Morphological and Photosynthetic Properties of Digitonin-treated Chloroplast Membranes from the Wild-type and ac-5 Strains of Chlamydomonas reinhardi

Chloroplast membranes of wild-type Chlamydomonas reinhardi, treated with digitonin, yield photosystem II-rich and photosystem I-rich fractions; this fractionation is accompanied by a separation of stacked (grana) lamella from unstacked (stroma) lamellae. Poor fractionation of the photosystems occurs when the treated chloroplast membranes derive from the ac-5 strain grown mixotrophically, whereas good fractionation occurs with ac-5 cells grown phototrophically; the mixotrophic cells possess only unstacked membranes, whereas the phototrophic cells possess stacked membranes. We concluded that digitonin fractionation is dependent on the stacked membrane configuration.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Conversion of everted thylakoids into vesicles of normal sidedness exposing the outer grana partition membrane surface

Inside-out spinach thylakoid vesicles can be isolated by aqueous polymer two-phase partition following mechanical disruption of spinach chloroplast lamellae (Andersson, B and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472) and a mechanism for their formation has been experimentally supported (Andersson B., Sundby, C. and Albertsson, P.-Å. (1980) Biochim. Biophys. Acta 599, 391–402). Upon disruption, inside-out vesicles may form under stacking conditions, e.g., in 5 mM MgCl₂ or 150 mM NaCl, while disruption under destacking conditions, i.e., low concentrations of monovalent cations, gives only right-side-out vesicles. This study deals with the sidedness stability of the isolated inside-out thylakoid vesicles when stored or disrupted by sonication in various ionic environments. The sidedness of thylakoid vesicles was determined by their partition behaviour in an aqueous polymer phase system, direction of proton translocation and aggregation response (stacking) upon addition of MgCl₂. The results show that no spontaneous change from everted to normal sidedness occurs upon storage of the inside-out thylakoids. In contrast, sonication of these vesicles under destacking conditions (5 mM NaCl) results in a nearly complete transformation to right-side-out orientation. Also, in the presence of 5 mM MgCl₂ or 150 mM NaCl, sonication induced a change in sidedness of the inside-out vesicles but to a lesser extent. The stabilizing effect on the everted sidedness by cations was shown to be a result of preventing vesicle fragmentation by maintaining internal thylakoid appresions rather than by influencing the membrane curvature during resealing. Once released from an appressed state by overcoming the stacking forces, an opened thylakoid membrane shows an absolute preference for turning right-side-out in all media tested. These results strongly support the proposed formation mechanism, in which pairs of neighbouring grana membranes after disruption reseal with each other promoted by their close proximity. Since the inside-out vesicles derive from the grana appressions, their transformation back to normal sidedness exposes the outer membrane surface of appressed thylakoids. This region of the thylakoid membrane is normally hidden in the grana appressions and removal of grana leads concomitantly to lateral intermixing with non-appressed thylakoid components. Thus the current isolation of right-sided vesicles derived from the grana appressions should be a new tool for studies on the molecular organization of the thylakoid membrane.     

Control of puffing activity in three chromosomal segments of explanted salivary gland cells of Chironomus thummi by variation in extracellular Na+,K+ and Mg2+

Salivary glands of Chironomus thummi larvae were incubated in media composed of those $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{or}\text{MgCl}{}_2\text{KCl}$ combinations and at concentrations which they tolerated without visible damage. Resulting changes in puffing activity were recorded for three chromosomal segments. Within certain combinatorial ranges $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{and}\text{MgCl}{}_2\text{KCl}$ induced puffs in the three segments. Each inducing range is depicted as a ‘puff-inducing field’ for extracellular ion concentrations (IF^e) in a two-dimensional lattice. The IF^e_s are coherent, distinctly delineated and highly overlapping. At most places a transition from 0 to 100 % puff induction (induction of respective puff in each nucleus) depends on changes in media composition of 10–20 mM. Ion sensitivities of the three chromosomal segments were computed for NaCl/KCl/MgCl₂ combinations and were found to conform to actual puff-inducing capacities of selected NaCl/KCl/MgCl₂ media.     

The stacking of chloroplast thylakoids: Evidence for segregation of charged groups into nonstacked regions

Small particles derived from the digitonin treatment of chloroplast thylakoid membranes in either the stacked (grana-containing) or unstacked condition, as determined by cation concentration, have been used to study the aggregation of thylakoid membranes. At pH values above 5, the small particles from stacked chloroplasts do not aggregate in the presence of Mg²⁺ or other screening cations at concentrations sufficient to cause the restacking of thylakoids from low-salt chloroplasts. However, the small particles from stacked chloroplasts are aggregated either by lowering the pH to 4.6 or adding the binding cation La³⁺. In contrast, the small particles obtained on digitonin treatment of unstacked chloroplasts were aggregated by cations at neutral pH. Large particles (mainly grana) derived from digitonin treatment of stacked chloroplasts could not be unstacked by transfer to media of low cation concentration. It is concluded that the nonappressed regions of the chloroplast thylakoid membranes under stacking conditions carry higher than average negative surface charge densities under physiological pH conditions. Transfer of chloroplasts to media of low cation concentration causes a time-dependent lateral redistribution of charge between the appressed and nonappressed regions, but this redistribution is prevented by prior digitonin treatment of stacked chloroplasts.     

Arylsulphatases in human brain: assay, some properties, and distribution

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in human brain were studied using a highly sensitive fluorometric technique based on the use of 4-methyl-umbelliferone sulphate (MUS) as substrate. In the dialysed homogenate of human brain at least two enzymes could be distinguished on the basis of pH optima and substrate concentration. One MUS-sulphatase, of the ‘insoluble’ type, exhibited a pH optimum of 6–9 and an apparent K_m of 0.05 mM, whereas the second, belonging to the ‘soluble’ type, exhibited pH optimum of 6–0 and an apparent K_m of 6.25 mM. Pronounced activities of the two arylsulphatases were observed in the 18,000 g sediment. About 25 per cent of the total tissue activity of the ‘soluble’-type MUS-sulphatase was found in the soluble subcellular fraction. However, this enzyme was completely solubilized by extraction of acetone-dried human brain with acetate buffer.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Uptake of [3H]ouabain from the cell surface into the lysosomal compartment of HeLa cells

[3H]Ouabain specifically bound at sublethal concentrations to Na,K-ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5'-nucleotidase is found in both conventional sucrose-gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker beta-hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and beta-hexosaminidase to be codistributed in a particulate fraction that is homogeneous with respect to shear; extrapolation to zero-shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half-time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell-associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na,K-ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not "age."     

The endoplasmic reticulum of mung-bean cotyledons

Germination and seedling growth of mungbean (Vigna radiata (L.) Wilczek) are accompanied by the incorporation of radioactive amino acids, glycerol, galactose, and glucosamine in an organelle fraction of the cotyledons which co-equilibrates with NADH-cytochrome-c-reductase activity at 1.13 g·cm^–3 on isopycnic gradients containing 1 mM EDTA. Up to 20% of the newly synthesized proteins accumulate in this organelle fraction. The organelle fraction has been identified as rough endoplasmic reticulum (ER) on the basis of its increased density (1.16 g·cm^–3) when 3 mM MgCl₂ is included in all media. Seedling growth is also accompanied by a marked rise (more than 5-fold) in ER-associated NADH- and NADPH-cytochrome-c-reductase activity, and by the incorporation of⁵⁹Fe into ER-associated heme. Other manifestations of the reorganization of the ER in the cotyledons include a relative increase in membrane-associated RNA (from 12% of total RNA after 12 h of imbibition to 23% after 6 d of growth), and a change in the pattern of polypeptides associated with the ER. These results provide further evidence for the extensive reorganization of the ER of the cotyledons which accompanies seedling growth. The reorganization includes the simultaneous breakdown of the pre-existing tubular ER and the biosynthesis of new ER components.This is the fourth paper in a series on the endoplasmic reticulum of mung-bean cotyledons. The first three papers are referenced as Gilkes and Chrispeels (in press); Harris and Chrispeels 1980; Van der Wilden et al. (in press)     

Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg²⁺, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl₂–0.02% NaN₃, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg²⁺ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl₂ prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl₂ raised to 10 mM, reassembly occurs. These results suggest that Mg²⁺ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg²⁺ (10 μM–10 mM) increases reassembly whereas chelation of Mg²⁺ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl₂ do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated,diethystilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the ‘microsomes’ ($\text{13 000–80 000 x g}\text{sediment}$) from pea stem tissue is strongly influenced by the concentration of Mg²⁺ in the homogenization medium. The absence of Mg²⁺ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at $\text{80 000 × g}$ upon addition of 5–10 mM Mg²⁺ (or Mn²⁺, Ca²⁺, Zn²⁺) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl₂ the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg²⁺. Microsomes prepared with Mg²⁺ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein

Protein has been selectively extracted from isolated chicken erythrocyte nuclear envelope by (1) dilute MgCl₂/Triton X-100 followed by (2) concentrated MgCl₂/Triton X-100 solutions. Certain proteins appear to be selectively dissolved in the first solvent and may occur in the nuclear envelope primarily as lipoproteins. Among the proteins insoluble in the low MgCl₂/Triton X-100 wash, as well as in 500 mM MgCl₂ without Triton previously used in the preparation of the envelope fraction, the quantitatively major polypeptides dissolve in a combination of high MgCl₂ and Triton X-100. Further, much of this dissolved protein precipitates when the MgCl₂ concentration is lowered by dialysis. The insolubility of these proteins appears to result from a combination of ionic and hydrophobic interactions and may explain the resistance of nuclei to various manipulative procedures including nonionic detergent washes. The procedures described provide a route for gently and selectively dissolving representative proteins from the nuclear envelope lipoprotein matrix and from the envelope “residual” protein.     

A method for obtaining high recovery of purified subcellular fractions of rat liver homogenate

A method for obtaining highly purified subcellular fractions of rat liver is described. The recovery of each fraction approaches 100%. The method is based on differential centrifugation and the use of appropriate concentrations of Triton X-100 and MgCl₂ at certain specific steps in the fractionation procedure.     

Inhibition of Na+, K+-ATPase of intact mouse soleus muscle by Mg++

The effect of 10 mM MgCl₂ on the inhibition of respiration by ouabain was investigated with intact mouse soleus muscle preparations. Although ouabain caused a 19.7% inhibition of respiration of soleus muscle incubated in 1 mM MgCl₂ buffer, the response of respiration to ouabain was abolished upon incubation in buffer containing 10 mM MgCl₂. Initial respiration rates were significantly decreased in soleus muscle exposed to 10 mM, as contrasted to 1 mM, MgCl₂.     

Theory of proton flow along appressed thylakoid membranes under both non-stationary and stationary conditions

Under illumination thylakoids take up protons from the suspending medium, e.g., at Photosystem II which is located in appressed portions of stacked thylakoid membranes (partitions). The rise of alkalization in the suspending medium after one single turnover of Photosystem II is rather slow (typically, half-rise time 100 ms) in stacked thylakoids and fast (2.7 ms) in unstacked ones (Polle, A. and Junge, W. (1986) Biochim. Biophys. Acta 848, 257–264). We described the transient alkalization of the suspending medium of stacked thylakoids by the theory of evaporation from a cylinder. The calculated time-course fitted the experimentally observed one with a single fit parameter, namely the ‘effective’ diffusion coefficient of hydroxyl anions in the narrow domain between appressed membranes. Its magnitude was 10⁵-times lower than for diffusion of hydroxyl in water. This large decrease could be rationalized by the action of fixed buffers in this domain, which decreased the ‘effective’ diffusion coefficient (in Fick's second law), but left the ‘true’ diffusion coefficient (Fick's first law) unaffected. We also modeled the continuous flow of hydroxyl anions through the alkaline partitions which is required for steady ATP synthesis. For stacked thylakoids and with a diffusion coefficient as in bulk water we calculated a lateral pH drop of some 0.1 units between center and fringes of thylakoids. This provided a physical basis to understand quantitatively slightly different efficiencies of the two photosystems in ATP synthesis without necessity to invoke nebulous-localized coupling devices.