Biomedical subjects

L A Staehelin

Publications and source records attributed to L A Staehelin.

At least 73 records · Page 4Linked to original sources

PubMed1987

Phosphorylation of spinach chlorophyll-protein complexes. CPII, but not CP29, CP27, or CP24, is phosphorylated in vitro.

Previous studies have indicated that the reversible phosphorylation of a population of antenna complexes that can donate energy to PS II ('mobile LHC II') plays a regulatory role in the state 1-state 2 transition in thylakoid membranes. The relationship of phosphorylated LHC II to the multiple PS II-associated chlorophyll a/b-proteins resolvable on green gels is currently unclear. We have used a high resolution gel system to analyze thylakoids phosphorylated in vitro. The only PS II-associated antenna complex to become phosphorylated is CPII, indicating that this complex represents the mobile LHC II. The other putative PS II antenna complexes, CP29, CP24, and the new complex designated CP27 which comigrates with CPII, are not phosphorylated and are probably components of the bound 'LHC II' antenna.

Chlorophyll↗

PubMed1987

Structural localization of the O2-evolving apparatus to multimeric (tetrameric) particles on the lumenal surface of freeze-etched photosynthetic membranes.

Isolated appressed chloroplast membranes, highly enriched in photosystem II (PSII) activity, were examined by freeze-etch electron microscopy. The exposed surfaces of these Triton X-100 solubilized membrane fragments correspond to the lumenal or ESs surface of intact stacked thylakoid membrane regions (Dunahay, T. G., L. A. Staehelin, M. Seibert, P. D. Ogilvie, and S. P. Berg. 1984. Biochim. Biophys. Acta. 764:179-193). The sequential removal from this sample of three extrinsic proteins (17, 23, and 33 kD) associated with the O2-evolving apparatus and the concomitant loss of O2 evolution, was related to subtle changes in the height and substructure of characteristic multimeric (often tetrameric) particles that protrude from the ESs membrane surface. After removal of these proteins, the multimeric particles disappeared and dimeric particles of similar diameter but of lesser height (6.1 vs. 8.2 nm in the controls) were observed. Reconstitution of the depleted membrane fragments with the extrinsic proteins led to rebinding of the three proteins, to a 63% recovery of the control rates of O2 evolution, and to the reappearance of the larger multimeric particles. Analysis of the structural changes associated with the loss and rebinding of the extrinsic proteins is consistent with a stoichiometry of one PSII complex for either one or two copies of the 17-, 23-, and 33-kD proteins, and these are symmetrically arranged on the lumenal surface of the complex. These results demonstrate that the multimeric ESs particles correspond to part of the intact O2-evolving apparatus of PSII, thus confirming previous indirect studies relating these particles to PSII. The dimeric particles probably contain the rest of the O2-evolving complex.

Chlorophyll↗

PubMed1987

A second extensin-like hydroxyproline-rich glycoprotein from carrot cell walls.

The insoluble extensin matrix of dicot cell walls has been studied most fruitfully by examining the salt-extractable precursors to this matrix. Multiple extensin-like hydroxyproline-rich glycoproteins (HRGPs) have been isolated, or their existence inferred, from tomato, potato, bean, soybean, melon, carrot, and other plants. We and others previously have studied a carrot extensin which we call extensin-1. Here we report on the properties of extensin-2, a second salt-extractable hydroxyproline-rich glycoprotein from carrot. Like extensin-1, extensin-2 contains large amounts of hydroxyproline, serine, histidine, and lysine. In contrast, its tyrosine content is only about one-third that of extensin-1. Arabinose and galactose are the most abundant neutral sugars in both proteins, and nearly identical buoyant densities in CsCl suggest a similar proportion of carbohydrate in each. The size of extensin-2 is about half the size of extensin-1 based on: (a) the measured lengths of shadowed molecules (about 40 versus 84 nanometers); (b) the migration of extensin-2 in acid-urea gels relative to monomers, dimers, and trimers of extensin-1; and (c) the Stokes' radii of these molecules as determined by gel filtration chromatography. Electron microscopy of shadowed extensin-2 molecules indicates that they contain kinks, which may indicate the presence of intramolecular isodityrosine cross-links, but intermolecular cross-links, either with other extensin-2 molecules or extensin-1 molecules, are observed rarely if ever.

Journal Article↗

PubMed1987

Does gibberellic Acid induce the transfer of lipase from protein bodies to lipid bodies in barley aleurone cells?

We have examined the effect of gibberellic acid (GA(3)) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA(3) for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.

Journal Article↗

PubMed1987

Improved specimen support cups and auxiliary devices for the Balzers high pressure freezing apparatus.

A new design for interlocking specimen support cups that permits reliable and reproducible freezing under high pressures of a range of different biological specimens is described. A stronger holder in which the support cups are tightly held during pressurization and freezing, and a sliding wedge for separating the cups after freezing are also described. Used in conjunction with the Balzers HPM 010 high pressure freezing apparatus, these modifications permit routine freezing of tissue for freeze-fracture and freeze-substitution for electron microscopy.

Freeze Fracturing↗

PubMed1986

Spatial organization of the cytochrome b6-f complex within chloroplast thylakoid membranes.

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b6-f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3',5,5'-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b6-f antibodies; (4) agglutination of membrane fragments with anti-Cyt b6-f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b6-f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b6-f throughout all thylakoid membranes.

Chloroplasts↗

PubMed1986

Implications of cytochrome b6/f location for thylakoidal electron transport.

The cytochrome b6/f complex of higher plant chloroplasts is uniformly distributed throughout both appressed and nonappressed thylakoids, in contrast to photosystem II and photosystem I, the other major membrane protein complexes involved in electron transport. We discuss how this distribution is likely to affect interactions of the cytochrome b6/f complex with other electron transport components because of the resulting local stoichiometries, and how these may affect the regulation of electron transport.

Chloroplasts↗

PubMed1986

Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concomitant changes in intramembrane particle size during photoinhibition of Chlamydomonas reinhardtii.

Light-dependent reduction of the plastoquinone pool regulates the activity of the thylakoid-bound protein kinase which phosphorylates the light harvesting chlorophyll a,b-protein complex (LHC II) and regulates energy distribution between photosystems II (PS II) and I (Staehelin, L. A., and C. J. Arntzen, 1983, J. Cell Biol., 97:1327-1337). Since reduction of plastoquinone by PS II is abolished in photoinhibited thylakoids due to loss of the secondary electron acceptor QB protein (Kyle, D. J., I. Ohad, and C. J. Arntzen, 1984, Proc. Natl. Acad. Sci. USA, 81:4070-4074), it was of interest to examine the activity of the LHC II protein kinase system during photoinhibition and recovery of PS II activity. The kinase activity was assessed both in vivo and in vitro in Chlamydomonas cells exposed to high light intensity (photoinhibition) and recovery at low light intensity. The kinase activity was progressively reduced during photoinhibition and became undetectable after 90 min. The inactive LHC II-kinase system could not be reactivated in vitro either by light or by reduction of the plastoquinone pool following addition of reduced duroquinone (TMQH2). The LHC II polypeptides were dephosphorylated in vivo when cells, prelabeled with [32P]orthophosphate before exposure to high light intensity, were transferred to photoinhibiting light in the presence of [32P]orthophosphate. In vivo recovery of the LHC II-kinase activity, elicited by the addition of TMQH2 to the assay system, did not require restoration of QB-dependent electron flow or de novo protein synthesis, either in the cytoplasm or in the chloroplast. Mild sonication of thylakoids isolated from photoinhibited cells restored the ability of the LHC II protein kinase system to be activated in vitro by addition to TMQH2. Restoration of the light-activated LHC-II kinase required recovery of QB-dependent electron flow. At the structural level, photoinhibition did not affect the ratio of grana/stroma thylakoids. A reduction of approximately 20% of the 11-17-nm intramembrane particles and an equivalent increase in the number of 6-10.5-nm particles was observed on the E-fracture faces of stacked thylakoid membranes. Similar but smaller changes were observed also on the E-fracture faces of unstacked thylakoid membranes (more 10-14-nm and less 6-9-nm particles) and P-fracture faces of stacked thylakoid membranes (more 6-8- and less 9.5-13-nm particles). All these structural changes were reversed to normal values during recovery of PS II activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Chlamydomonas↗

PubMed1986

Isolation and Characterization of a New Minor Chlorophyll a/b-Protein Complex (CP24) from Spinach.

We have identified a new minor chlorophyll a/b-protein complex in the thylakoid membranes of spinach (Spinacia oleracea L.), which migrates as a green band below CPII on mildly denaturing polyacrylamide gels. This complex, designated CP24, was isolated from octyl glucoside/sodium dodecyl sulfate solubilized spinach grana membrane fractions by preparative gel electrophoresis and has been characterized as to its spectral properties and polypeptide composition. CP24 has a room temperature absorption maximum at 668 nanometers, a chlorophyll a/b ratio between 0.8 and 1.2, and contains three or four polypeptides between 20 and 23 kilodaltons. CP24 was also identified in grana membrane preparations from peas (Pisum sativum) and barley (Hordeum vulgare). We postulate that CP24 functions as a linker component in photosystem II, acting to orient the photosystem II light harvesting components to ensure efficient energy transfer to the reaction center.

Journal Article↗

PubMed1986

Cross-linking patterns in salt-extractable extensin from carrot cell walls.

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as "extensin-1" to distinguish it from "extensin-2," a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated ( congruent with84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.

Journal Article↗

PubMed1986

The role of carbohydrate in maintaining extensin in an extended conformation.

Monomers of the plant cell wall glycoprotein extensin are secreted into the wall where they become cross-linked to each other to form a rigid matrix. Expression of the extensin matrix is correlated with the inhibition of further cell elongation during normal development, with increased resistance to virulent pathogens and with other physiological responses characterized by wall strengthening. Carbohydrates make up about two-thirds of the mass of extensin. Arabinose oligomers linked to hydroxyproline residues represent 95% of the total carbohydrate with the remainder occurring as single residues of galactose linked to some serine residues. Electron microscopy of shadowed extensin shows the glycosylated form to be an easily visualized and highly elongated molecule. In contrast, extensin that has been deglycosylated with anhydrous hydrogen fluoride is difficult to resolve in the EM. Glycosylated extensin elutes from a gel filtration column much more rapidly than does the deglycosylated form, and from this analysis we have calculated respective Stokes' radii of 89 and 11 Angstroms for these molecules. Others have shown that inhibition of extensin glycosylation has no effect on its secretion or insolubilization in the cell wall, but that this extensin cannot inhibit cell elongation. It is likely that carbohydrate moieties keep extensin in an extended conformation and that extensin must be in this conformation to form a cross-linked matrix that can function properly in vivo.

Journal Article↗

PubMed1986

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

Journal Article↗

PubMed1985

Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor.

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg2+ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg2+. We also tested for the effects of Mg2+ on detergent extractability of pigment-protein complexes from intact membranes. Mg2+ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.

Adhesiveness↗

PubMed1985

Reconstitution of light-harvesting complexes and photosystem II cores into galactolipid and phospholipid liposomes.

Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.

Chlorophyll↗

PubMed1985

Lateral Distribution of the Cytochrome b(6)/f and Coupling Factor ATP Synthetase Complexes of Chloroplast Thylakoid Membranes.

We have visualized directly the distribution of the cytochrome b(6)/f and coupling factor ATP synthetase complexes in thylakoid membranes of embedded, thin-sectioned, intact chloroplasts by using rabbit antibodies directed against each complex, followed by ferritin-conjugated goat anti- (rabbit immunoglobulin G) antibodies. The labeling patterns indicate that in spinach (Spinacia oleracea) chloroplasts the cytochrome b(6)/f complex is distributed laterally throughout both stacked grana and unstacked stroma membrane regions, whereas the coupling factor ATP synthetase complex is found exclusively in stroma thylakoids and in the marginal and end membranes of grana.

Journal Article↗

PubMed1985

Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids : characterization and reconstitution into liposomes.

We have used the nonionic detergent octyl-beta-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P(700), and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI(*), is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.In addition, the PSI reaction center complex CPI isolated from preparative gels and PSI-B were reconstituted into lecithin liposomes for structural analysis using freeze-fracture electron microscopy. The results suggest that the native PSI complex produces 12- to 13-nanometer particles, while the PSI reaction center, depleted of LHCI and peripheral proteins, produces particles with an average diameter of 10 nanometers.

Journal Article↗

PubMed1985

Pigment-protein complexes from Rhodopseudomonas palustris: isolation, characterization, and reconstitution into liposomes.

We have employed detergent solubilization and sucrose density gradient centrifugation to obtain pigment-protein complexes from Rhodopseudomonas palustris. Two types of detergent buffers were used, containing either octyl-beta-glucopyranoside (OG) plus sodium dodecyl sulfate (SDS) or OG alone. The fractions thus obtained were analyzed spectrophotometrically and by polyacrylamide gel electrophoresis to determine their pigment and protein composition. OG-SDS solubilization yields four fractions. The least dense of these fractions (OG-SDS a and b) are nonspecific mixtures of peptides and pigments. The next fraction, OG-SDS c, is an accessory light-harvesting complex, LHII, called B800-850. The largest particle, OG-SDS d, is a combination of reaction center (RC) and primary light-harvesting complex (LHI), B880. Solubilization using OG alone yields one fraction, a single large complex consisting of RC, LHI, and LHII. We have inserted the two large OG-SDS complexes and the OG complex into phospholipid liposomes to determine the size of such complexes in freeze-fractured membranes. On the basis of morphological, biochemical, and available biophysical data, we propose the following models for pigment-protein complexes in R. palustris membranes: 5-nm particles as free RC or LHI tetramers, 7.5-nm particles as LHI or LHII octamers (or both); 10-nm particles as RC-LHI core complexes (1 RC plus 12 LHI) or large LHII oligomers (or both), and large particles of 12.5 and 15 nm and LHII associated with the RC-LHI core complex.

Bacterial Proteins↗

PubMed1984

Correlation of structure and function of chloroplast membranes at the supramolecular level.

Freeze-fracture electron microscopy has revealed that different size classes of intramembrane particles of chloroplast membranes are nonrandomly distributed between appressed grana and nonappressed stroma membrane regions. It is now generally assumed that thylakoid membranes contain five major functional complexes, each of which can give rise to an intramembrane particle of a defined size. These are the photosystem II complex, the photosystem I complex, the cytochrome f/b6 complex, the chlorophyll a/b light-harvesting complex, and the CF0 -CF1 ATP synthetase complex. By mapping the distribution of the different categories of intramembrane particles, information on the lateral organization of functional membrane units of thylakoid membranes can be determined. In this review, we present a brief summary of the evidence supporting the correlation of specific categories of intramembrane particles with known biochemical entities. In addition, we discuss studies showing that ions and phosphorylation of the membrane adhesion factor, the chlorophyll a/b light-harvesting complex, can affect the lateral organization of chloroplast membrane components and thereby regulate membrane function.

ATP Synthetase Complexes↗