Structural and functional stability of isolated intact chloroplasts

The effect of in vitro ageing on the ultrastructure, electron transport, thermoluminescence and flash-induced 515 nm absorbance change of isolated intact (type A) chloroplasts compared with non-intact (types B and C) chloroplasts was studied.When stored in the dark for 18 h at 5°C, the structural characteristics of intact and non-intact chloroplasts were only slightly altered. The most conspicuous difference between the two was in the coupling of the electron transport which was tighter and more stable in intact chloroplasts. Under dark-storage the activity of PS 2* decreased and the -20°C peak of thermoluminescence increased at the expense of the emission at +25°C. These changes were less pronounced in the intact chloroplasts. PS 1 activity and the flash-induced 515 nm absorbance change were not affected by dark-storage.When kept in the light (80 W m^-2 (400–700 nm) for 1 h at 5°C), the thylakoid system of chloroplasts rapidly became disorganized. Although the initial activity of electron transport was much higher in intact chloroplasts, after a short period of light-storage the linear electron transport and the electron transport around PS 2 decreased in both types of preparations to the same low level. These changes were accompanied by an overall decrease of the intensity of thermoluminescence. PS 1 was not inhibited by light-storage, while the flash-induced 515 nm absorbance change was virtually abolished both in preparations of intact and non-intact chloroplasts.The data show that in stored chloroplast preparations intactness cannot be estimated reliably either by the FeCy test or by inspection under the electron microscope. These tests should be cross-checked on the level and coupling of the electron transport.     

Structural and functional consequences of methionine oxidation in thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.     

Structural and functional consequences of galactolipids on thylakoid membrane organization

Photosynthetic membranes of higher plant chloroplasts are composed primarily of polar, but uncharged, galactolipids unlike most mammalian membranes which contain large amounts of phosphatidylcholine. It is unclear what role(s) the galactolipids play in maintaining the differentiated thylakoid membranes, or in stabilizing the photosynthetically active enzyme complexes. Some of the membrane complexes show no lipid selectivity for maintaining structural or functional integrity. Others are poisoned or dissociated in the presence of high concentrations of a trace lipid class. The efficiency of energy transfer and the reconstitution of protein complexes into liposomes are dependent on the lipid class employed. The lipids are asymmetrically arranged along and across the thylakoid membranes but not as distinctly as the proteins.Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SQDG sulfoquinovosyldiglyceride - PG phosphatidylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PSI photosystem I - PSII photosystem II - LHC chlorophylla/b lightharvesting complex - cytb ₆ f cytochromeb ₆ f complex - CF₀/CF₁ coupling factor ATPase - DCIP 2,6-dichlorophenolindophenol - LRa galactolipase fromRhizopus arrhis     

Structural and functional correlates of sucrase-alpha-dextrinase in intact brush border membranes

The structure and catalytic function of rat intestinal sucrase-alpha-dextrinase (sucrase-isomaltase) were characterized in intact brush border membranes by differential denaturation in 1% SDS at 4, 37, 45, 55, and 100 degrees C, analysis by acrylamide electrophoresis, and subsequent renaturation by transfer to nitrocellulose and in situ analyses of immunoactivity and catalytic activity (immunoblotting and catalytic blotting). Both the sucrase and alpha-dextrinase activities were associated with two mature oligomers, with sucrase predominantly in a 250-260-kDa unit and dextrinase in a 330-350-kDa unit. While sucrase activity declined progressively in response to increasing temperature to 45 degrees C due to loss of active sites, alpha-dextrinase activity increased reciprocally (Vmax +176%). Three principal monomeric products of postinsertional processing comprise the oligomers: alpha, 140 kDa, which carries the sucrase active site; beta, 125 kDa, harboring the dextrinase active site; and gamma, 110 kDa, produced by removal of 185 amino acid residues from the N-terminus of the alpha. Rather than being a simple hybrid dimer, membrane-associated sucrase-alpha-dextrinase appears to consist of two major oligomeric forms having complex structural associations that dramatically affect the availability of the active catalytic sites at the brush border membrane surface.     

Structural and functional consequences of altering a peptide MHC anchor residue

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.     

Structural and functional changes associated with modification of the ubiquitin methionine

The effects of oxidation and cleavage of Met-1 of ubiquitin on conformation and biological activity were individually investigated. Proton NMR studies demonstrated that oxidation to the sulfone led to restricted structural perturbations at neutral pH, particularly in the vicinity of Ile-61. Below pH 3, in the presence of acetic acid, oxidation to the sulfone facilitated a conformational expansion demonstrable by retardation on gel electrophoresis and CD changes below 210 nm. The predominant phase of the low-pH transition did not involve significant changes in alpha-helix content, indicating the capacity of ubiquitin for limited structural transitions. Cleavage of Met-1 by CNBr, on the other hand, was associated with a global unfolding transition below pH 4 that involved a major loss of alpha-helix. Differences in the behavior of the native and des-Met proteins at low pH indicate that Met-1 contributes a minimum of 3.4 kcal/mol to the stability of the native conformation. Two Met-1 sulfoxide isomers, of markedly different conformational stability, were formed by treatment of ubiquitin with H2O2. One isomer was similar in stability to the sulfone, while the other was intermediate in stability between the sulfone and des-Met proteins, the differences potentially interpretable in terms of the geometry of the Met-1-Lys-63 hydrogen bond. The overall activities of the oxidized and des-Met derivatives in ATP-dependent proteolysis differed subtly from that of native ubiquitin. The unresolved sulfoxides exhibited an approximately 50% increase in activity, while the sulfone and des-Met proteins exhibited a 50% decrease in activity at low concentrations and normal activity at higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c₁ and a + a₃, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c1 and a + a3, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Structural and functional organization of mitochondria in soybean root statocytes under microgravitation

The effects of microgravity and ethylene on morphology and ultrastructural organization of mitochondria in root statocytes of soybean seedlings grown for 6 days on the board of the space shuttle Columbia during the STS-87 mission were investigated. The spaceflight seedlings and the ground-grown control seedlings were grown in BRIG (Biological Research in Canister) in the presence of KMnO4 to remove ethylene. It was revealed that irrespectively of KMnO4 treatment the mitochondria in the spaceflight seedlings were characterized by round or oviform and by low electron density of organelle matrix, whereas the organelles in the ground controls were polymorphic in shape and had higher electron density of matrix. The possible mechanisms of morphological and ultrastructural rearrangements of mitochondria that may be involved in adaptation processes of soybean seedlings to microgravity conditions are discussed.     

Structural and functional properties of rat liver mitochondria in hexachlorobenzene induced experimental porphyria

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.     

Structural and functional features and significance of the physical linkage between ER and mitochondria

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER-mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are approximately 10 nm at the smooth ER and approximately 25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short "synthetic linker" (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.     

Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria

Single giant mitochondria isolated from mice fed cuprizone were assayed for their metabolic viability. Two tests were devised. One test optically detected the accumulation of calcium phosphate within the mitochondria under massive loading conditions (including the presence of succinate and ATP). The accumulation corresponds to a test of energy coupling from either electron transport or the hydrolysis of ATP since it is blocked by either antimycin A or oligomycin. The other assay tested for the production of ATP from ADP and Pi, using myofibrils. Myofibrils prepared from glycerinated rabbit psoas muscle contract only in the presence of ATP and not in the presence of ADP. Myofibrillar contraction is unaffected by the presence of antimycin A or oligomycin. However, myofibrils in the presence of mitochondria that are phosphorylating ADP to ATP do contract. This contraction is blocked by antimycin A and/or oligomycin. Hence, the ATP which causes myofibrillar contraction is produced by oxidative phosphorylation. At low mitochondrial concentration, only the myofibrils in close proximity with mitochondria contract in the presence of ADP. Therefore the assay can be used to test the viability of individual mitochondria. Individual giant mitochondria were found to be viable, using both of these assays. Comparable results were obtained in mitochondria impaled with microelectrodes. The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation.     

Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain

The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.     

Structural and functional changes associated with cyanogen bromide treatment of wheat germ agglutinin

The lectin, wheat germ agglutinin (WGA), has been shown to have a significant larvicidal effect on the European corn borer, a major insect pest of corn. In order to characterize this toxic effect, we have undertaken structure-function studies on WGA. To this extent, the effect of cyanogen bromide (CNBr) on the conformation, subunit interactions, and biological activity of WGA has been investigated. The CNBr-modified lectin exhibits no toxicity to the ECB, cannot dimerize, does not bind to N-acetylglucosamine or its polymers, has no or vastly reduced hemagglutinating activity against red blood cells of different animals, and shows loss of an antigenic determinant by immunodiffusion. The CD spectrum of CNBr-WGA is not significantly different from that of native WGA, although the intrinsic fluorescence shows about 30% quenching. Our results suggest that the integrity of the N-terminal domain of WGA is essential for dimer formation. Furthermore, toxicity of WGA to ECB may be intrinsically related to its ability to dimerize and bind to sugar residues.     

Structural and functional features of mitochondria in statocytes of soybean root under microgravity conditions

A study of the influence of microgravity and ethylene on the morphology and ultrastructural organization of mitochondria in root apex statocytes of 6-day old soybean seedlings that traveled on the Columbia space station on the STS-87 mission is presented. The seedlings were grown in hermetically sealed canisters in the presence of KMnO₄ to neutralize ethylene. It is established that, independent of KMnO₄ treatment, most of the mitochondria in the cells of the spaceflight seedlings were characterized by a round or oval shape and electronically transparent matrix, whereas in the seedlings in the ground controls the organelles were distinguished by a characteristic polymorphism, large dimensions, and higher electronic density of the matrix. One possible mechanism that could be responsible for the morphological and ultrastructural arrangement of the organelles in the process of adaptation of the seedlings to conditions of microgravity is discussed.