Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Chloroplast membrane conformational changes measured by chemical modification

The chemical modification reagents iodoacetic acid (primarily sulfhydryl group directed) and acetic anhydride (primarily amino group directed) were used to monitor chloroplast thylakoid membrane conformational changes. The incorporation of [³H]-iodoacetate and [³H]acetic anhydride showed the following pattern: (i) There was an increased level of binding of iodoacetate in the light compared to the dark or light plus 2,4-dichlorophenyl-1,1-dimethyl urea (DCMU) conditions. A 30 to 50% increase, from about 1.0 to 1.3–1.5 nmol/mg of Chl in iodoacetate incorporation, was found; 30–50% less acetic anhydride was bound in the light than in the dark or light plus DCMU state, typical values being near 15 nmol of acetic anhydride bound/mg of Chl in the dark and 10 nmol/mg of Chl in the light, (ii) The incorporation pattern for both reagents indicated that Photosystem II-dependent proton release is required to elicit the differential binding. Evidence for this is: (a) Cyclic electron flow and proton accumulation, mediated by phenazine methosulfate in the presence of a Photosystem II inhibitor (DCMU), did not induce either the extra binding of iodoacetate or the decrease in binding of acetic anhydride; (b) in chloroplasts made deficient in water oxidation by NH₂OH treatment, electron flow from I^?, an alternate Photosystem II electron donor, to methyl viologen did not induce the differential binding, whereas with the proton-donating donor, diphenyl carbazide, Photosystem II electron flow did elicit the differential binding, (iii) Uncouplers of phosphorylation (nigericin plus valinomycin) had no affect on the differential binding of either reagent, consistent with the hypothesis that it is not simply a transmembrane proton gradient that potentiates the conformational change, but rather an intramembrane reaction between protons released by Photosystem II and certain membrane components. The lack of uncoupler effect also suggests that the conformational change does not involve the coupling factor complex, at least not in the same sense as for the coupling factor conformational changes detected by tritium exchange (I. J. Ryrie and A. T. Jagendorf, 1971, J. Biol. Chem.246, 582–588) or N-ethyl maleimide binding (R. E. McCarty et al., 1972, J. Biol. Chem.247, 3048–3051). (iv) The decrease in acetic anhydride binding in the light was independent of the structural state of the chloroplast. Stacked and unstacked (by low salt) grana membranes showed similar light-dependent decreases in acetic anhydride binding. The results with these modification reagents support earlier conclusions about a Photosystem II-linked conformational change based on work with diazonium benzenesulfonic acid (R. Giaquinta et al., 1975, Biochemistry14, 4392–4396).     

3-D modelling of chloroplast structure under (Mg) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.     

PROTONATION AND CHLOROPLAST MEMBRANE STRUCTURE

Light changes the structure of chloroplasts. This effect was investigated by high resolution electron microscopy, photometric methods, and chemical modification. (a) A reversible contraction of chloroplast membrane occurs upon illumination, dark titration with H⁺, or increasing osmolarity. These gross structural changes arise from a flattening of the thylakoids, with a corresponding decrease in the spacing between membranes. Microdensitometry showed that illumination or dark addition of H⁺ resulted in a 13–23% decrease in membrane thickness. Osmotically contracted chloroplasts do not show this effect. (b) Rapid glutaraldehyde fixation during actual experiments revealed that transmission changes are closely correlated with the spacing changes and therefore reflect an osmotic mechanism, whereas the light scattering changes have kinetics most similar to changes in membrane thickness or conformation. (c) Kinetic analysis of light scattering and transmission changes with the changes in fluorescence of anilinonaphthalene sulfonic acid bound to membranes revealed that fluorescence preceded light scattering or transmission changes. (d) It is concluded that the temporal sequence of events following illumination probably are protonation, changes in the environment within the membrane, change in membrane thickness, change in internal osmolarity accompanying ion movements with consequent collapse and flattening of thylakoid, change in the gross morphology of the inner chloroplast membrane system, and change in the gross morphology of whole chloroplasts.     

Evidence for light-dependent reversible conformational change in spinach chloroplast CF1

We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF₁) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg²⁺-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the K_m of CF₁ for both ADP and P_i. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF₁ may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF₁ which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination.     

Flash Inactivation of Oxygen Evolution: IDENTIFICATION OF S(2) AS THE TARGET OF INACTIVATION BY TRIS

Brief saturating light flashes were used to probe the mechanism of inactivation of O₂ evolution by Tris in chloroplasts. Maximum inactivation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting O₂-evolving catalyst of photosystem II (S₂-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequilibration caused a three-flash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S₂-states were similar; and (c) reagents accelerating S₂ deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II_s, as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH₃ was shown to be a competitive inhibitor of the Tris-induced inactivation. S₂-dependent inactivation was inhibited by glutaraldehyde fixation of chloroplasts, possibly suggesting that inactivation proceeds via conformational changes of the S₂-state.     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.     

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.     

ACTION OF TRITON X-100 ON CHLOROPLAST MEMBRANES : Mechanisms of Structural and Functional Disruption

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100–200 µM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

Effects of cations upon chloroplast membrane subunit interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under “low salt” conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll · protein complex (chlorophyll $\text{a}\text{b}$ ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll $\text{a}\text{b}$ protein and which acts as a light-harvesting antenna primarily for Photosystem II.Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll b and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment · protein, the combined complexes pellet as a “heavy” membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a “light” submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation.Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment · protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H⁺-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H⁺-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H⁺ membrane permeability on the Δψ buildup was demonstrated by manipulating the H⁺ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H⁺-ATPase-mediated Δψ buildup.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion

Background

Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50–90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP_1,2) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP_1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP_1,2 trimeric, viral surface spike. After entry into host endosomes, GP_1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.

Principal Findings

We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP_1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP_1,2. Further, ELISA binding data of primed GP_1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.

Significance

The results reveal that the ebolavirus GP_1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP_1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.     

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.

Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.

These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

     

Chloroplast maintenance and partial differentiation in vitro

Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control.     

Further Studies on Factors Affecting the Efflux of Betacyanin from Beet Root: A Note on Thermal Effects

As judged by betacyanin efflux, beet root tissue differs in stability toward O₂ at low and high temperatures (45–60°C and 60–100°C respectively). The effect of temperature itself can he divided into a high activation energy (93 Kcal mol^?1) process in the lower temperature range and a low activation energy process (19 Kcal mor^?1) in the higher range (> 60°C). From these data it is suggested that initially, elevating temperatures bring about reversible conformational changes in the membrane. With continuing increase in temperature in the presence of O₂, membrane chemical groups susceptible to oxidation are exposed and, upon oxidation, render conformational changes irreversible.     

Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

Sequential bleaching in the presence of hydroxylamine and subsequent regeneration of the purple membrane of Halobacterium halobium was studied by concomitant monitoring of its absorption and circular dichroic spectra in order to ascertain its effects on protein interaction(s) (which may result in possible excitonic interaction between the retinal chromophores), chromophore-apoprotein interaction(s), and protein conformational stability in the membrane. It was concluded that (a) although experimental results are consistent with an exciton mechanism for the interaction between retinal π - π* (NV₁) transition movements in the purple membrane, no evidence for such a mechanism for interaction between retinaloxime transition moments is apparent in the case of the bleached membrane; (b) the bacteriorhodopsin molecules organized in clusters of three in the membrane appear to bleach simultaneously; (c) the retinaloxime produced on bleaching the purple membrane in the presence of hydroxylamine is strongly optically active, because of dissymmetry-inducing and/or -selecting constraints on the chromophore by a component of the membrane (most likely the apoprotein), and when the membrane is regenerated by the addition of retinal, these constraints are lost; and (d) evidence from ultraviolet absorption and circular dichroic spectra suggests that the membrane apoprotein undergoes appreciable conformational changes involving tertiary structure on bleaching with no significant secondary structure involvement. These results are compared with recently reported results from this laboratory on the effects of bleaching on the bovine rod outer segment disk membrane structure.     

Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol, and mitochondria

A technique is presented for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the mitochondrial matrix of wheat (Triticum aestivum, var `Timmo') leaf protoplasts, in which membrane filtration is used to prepare fractions enriched in the different subcellular fractions within 0.1 seconds after disruption of the protoplasts. By closing a syringe, protoplasts are forced through a net and disrupted, diluting the cytosol into the medium and also releasing intact chloroplasts and mitochondria which can then be immediately removed on membrane filters placed behind the nylon net. By varying the membrane filters, different filtrates are obtained corresponding to (a) mainly cytosol, or (b) cytosol and mitochondria with only low levels of chloroplasts; alternatively, (c) the entire protoplast contents are obtained by omitting the filters. The filtrates are immediately split, half flowing into HClO₄ where they are immediately quenched for subsequent metabolite analyses; the other half flows into detergent and is used to monitor the exact distribution of marker enzymes in each individual fractionation. Using the measured distributions of metabolite and of marker enzymes in the three filtrates, the subcellular distribution of the metabolite can be algebraically calculated. The method is presented using ATP as an example.

The quench time (0.1 second) made possible by membrane filtration is considerably faster than has been possible in the previously developed techniques using silicone oil centrifugation for chloroplasts (1 second) or mitochondria (1 minute). This rapid quench makes it possible to investigate subcellular pools which have a rapid turnover, like the adenine nucleotides.