neither required for nor stimulatory to uricolysis by strain UAD-50. Studies of UA catabolism by Citrobacter strains were limited, because only small amounts of UA were metabolized by cells in liquid medium. Uricolytic activity of such bacteria in situ could be important to the carbon, nitrogen, and energy economy of R. flavipes.     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

The interconversion of glycine and serine by plant tissue extracts

1. Extracts prepared from a variety of higher-plant tissues by ammonium sulphate fractionation were shown to catalyse the interconversion of glycine and serine. This interconversion had an absolute requirement for tetrahydrofolate and appeared to favour serine formation. 2. The biosynthesis of serine from glycine was studied in more detail with protein fractionated from 15-day-old wheat leaves. Synthesis of [¹⁴C]serine from [¹⁴C]glycine was not accompanied by labelling of glyoxylate, glycollate or formate. 3. The synthesis of serine from glycine was stimulated by additions of formaldehyde, and [¹⁴C]formaldehyde was readily incorporated into C-3 of serine in the presence of tetrahydrofolate. 4. The results are interpreted as indicating that serine biosynthesis involves a direct cleavage of glycine whereby the α-carbon is transferred via N⁵N¹⁰-methylenetetrahydrofolate to become the β-carbon of serine.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

Generation and maintenance of concentration gradients between the mesophyll and bundle sheath in maize leaves

(1) Experiments have been carried out to test the proposal that intercellular transport of carbon occurs by diffusion during photosynthesis in C-4 plants. (2) The intercellular distribution of metabolites has been compared in different conditions. A partial separation of the mesophyll and bundle sheath was obtained by homogenisation in liquid N₂, followed by filtration through nylon nets with differing aperture. (3) Concentration gradients between the bundle sheath and mesophyll were found for 3-phosphoglycerate, triose phosphates, malate and pyruvate during photosynthesis. These gradients are shown to be large enough to allow rapid intercellular transport by diffusion. They disappear when photosynthesis is prevented by removal of light or CO₂. (4) The concentration gradients for triose phosphates and 3-phosphoglycerate are due to the differing capacity of the bundle sheath and mesophyll to reduce 3-phosphoglycerate. (5) The distribution of carbon between the malate/pyruvate and 3-phosphoglycerate/triose phosphate shuttles is flexible, and may be controlled by phosphoenolpyruvate carboxylase. (6) The maintenance of these large concentration gradients has consequences for the regulation of sucrose synthesis and the Calvin cycle.     

Plastidic Isoprenoid Synthesis during Chloroplast Development : Change from Metabolic Autonomy to a Division-of-Labor Stage

The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO₂ → pyruvate → acetyl-coenzyme A (C₃ → C₂) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C₃ → C₂ metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO₂-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH¹⁴CO₃ decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A.     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : II. THE ACTION OF THE CHLORIDES OF LEAD,MERCURY, COPPER,IRON, AND ALUMINUM ON THE PROTOPLASM OF AM?BA PROTEUS.

I. Plasmalemma. 1. The order of toxicity of the salts used in these experiments on the surface membrane of a cell, taking as a criterion viability of amebæ immersed in solutions for 1 day, is HgCl₂, FeCl₃> AlCl₃> CuCl₂> PbCl₂> FeCl₂. Using viability for 5 days as a criterion, the order of toxicity is PbCl₂> CuCl₂> HgCl₂> AlCl₃> FeCl₃> FeCl₂. 2. The rate of toxicity is in the order FeCl₃> HgCl₂> AlCl₃> FeCl₂> CuCl₂> PbCl₂. 3. The ability of amebæ to recover from a marked tear of the plasmalemma in the solutions of the salts occurred in the following order: AlCl₃> PbCl₂> FeCl₂> CuCl₂> FeCl₃> HgCl₂. II. Internal Protoplasm. 4. The relative toxicity of the salts on the internal protoplasm, judged by the recovery of the amebæ from large injections and the range over which these salts can cause coagulation of the internal protoplasm, is in the following order: PbCl₂> CuCl₂> FeCl₃> HgCl₂> FeCl₂> AlCl₃. 5. AlCl₃ in concentrations between M/32 and M/250 causes a marked temporary enlargement of the contractile vacuole. FeCl₂, FeCl₃, and CuCl₃ produce a slight enlargement of the vacuole. 6. PbCl₂, in concentrations used in these experiments, appears to form a different type of combination with the internal protoplasm than do the other salts. III. Permeability. 7. Using the similarity in appearance of the internal protoplasm after injection and after immersion to indicate that the surface is permeable to a substance in which the ameba is immersed, it is concluded that AlCl₃ can easily penetrate the intact plasmalemma. CuCl₂ also seems to have some penetrating power. None of the other salts studied give visible internal evidence of penetrability into the ameba. IV. Toxicity. 8. The toxic action of the chlorides of the heavy metals used in these experiments, and of aluminum, is exerted principally upon the surface of the cell and is due not only to the action of the metal cation but also to acid which is produced by hydrolysis.     

Chlorophyll Formation in Greening Bean Leaves during the Early Stages

In the evolution of the absorption spectrum of etiolated bean leaves (Phaseolus vulgaris L.) following illumination, a rapid photoconversion of 50% or more of the active protochlorophyllide at room temperature is followed by a shift of the chlorophyll(ide) absorption maximum: C₆₇₈→ →C₆₈₄→C_{672 nm}. Kinetic studies at 2 C and the absence of an isosbestic point provide evidence for an intermediate between C₆₇₈ and C₆₈₄. A dramatically different evolution is observed following the photoconversion of only 5 to 30% of the active protochlorophyllide at room temperature. C₆₇₂ appears within 30 seconds, and no subsequent dark shift occurs during the following 90 minutes. At 0 C, conversion of 5% of the active protochlorophyllide produces a new species, C₆₇₆, which converts progressively to C₆₇₂ within 10 minutes. We interpret the results in terms of two photochemical steps operating in series for the complete conversion of active protochlorophyllide. Furthermore, there appears to be competition between an irreversible, terminal dark shift and the second light reaction. We propose a scheme based on dimers of protochlorophyllide reduced stepwise to dimers of chlorophyllide in two successive light reactions. The intermediate mixed protochlorophyllide-chlorophyllide dimer absorbs at 676 nm and displays a much faster dissociation to monomers than does the chlorophyllide-chlorophyllide dimer.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Photosynthesis in the Higher Plant Vicia faba: V. Role of Malate as a Precursor of the Tricarboxylic Acid Cycle

In the higher plant Vicia faba, anomalous labeling patterns in the organic acids and related amino acids of the tricarboxylic acid cycle which result from photosynthetic ¹⁴CO₂ fixation (in conjunction with an enzyme localization pattern unique to plant mitochondria) suggest that the tricarboxylic acid cycle functions primarily as a pathway leading to glutamic acid biosynthesis during autotrophic growth. The distribution of isotope in citrate indicates little recycling of oxaloacetate for the resynthesis of citrate. Rather, malate appears to provide both the C₂ and C₄ fragments for the synthesis of citrate, and [³H]formate and ¹⁴CO₂-labeling patterns implicate serine as the ultimate C₃ precursor of malate.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Stimulation,Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E₁-E₂ conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E₂, but their detailed mechanisms of action are different. PE accelerates the rate of E₁ → E₂P but does not affect E₂(2K)ATP → E₁3NaATP, whereas sphingomyelin inhibits the rate of E₂(2K)ATP → E₁3NaATP, with very little effect on E₁ → E₂P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.     

Bacteriorhodopsin wildtype and variant aspartate-96 → aspargine as reversible holographic media

Air dried films of purple membranes (PM) from Halobacterium halobium containing the photochromic protein bacteriorhodopsin (BR) were prepared and the BR-photocycle of this material analyzed. The absorption maxima of the initial state B (λ_max = 570 nm) and the photochemical intermediate M (λ_max = 412 nm), which is the longest living intermediate in suspension (τ ≈ 10 ms), were spectrally well separated. Light-induced population gratings between B and M were used for reversible holographic recording in these dry PM films. The resolution (>5,000 lines/mm) of PM films was comparable to the corresponding values of conventional photochromic recording materials. The longterm stability toward photochemical degradation of PM films is excellent (> 100.000 recording cycles). The spectral bandwidth (400-680 nm) of such films covers nearly the whole visible spectrum. Both the photochemical transition from B → M with wavelengths in the green-red range and from M → B with blue light were utilized for holographic recording. The latter possibility (M → B) seems to be advantageous for several applications because the holographic grating is only formed during reconstruction. Higher reading intensities lead to higher population of the M-state and result in an increase of the fringe contrast instead of decreasing it. New possibilities for the further development of holographic media based on bacteriorhodopsin are raised by the availability of PM variants with modified optical properties. By the use of the variant BR-326, which differs from the wildtype PM by a single amino acid exchange (aspartate-96 → asparagine), the sensitivity of PM films is increased by ~50% from 12 cm²/J to 19 cm²/J for recording with 568 nm. The sensitivity for recording with 413 nm (33 cm²/J) is not influenced by the amino acid exchange. The observed diffraction efficiency η of PM films with BR-326 is twice that of BR-wildtype (BR-WT) films and is in the range of conventional organic photochromics (≈ 1%). In dried films of both BR-WT and BR-326 the M-decay was shown to be at least biexponential.