On the nature of the photosynthetic energy storage monitored by photoacoustic spectroscopy

The photosynthetic energy storage yield of uncoupled thylakoid membranes was monitored by photoacoustic spectroscopy at various measuring beam intensities. The energy storage rate as evaluated by the half-saturation measuring beam intensity (i₅₀) was inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea, by heat inactivation or by artificial electron acceptors specific for photosystem I or photosystem II; and was activated by electron donors to photosystem I. The reactions involving both photosystems were all characterized by a similar maximal energy storage yield of 16±2 percent. The data could be interpreted if we assumed that the energy storage elicited by the photosystems at 35 Hz is detected at the level of the plastoquinone pool.Abbreviations PS photosystem - Tes N-Tris [hydroxymethl] methyl-2-aminoethanesulfonic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - FeCN potassium ferricyanide - DCBQ 2,5-dichlorobenzoquinone - TMPD N,N,N-tetramethyl-p-phenilenediamine     

The Supramolecular Organization of Photosynthetic Membranes of the Thallus Stage of Porphyra leucosticta Thuret. (Bangiales,Rhodophyta) Visualized by Freeze-Fracture

The supramolecular organization of the thylakoid membranes of the thallus stage in the red alga Porphyra leucosticta is studied in replicas of rapidly frozen and fractured cells. Freeze-fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF), because the lamellae in red algal chloroplasts are not stacked. The PF reveals numerous, tightly packed, but randomly distributed particles (density range from 2970 to 3550 particles/μm²). In contrast, the EF particles appear organized into parallel rows, the spacing of which is about 60–70 nm (about 8–9 particles occur along 100 nm of the line that is formed). Significant numbers of single EF particles are randomly distributed between the EF particle rows. The particles on both fracture faces (PF and EF) fall into two size classes: 10 to 11 nm (major size class) and 14 to 15 nm (minor size class).     

Platelet calcium pump activity in essential hypertensives and their first-degree relatives

Intracellular free Ca²⁺ concentration has been shown to be elevated in platelets of patients with essential hypertension. This study was designed to characterize Ca²⁺-pump activity of the platelet membranes (surface and intracellular) in these patients. A double-blind study was carried out. Untreated and treated (on R-blockers) essential hypertensives were studied in comparison with normotensive control subjects. First degree blood relatives of essential hypertensives were also studied. The Ca²⁺-activation kinetics of the enzyme showed a significant decrease in the V_max. (for the plasma- and intracellular membranes) and Km (for the intracellular membranes) in the essential hypertensive patients. Increased platelet membrane cholesterol content was observed in these patients. Lowered Ca²⁺-efflux by Ca²⁺-ATPase may lead to elevated intracellular free Ca²⁺-levels in platelets of essential hypertensives. A lowered Ca²⁺-ATPase activity may emerge as a marker for essential hypertension.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

玉米根细胞中对钒酸盐敏感的H~＋－泵的研究

用奎吖咽（ｑｕｉｎａｃｒｉｎｅ）作荧光指标剂,测定玉米（ＺｅａｍａｙｓＬ．）根尖微粒体（ＭＩＣ）膜囊泡的Ｈ~＋－泵活性,结果表明１ｍｍｏｌ／ＬＮａＮ_３仅抑制该泵活性约８％,而０．８ｍｍｏｌ／Ｌ钒酸盐（Ｖａｎ）则可抑制其活性达８０％,说明ＭＩＣ制剂中Ｈ~＋－泵活性主要由质膜（ＰＭ）Ｈ~＋－ＡＴＰａｓｅ产生。此泵活性严格需要Ｍｇ~（２＋）,二价阳离子作用大小的顺序为Ｍｇ~（２＋）＞Ｍｎ~（２＋）＞Ｚｎ~（２＋）＞Ｃａ~（２＋）＝０;阴离子作用大小的１顺序为Ｂｒ~－＞Ｃｌ~－＞ＮＯ_３~－＞ＳＯ_４~（２－）,并初步证实当质膜同侧发生电子传递时,没有跨膜Ｈ~＋梯度（△μＨ~＋）生成。     

Hormone- and growth factor-stimulated NADH oxidase

An NADH oxidase activity of animal and plant plasma membrane is described that is stimulated by hormones and growth factors. In plasma membranes of cancer cells and tissues, the activity appears to be constitutively activated and no longer hormone responsive. With drugs that inhibit the activity, cells are unable to grow although growth inhibition may be more related to a failure of the cells to enlarge than to a direct inhibition of mitosis. The hormone-stimulated activity in plasma membranes of plants and the constitutively activated NADH oxidase in tumor cell plasma membranes is inhibited by thiol reagents whereas the basal activity is not. These findings point to a thiol involvement in the action of the activated form of the oxidase. NADH oxidase oxidation by Golgi apparatus of rat liver is inhibited by brefeldin A plus GDP. Brefeldin A is a macrolide antibiotic inhibitor of membrane trafficking. A model is presented where the NADH oxidase functions as a thiol-disulfide oxidoreductase activity involved in the formation and breakage of disulfide bonds. The thiol-disulfide interchange is postulated as being associated with physical membrane displacement as encountered in cell enlargement or in vesicle budding. The model, although speculative, does provide a basis for further experimentation to probe a potential function for this enzyme system which, under certain conditions, exhibits a hormone- and growth factor-stimulated oxidation of NADH.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes

A membrane preparation from rat brain catalyzed the hydrolysis of [2-³H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca²⁺ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca²⁺. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.Abbreviations PI phosphatidylinositol - lysoPI lysophosphatidylinositol - lysoPI-PLC lysophosphoinositide-specific phospholipase C - PI-PLC phosphoinositide-specific phospholipase C - MG monoacylglycerol - PLC phospholipase C To whom to address reprint requests.