Claes Weibull

Separation and characterization of stroma and grana membranes: evidence for heterogeneity in antenna size of both photosystem I and photosystem II

A rapid procedure to fractionate the thylakoid membrane into two well-separated vesicle populations, one originating from the grana and the other from the stroma-membrane region, has been developed. This was achieved by sonication of thylakoids present in an aqueous two-phase system followed by partitioning either by countercurrent distribution or by a batch procedure in three steps. The membrane populations were analysed according to their composition and photochemical activities. The grana membranes comprise, on chlorophyll basis, about 60% of the thylakoid material and are enriched in PS II, but also contain some PS I, while the stroma membranes comprise about 40% and are enriched in PS I, but also contain some PS II. Cytochrome f was slightly enriched in the grana-derived vesicle fraction. The properties of both PS I and PS II differ between the two populations. The PS I of the grana fraction (PS Iα) reached half-saturation at about half the light intensity of the PS I in the stroma-membrane fraction (PS Iβ). The rate of P-700 photooxidation under low light illumination was higher for PS Iα than for PS Iβ (30% larger rate constant), showing that PS Iα has a larger antenna. The PS II of the grana fraction (PS IIα) reached half-saturation at half the light intensity compared to the PS II of the stroma-membrane fraction (PS IIβ). The results show that the grana-derived membranes contain PS Iα and PS IIα which have larger functional antenna sizes than the corresponding PS Iβ and PS IIβ of the stroma membranes. The results suggest that the photosystems of the grana are designed to allow effective electron transport both at low and high light intensities, while the stroma-membrane photosystems mainly work at high light intensities as a supplement to the grana systems.

Extraction of membrane lipids during fixation, dehydration and embedding of Acholeplasma laidlawii-cells for electron microscopy

The aim of the present investigation was to study the extent to which lipids are extracted from biological membranes during dehydration and embedding procedures carried out at high or low temperatures. Cells of Acholeplasma laidlawii were used as experimental material, since the lipids of this bacterium easily can be radioactively labelled without labelling the rest of the cell, and the lipids are almost entirely located in the cytoplasmic membrane. The cells were fixed at 277 K with glutaraldehyde, sequentially with this reagent and osmium tetroxide, or with glutaraldehyde, osmium tetroxide and uranyl acetate in that order. Loss of lipid during these procedures was negligible.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy

The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde‐fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction.

Extraction of lipids during freeze-substitution of Acholeplasma laidlawii-cells for electron microscopy

Cells of the bacterium Acholeplasma laidlawii were rapidly frozen against a copper block cooled by liquid helium. The frozen cells were transferred to liquid nitrogen and subsequently to acetone or methanol at 183 K. After 24 h the cells were infiltrated at 203 K with a non‐polar methacrylate resin of the same type as Lowicryl HM20. The resin was cured at the same temperature. Acetone extracted approximately 5% of the lipid content of the cells, methanol 15–45% and the resin only negligible amounts. Similar results were obtained with A. laidlawii‐ghosts. The cells appeared well preserved when examined in the electron microscope.

Further characterization of the chloroplast grana margins: the non-detergent preparation of granal Photosystem I cannot reduce ferredoxin in the absence of NADP+ reduction

The chloroplast grana margins of spinach thylakoids were isolated by sonication and aqueous-two-phase partitioning and their electron transport properties examined. Photosystem II and I electron transport activities were measured and compared to the appressed and non-appressed grana core and stroma lamellae, respectively, as well as to whole thylakoids. The results show that the PS II complexes in the grana margins are of the PS II subtype with respect to antenna size, but are QB reducing with respect to the acceptor side properties, while the PS I centers in the grana margins have slightly larger antennae as compared to the PS I centers in the stroma lamellae and are more like the PS I centers located in the grana domain. The ability to reduce ferredoxin and NADP+ was also tested and it was found that the grana margin membrane fraction was unable to reduce ferredoxin, even in the presence of added artificial electron donors. The stroma lamellae and whole thylakoid fractions both reduced ferredoxin at high rates. However, the grana margins could catalyze the reduction of NADP+ when supplied with the necessary components (ferredoxin, ferredoxin:NADP+ oxidoreductase, and an electron source). The results suggest that the PS I populations located in the margins of the grana domain are functionally different from the PS I centers located in the stroma lamellae. The data support a model whereby the PS I centers in the grana are primarily involved in non-cyclic electron transport, while the PS I centers located in the stroma lamellae are capable of participating in both cyclic and non-cyclic electron transport. (Less)

Fractionation of membranes from Acholeplasma laidlawii A on the basis of their surface properties by partition in two-polymer aqueous phase systems

Acholeplasma laidlawii A consists of pleomorphic cell clusters surrounded by a single membrane. When lysed, a cell gives rise to several membrane fragments which cannot be separated from each other by isopycnic sucrose gradient centrifugation. A heterogeneous lateral organization of the cell membranes was detected by countercurrent distribution of membrane fragments in a two-polymer aqueous phase system. It revealed that the membranes consist of at least two subpopulations with respect to surface properties. Changes in the fatty acid and cholesterol content of the membranes revealed that the resolution of different subpopulations was predominantly due to a critical ratio of monoglucosyldiglyceride to diglucosyldiglyceride. The heterogeneity of the membrane probably depends on lipid-lipid and lipid-protein steric interactions. Charged lipids, an apolar monoglucolipid and the ratio between lipids and proteins also affect membrane partition. The differences in the subpopulations were further reflected by different specific activities of NADH dehydrogenase, NADH oxidase and ATPase. These activities varied independently. Minor quantitative differences in the protein patterns of different subpopulations were apparent. The origin and the preservation of the membrane subpopulations are discussed in terms of lipid-lipid and lipid-protein interactions, their age and energy metabolism.

Low-temperature embedding procedures applied to chloroplasts

Pea and spinach chloroplasts were fixed with glutaraldehyde and dehydrated with ethylene glycol at 0°C. In some experiments dehydration was achieved by the resin itself. The material was infiltrated at temperatures between −35 and 0°C with mixtures of hydroxypropylmethacrylate and butylmethacrylate or a mixture of methacrylates and acrylates containing crosslinking agents. Polymerization was achieved, at the same temperature as the infiltration, using uv light and benzoin methyl ether as the catalyst. In uranyl acetate stained sections of specimens embedded at −35°C the thylakoid membranes appeared light between the strongly and evenly stained partition gaps. Dark spots of various shapes appeared in the middle of each thylakoid intercalated at irregular intervals by closely apposed membrane segments. In specimens embedded at −18 or 0°C the dark spots were replaced by a continuous dark layer identical to the partition gaps. The patterns seen in sections of embedded chloroplasts also appeared in specimens prepared by cryoultramicrotomy. The structural patterns described deviate markedly from those obtained when a conventional procedure is applied to the material mentioned.

Fractionation of the thylakoid membrane from Dunaliella salina – heterogeneity is found in Photosystem I over a broad range of growth irradiance

Thylakoids from the green alga, Dunaliella salina, were fragmented by sonication and the appressed grasna membranes separated from stroma lamellae by partitioning in aqueous two-phase systems. The concentration and antenna size of Photosystem I in the two membrane domains were determined for cultures grown at three different light intensities. Although the antenna size of both PS is decreased with increasing growth irradiance, the antenna size of Photosystem I in the grana was approximately 25-30% greater than the antenna size of Photosystem I in the stroma lamellae. Counter-current distribution analysis of sonicated thylakoids revealed that the amount of stroma lamellae increased whereas the amount of the stacked membranes decreased at higher growth irradiance. The overall decrease in the antenna size of PS I, in D. salina, at higher light intensities can therefore be explained by the combined effect of a decrease in the antenna size of Photosystem I, both in the grana and the stroma lamellae, and a relative increase in the amount of stroma lamellae which has smaller Photosystem I antennae than the Photosystem I centers found in the grana. Light-induced protein phosphorylation increased the relative amount of the stroma lamella fraction. This is interpreted as a result of partial unstacking of the grana. It is suggested that this may be a mechanism for increasing the cyclic electron transport around Photosystem I. (Less)

Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL)

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X‐ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 Å, with α = 82°, β = 95° and γ = 107°. The space group is P1 and the crystals diffract to a maximum of 7 Å when using CuKα X‐rays from a rotating anode. Both electron microscopy and non‐denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The V M value for two cpn60 molecules per unit cell is 3.5 Å3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

Temperature rise in lowicryl resins during polymerization by ultraviolet light

The temperature rise during polymerization of the resins Lowicryl K4M, K11M, HM20, and HM23 by uv light was studied. Polymerization was performed in 1.7-ml (in a few experiments 0.7-ml) polyethene tubes equipped with a lid. The temperature rise was considerable at room temperature and at 5°C when the tubes were kept in air, but slight (2–4°C) when they were immersed in a water bath. At −35°C and below, the temprature rise was less than 2°C in samples that were surrounded by air during the polymerization. When such samples were allowed to warm up to 20°C a small, transient rise (about 5°C) of the resin temperature above ambient occurred.