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E Skriver

Publications and source records attributed to E Skriver.

35 records · Page 2Linked to original sources
PubMed1988

Assembly of two-dimensional membrane crystals of Na,K-ATPase.

The assembly of vanadate-induced two-dimensional membrane crystals of Na,K-ATPase was analyzed by electron microscopy and image processing. Electron micrographs of negatively stained linear arrays of protein molecules were recorded and processed by correlation averaging methods. The arrays were compared with fully developed p21 crystals of the enzyme. On the basis of similarity in protein form, symmetry, and packing arrangement it was concluded that the fully developed crystals are built of tightly packed ribbons. Assembly pathways for two-dimensional membrane crystals of Na,K-ATPase are proposed.

Animals↗
PubMed1985

Three-dimensional structure of renal Na,K-ATPase determined by electron microscopy of membrane crystals.

The three-dimensional structure of Na,K-ATPase was determined by electron microscopy and image processing. Tilt series of negatively stained membrane crystals were recorded. The projections were analyzed by Fourier methods and the data combined to a 3-D model. The unit cell contains two rod-shaped stain-deficient regions interpreted as alpha beta-promoters of Na,K-ATPase. The rods are related by dyad axes oriented perpendicular to the membrane. Outside the lipid bilayer the rods contact different protein units on the two sides of the membrane.

Animals↗
PubMed1985

Quantitative ultrastructure of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis).

Surgically removed perfusion-fixed human kidneys with chronic renal disease (hydronephrosis) were studied by electron microscopy in order to determine whether there is a quantitative relationship between ultrastructural changes in proximal tubules in atrophy and changes in the surrounding cortical interstitium. Morphometric techniques were applied to montages of electron micrographs each covering several tubular profiles in the cortical labyrinth and to montages representing cross-sections of individual proximal convoluted tubules at a higher magnification. In order to enable a quantification of the spatial relations between individual tubular cross-sections and adjacent peritubular capillaries a tubulo-capillary index (TCI) was defined. This index was based on the mean distances between individual tubular cross-sections and adjacent peritubular capillaries and on the fraction of tubular circumference facing capillaries. Normal tissue from similarly fixed human nephrectomy specimens, which had been removed mainly because of neoplastic disorders, served as control material. In the hydronephrotic kidneys the relative volume of cortical interstitium (excluding capillaries) covered a range from 19.2-70.3%. Inverse correlations were demonstrated between the relative volume of cortical interstitium and various structural variables of proximal convoluted tubules, including tubular wall volume, the volume of mitochondria and the surface area of basolateral membranes. The TCI showed positive correlations with these tubular variables. No significant correlation was found between the volume fractions of cortical interstitium and capillaries. Finally, it was found that an increase in the volume fraction of the cortical interstitium from 16.2% in controls to 24.7% in cortical areas of hydronephrotic kidneys was associated with a 40-50% reduction in the volume of mitochondria and in the surface area of basolateral membranes in proximal tubules. The results are consistent with a pathogenic interrelationship between tubular and interstitial changes. An important factor in this relationship might be disturbed topographic associations between tubules and blood capillaries caused by the increase in cortical interstitium. The results further show that even slight increases in the cortical interstitial volume are associated with significant quantitative changes in tubular fine structure suggesting impaired tubular functions.

Adult↗
PubMed1984

Ultrastructural analysis of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis).

A systematic ultrastructural analysis of proximal tubule atrophy and cortical interstitial changes was carried out in human chronic nephropathy. The investigation was based on human hydronephrotic kidneys, which had been surgically removed and subsequently perfusion-fixed for light and electron microscopy. Normal kidney tissue, which was derived from nephrectomy specimens with pathological changes confined to part of the kidney or to the renal pelvis, was used for control material. A slight degree of proximal tubule atrophy was characterized by reduction of mitochondria and basolateral membranes, enlargement of large endocytic vacuoles and increased numbers of lysosomes containing lamellar material. In moderate atrophy these changes were further accentuated, and in addition there was an increasing loss of microvilli and a reduction of endocytic invaginations and small endocytic vacuoles. In severe atrophy all types of organelles were sparse and the architecture of the tubule cells greatly simplified. A distinctive feature of atrophic tubules was the presence in the tubule cells of large bundles of actin-like filaments, which were often associated with outpouchings of basal cell parts and basement membrane. The reduction of mitochondria and basolateral cell membranes and the changes of endocytic vacuoles and lysosomes indicate that proximal tubule atrophy also in early stages may be associated with impairment of tubular transport processes. Comparisons with previous observations in various types of experimentally induced tubule cell degeneration and with the ultrastructure of regenerating proximal tubule cells provide some evidence that degenerative changes as well as imperfect regeneration of tubule cells may contribute to the alterations of ultrastructure in tubular atrophy. It is suggested that changes of the cortical interstitium may be of pathogenic importance for the progression of tubular atrophy by altering the spatial relationships between tubules and capillaries.

Adult↗
PubMed1983

Reconstitution of sarcoplasmic reticulum Ca2+-ATPase with excess lipid dispersion of the pump units.

Sarcoplasmic reticulum Ca2+-ATPase has been reconstituted with excess lipid (25-150 g egg phosphatidylcholine per g sarcoplasmic reticulum protein) by a procedure combining the use of a non-ionic detergent with cholate dialysis. The reconstituted vesicles were analyzed by sucrose density fractionation and freeze-fracture electron microscopy. At the lowest lipid to protein ratios some vesicles containing aggregated protein were observed. At a lipid to protein ratio of 150:1 (w/w) only 30-40% of the reconstituted protein sedimented through 7% (w/v) sucrose. The remainder of the latter preparation was characterized by a high Ca2+-uptake capacity and a coupling ratio of 1.6 mol Ca2+ transported per mol ATP hydrolyzed. Intramembranous particles in this preparation occurred isolated in the membrane. In most cases only one particle could be seen on a fracture face. Cross-linking with cupric phenanthroline indicated that protein-protein contacts were drastically reduced by reconstitution. It is concluded that aggregation of intramembranous particles is not required for optimal Ca2+-transport function. The dispersed preparation obtained by a combined reconstitution and sucrose density fractionation procedure is useful for further characterization of the Ca2+ pump.

Animals↗
PubMed1982

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase.

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.

Animals↗
PubMed1982

Ultrastructure of the Na, K-ion pump.

Na, K-ATPase has been analysed by electron microscopy to obtain information about the structure of the enzyme and its organization within the membrane. Following negative staining the membrane-bound enzyme was observed as surface particles which on the basis of their size and frequency and the enzymatic and chemical composition of the membranes are interpreted as protomers (alpha beta-units). Freeze-fracture electron microscopy revealed the enzyme as intramembrane particles. Quantitative electron microscope studies suggested that the intramembrane particles are oligomers of the protein unit that forms the surface particles. Following reconstitution of the enzyme into phospholipid vesicles it was demonstrated that similar intramembrane particles represent a protein unit which transports sodium and potassium. Vanadate and magnesium induced the formation of two-dimensional crystals in the membrane fragments of the purified Na, K-ATPase. Further information regarding the shape and dimensions of the protomer was obtained through analysis of electron micrographs of negatively stained crystals with optical diffraction and image reconstruction methods.

Animals↗
PubMed1980

Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase.

Purified Na,K-ATPase after reconstitution into phospholipid vesicles catalyzed an active coupled transport with a ratio close to 3Na/2K. A uniform population of closed vesicles with average diameters close to 900 A are observed after freeze-fracture and thin sectioning. After freeze-fracture intramembranous particles with diameters of 80-100 A are observed. The data suggest that these particles correspond to Na,K-ATPase molecules.

Animals↗
PubMed1980

Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.

To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture electron microscopy after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 A. Also, freeze-fracture electron microscopy demonstrated uniform vesicles with diameters in the range of 700-1,100 A and an average value close to 900 A. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 A and similar to our previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles per vesicle. The data therefore show that active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close fo 90 A in the freeze-fracture micrographs.

Animals↗
PubMedDate not supplied

Structure of two-dimensional crystals of membrane-bound Na,K-ATPase as analyzed by correlation averaging.

The structure of two-dimensional crystals of membrane-bound Na,K-ATPase from rabbit kidney has been analyzed with a correlation averaging procedure. Two principally different crystal forms are observed with p1 and p21 symmetry, respectively. In the p1 form the averaged projection structure shows a triangular shaped protein domain interpreted as a protomer (alpha beta-unit) of Na,K-ATPase. In the p21-form the stain-deficient area is extended toward a twofold symmetry axis. The results are in good agreement with a previous analysis where Fourier methods were applied to well ordered crystals of pig kidney Na,K-ATPase and illustrate that the correlation averaging procedure can be used for the analysis of membrane crystals of Na,K-ATPase showing curved lattice lines.

Animals↗
PubMedDate not supplied

Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP.

The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.

Adenosine Triphosphate↗
PubMedDate not supplied

Perfusion--fixation of human kidneys for ultrastructural analysis.

Surgically removed human kidneys were fixed by vascular perfusion to improve the preservation of renal tissue and, in particular, the proximal tubules for electron microscopic analysis. Fifty kidneys with various pathologic changes were perfusion-fixed with glutaraldehyde under pressure control immediately after nephrectomy. A Tyrode solution containing a spasmolytic drug initiated the perfusion. The results demonstrate that large volumes of ultrastructurally well-preserved kidney tissue can be obtained in normal (nontumorous) parts of tumor kidneys as well as in kidneys with chronic nephropathy. Several factors improved the quality of fixation: (1) start of perfusion-fixation within 15 min after clamping of the renal vessels, (2) careful handling of the kidney during nephrectomy, (3) simultaneous clamping of the main renal vessels, and (4) employment of a spasmolytic drug. In comparison with tissue from immersion-fixed renal biopsies, perfusion-fixed kidneys represented an improved basis for ultrastructural investigations of normal or pathologically changed human kidney tissue.

Histological Techniques↗