Elisabeth Skriver

Formation of two-dimensional crystals in pure membrane-bound Na+,K+-ATPase

The aim of this work has been to induce twodimensional crystals in preparations of purified membrane-bound Na+,K+-ATPase since information about the structure of integral membrane proteins can be obtained by electron microscopy of membrane crystals [1,2]. Na*,K+-ATPase is responsible for active Na+,K÷-transport in kidney tubules and it can be purified in membrane-bound form without perturbing lipoprotein associations [3]. The organization of its proteins in the membrane had been examined by electron microscopy after negative staining and freeze-fracture [4-6] . The enzyme is asymmetrically oriented in the membrane and observed as protein units protruding above the plane of the membrane bilayer after negative staining. Here, we have used negative staining to monitor the aggregation of the protein units during exposure to different combinations of the specific ligands of Na+,K÷-ATPase. We report the induction of two-dimensional crystals in membrane fragments of purified Na+,K+-ATPase during incubation with vanadate and magnesium, while crystalline arrays are rare with other ligand combinations. Information is also presented on the mode of assembly of the membrane crystals. It is suggested that transition to the vanadate-bound Erform of the enzyme protein favours immobilization of the units in crystalline arrays.

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.

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 αβ‐protomers 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.

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.

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.

Two-dimensional crystalline arrays of Na,K-ATPase with new subunit interactions induced by cobalt-tetrammine-ATP.

Purified membrane-bound Na,K-ATPase incubated with cobalt-tetrammine-ATP [Co(NH3)4ATP], which is a stable MgATP complex analog, shows two new types of membrane crystals, a new p21 form and a p4 form. The building blocks of the crystalline arrays correspond to (alpha beta)2 dimers of the enzyme protein suggesting that alpha-alpha interaction may be important in the pumping process.

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.

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.

Three-dimensional structure of renal Na, K-ATPase determined from two-dimensional membrane crystals of the p1 form

Electron microscopy and image processing were used to reconstruct a three-dimensional model of membrane-bound monomeric renal Na,K-ATPase from negatively stained two-dimensional crystals of the p1 type. Correlation methods were applied to obtain projection averages which were aligned by a phase difference minimization procedure. The self-consistency of the reconstruction process was high as determined by correlation between experimental projections and projections of the calculated model. The three-dimensional model of the Na,K-ATPase promoter in the p1 crystal form contains three characteristic domains, a protein dense ellipsoid, a small globular stain deficient domain, and a connecting low-contrast region. The latter is thought to correspond to the lipid-penetrating part of the Na,K-ATPase promoter. The location of this domain gives the protein an asymmetric distribution in the bilayer so that it is exposed primarily on one side proposed to correspond to the intracellular face.

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.