Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.     

The Staphylococcus aureus alpha-toxin channel complex and the effect of Ca2+ ions on its interaction with lipid layers.

Using the techniques of two-dimensional crystallization on supported lipid bilayers together with computer image processing, two distinct two-dimensional crystal types of staphylococcal alpha-toxin complex are formed depending on the presence or absence of Ca2+ ions. Without Ca2+, these are hexagonally packed (in A, a = b = 89.5 +/- 2.5 A; theta = 119.7 degrees) With Ca2+ present, rectangular crystal packing is seen (in A, a = 114.8 +/- 1.6 A, b = 140.2 +/- 0.7 A; theta = 89.1 degrees). A third, banded crystal type is also seen which is interpreted as a side-to-side packing of regular tubules. We use these tubular crystals for cross-correlation searches with top and side-on views of the complex from single particle reconstructions, and with the repeating units from the two-dimensional crystal types. The results lead us to propose a model in which the different two-dimensional crystal types are formed as a result of alpha-toxin hexamers packing in different orientations. In the hexagonal crystals the hexamers lie end-on with a 6-fold axis in projection. On the addition of Ca2+, the hexamers reorient to lie tilted with respect to the support, thus giving rise to a rectangular projection.     

Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein.

We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stained membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm)-1. Furthermore, we have core-constituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with maltoporin. One of the hexagonal arrays was found to bind maltose-binding protein molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.     

Crystallization properties and structure of Panulirus interruptus haemocyanin

Electron-microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close-packed two-dimensional crystals whereas homohexamers of subunit c form open two-dimensional arrays. We applied computer-image analysis to these arrays and studied the differences in crystallization properties by combining the electron-microscopic data with amino acid sequence information and the X-ray diffraction model of subunit a.     

Observations on crystals found in the intestinal cells of Haemonchus contortus and in the intestinal lumen of Ostertagia ostertagi.

Bird A. F., Waller P. J., Dash K. M. and Major G. 1978. Observations on crystals found in the intestinal cells of Haemonchus contortus and in the intestinal lumen of Ostertagia ostertagi. International Journal for Parasitology8: 69–74. Crystals are described which are located in the distal intestinal cells of the fourth stage larva of Haemonchus contortus and in the distal intestinal lumen of both larvae and adults of Ostertagia ostertagi.The crystals are thought to be by-products of degenerative processes in these nematodes. They are irregular hexagonal rod-shaped structures which measure approx. 2.5 × 15 um in H. contortus. In O. ostertagi they are also irregular hexagonal structures commonly reaching dimensions of 25 × 50 μm, but they vary considerably in size and are not rod-shaped.Histochemical tests. X-ray analysis and electron microscope studies reveal that both types of crystal contain protein and the element sulphur and are of uniform composition, being neither hollow nor having a core of different refractive index.They differ in that the crystals of O. ostertagi contain more sulphur, exhibit birefringence under polarized light, stain with methylene blue, toluidine blue and osmium tetroxide. The crystals from H. contortus on the other hand are not birefringent and do not stain with the dyes mentioned above.     

Starch grain morphology of the seagrasses Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum

Starch grains are a ubiquitous component of plants that have been used in tandem with phytoliths, pollen, and macrofossils to reconstruct past floral diversity. This tool has yet to be fully explored for aquatic plants, specifically seagrasses, which lack phytoliths and are rarely preserved as macrofossils or pollen. If starch grains in seagrasses are morphologically distinct, this method has the potential to improve seagrass identification in the fossil record in such cases where its starch is preserved (e.g. scratches and occlusal surfaces of tooth enamel from seagrass consumers). The goals of this study were twofold: (1) to determine if starch is present in seagrass material and (2) to assess how starch grain morphology differs between different seagrasses.This study focused on four abundant and ecologically distinct seagrasses from the Caribbean: Halodule wrightii, Ruppia maritima, Syringodium filiforme, and Thalassia testudinum. Starch grains were observed in all species except S. filiforme. Grains from H. wrightii are typically observed in side-on orientation, are sub-round to angular, and are fairly small (3-19 μm, end-on). Grains of R. maritima are small spherical grains (4-8 μm) that have a centric hilum and a straight extinction cross with a median angle between the arms of 90°. Grains from T. testudinum are large (9-31 μm, end-on), conical in side-on and round/sub-round in end-on orientation, have a slightly eccentric hilum with an obvious particle, and prominent lamellae.Visual assessment and comparative statistics demonstrate that the morphology of starch grains from T. testudinum, R. maritima, and H. wrightii are significantly different. With more extensive research, there is potential for the positive identification of starch grains from an unknown seagrass. The ability to identify seagrass from starch grains could facilitate the identification of seagrasses in the fossil record and supply information on seagrass evolution and distribution, climate effects on seagrass distribution, and the diets of seagrass consumers.     

Preliminary Crystallographic Analysis of a Cruciferin Protein from Seeds of Moringa oleifera

A 55 kDa cruciferin protein has been purified and characterized from seeds of Moringa oleifera plant. Protein blast of N-terminal amino-acid sequence showed 60 % sequence similarity with cruciferin from Brassica napus. The M. oleifera protein has been crystallized applying the sitting drop method using 5 % polyethylene glycol 8,000, 38.5 % 3-methyl-1,5-pentanediol and 0.1 M sodium cacodylate pH 6.5. The crystals belonged to the P6322 hexagonal space group with cell dimensions, a = b = 98.4, c = 274.3 Å. Initial diffraction data have been collected to a resolution of 6 Å.     

Beef liver esterase. I. Isoelectric point and molecular weight

A sample of carboxylesterase has been purified from beef liver by the method of Runnegar et al. [Biochemistry8, 2013 (1969)]. The protein isoelectric point, pI was found to be 5.5, using the isoelectric focusing method. The molecular weight was found by equilibrium sedimentation to be 5.5 × 10⁴. Sedimentation velocity combined with diffusion gave a similar value. In high protein concentrations aggregation was not detected.     

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.     

How hollow melanosomes affect iridescent colour production in birds

Developmental constraints and trade-offs can limit diversity, but organisms have repeatedly evolved morphological innovations that overcome these limits by expanding the range and functionality of traits. Iridescent colours in birds are commonly produced by melanin-containing organelles (melanosomes) organized into nanostructured arrays within feather barbules. Variation in array type (e.g. multilayers and photonic crystals, PCs) is known to have remarkable effects on plumage colour, but the optical consequences of variation in melanosome shape remain poorly understood. Here, we used a combination of spectrophotometric, experimental and theoretical methods to test how melanosome hollowness—a morphological innovation largely restricted to birds—affects feather colour. Optical analyses of hexagonal close-packed arrays of hollow melanosomes in two species, wild turkeys (Meleagris gallopavo) and violet-backed starlings (Cinnyricinclus leucogaster), indicated that they function as two-dimensional PCs. Incorporation of a larger dataset and optical modelling showed that, compared with solid melanosomes, hollow melanosomes allow birds to produce distinct colours with the same energetically favourable, close-packed configurations. These data suggest that a morphological novelty has, at least in part, allowed birds to achieve their vast morphological and colour diversity.     

Hexagonal crystalline arrays of adenovirus type 1 hexons

Separated, highly purified and concentrated adenovirus type 1 soluble hexon capsomers were crystallized by dialysis against 0.5 M acetate buffer. The crystallization process was followed electron microscopically. In the early phase of the crystallization, groups of a few hexons began to appear, then the two-dimensional crystal lattices grew gradually to a size of 1-2 micron. Simultaneously three-dimensional crystals of tetrahedral and prismatic shapes developed. The hexons in the two-dimensional crystal lattice formed regulator dense arrays corresponding to the hexagonal packing. Analysis of the crystal structure revealed 15-20% local irregularity (short range disorder) and about 10% deviation in the values of the lattice constant if determined from three different directions. The average lattice constant values showed considerable differences in different preparations. Angles formed by non-parallel hexon rows deviated by a few degrees from the regular hexagonal order. Consequently, the position of the hexons in dense two-dimensional crystals was found slightly skew and irregular, although each unit stayed within a certain distance as compared to its equilibrium position defined theoretically in the network. Dislocations were frequently found to disturb the regular arrays. The extra hexon row developing between two rows deverted them from their original direction. At these sites the crystal lattice slanted and the dense array of the hexons loosened. High resolution electron microscopy revealed fine linking structures between the hexons. In several cases the aggregated hexons failed to show a ring-like appearance, they were situated in lying--profile--position and the hexon-building polypeptide fibres became visible. The diameters of the hexons and the distance between them were measured in three directions and the size of the hexon-building polypeptides was determined as well.     

The negative staining-carbon film procedure: Technical considerations and a survey of macromolecular applications

The development and initial virological applications of the negative staining-carbon film (NS-CF) procedure are described and followed by a survey of some recent macromolecular applications of the technique. Electron microscopical data are shown which indicate the capability of the technique for the production of two-dimensional (2-D) crystals from several different soluble proteins (human erythrocyte catalase, human erythrocyte, Xenopus and Thermoplasma acidophilum cylindrin/multicatalytic proteinase, yeast glutamine synthetase and keyhole limpet haemocyanin). Instances in which ordered two-dimensional close-packing of molecules is produced, rather than true two-dimensional crystallization, are also indicated. In addition, data obtained from the application of the NS-CF procedure to a number of fibrillar proteins are briefly presented. Examples include bovine lens vimentin, thrombin-cleaved vimentin and bacterially expressed Xenopus lamin-A. These elongated proteins form higher order filamentous structures, networks and, in the case of lamin-A, large paracrystalline structures. A general discussion of the achievements and future potential of the NS-CF procedure is presented.     

Observations on the occurrence of crystal-like structures in nematode parasites of sheep and cattle

Waller P. J., Dash K. M. and Major G. W. 1979. Observations on the Occurrence of crystal-like structures in nematode parasites of sheep and cattle. Internationl Journal for Parasitology9: 147–151. Intracellular rod-like inclusions were found in a high percentage of inhibited fourth-stage larvae of H. contortus in sheep with naturally acquired infections. Artificial infections showed inclusions occurred in developing as well as inhibited fourth-stage larvae. The presence of these structures was associated with degenerative changes of the parasites and serial worm counts showed that larvae with large numbers of inclusions failed to persist within the host.Large refractive hexagonal crystals were observed in the gut lumen of fourth-stage and adult O. ostertagi acquired by previously worm-free lambs grazed on cattle pastures. The crystals tended to accumulate in the posterior gut region where they may cause gut blockage and lead to early mortality of the parasites in the abnormal host. No inclusions or crystals were observed in O. ostertagi from cattle or goats, or in O. circumcincta from sheep.     

The molecular architecture of the extracellular hemoglobin of Ophelia bicornis: Analysis of two-dimensional crystalline arrays

The double-layered hexagonal disks of the extracellular hemoglobin of the annelid worm Ophelia bicornis form two types of two-dimensional crystalline arrays. The hexagonal type exhibited a typical honeycomb pattern of top views with a center-to-center distance of 26.2 nm. Laterally oriented molecules formed rectangular crystals with lattice constants a = 26.7 run and b = 19.8 nm. The three-dimensional structure was determined from both crystal forms by reconstruction from images of tilt series. At the resolutions obtained, 1.8 nm for the hexagonal form and 2.5 nm for the rectangular form, flattening of the hemoglobin molecules against the support was observed. Nevertheless the two independent reconstructions provided information about the mass distribution within the main subunit and the connectivity between different parts of the molecule.     

Transfer of humoural immunity over two generations in urban pigeons

Maternal antibodies (MatAb) are known to provide passive protection early in life for young vertebrates but their effects on the development of offspring immune response across generations are still unknown. Here, we investigated the effects of antigen exposure (keyhole limpet haemocyanin, KLH) experienced by urban pigeon (Columba livia) females on the amount of antigen-specific antibodies (Abs) transferred into the egg yolk of their daughters and on the humoural immune response towards this same antigen in their grandchildren. We found that chicks from KLH-injected maternal grandmothers had a higher humoural response than chicks from sham-injected grandmothers. However, we did not detect a significant effect of female KLH exposure on the ability of their daughters to transmit anti-KLH Abs into their eggs. These results suggest that antigen exposure at one generation may shape the immune profile of offspring over two next generations, although the underlying mechanisms remain to be investigated.     

Cellular immunity in man: correlation of leukocyte migration inhibition factor formation and delayed hypersensitivity

The formation of leukocyte migration inhibition factor (MIF) by the lymphocytes of 13 normal persons immune to the protein antigen keyhole limpet hemocyanin (KLH) has been investigated. KLH-induced MIF formation expressed as percent migration was compared with delayed hypersensitivity, antibody, and in vitro lymphocyte blastogenic responses to this antigen. Individuals were studied 404–840 days (median 540 days) after their last exposure to KLH. Nine persons had delayed hypersensitivity to KLH and 10 had circulating KLH antibody. The lymphocytes of 11 showed an in vitro blastogenic response to KLH stimulation, while the lymphocytes of nine produced MIF after KLH stimulation. The mean percent migration for the subjects with KLH delayed hypersensitivity was 48.2 (range 20.4–70.4) compared with 133 (range 120–161) for the four persons who did not have KLH delayed hypersensitivity (P < 0.05). The correlation coefficient between the precent migration and delayed hypersensitivity was ?0.78 (P < 0.01). No correlation was demonstrated between migration inhibition and the other parameters of immunity.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.     

Evidence for a charge variant of Cd-BP 14, a cadmium-binding protein from Allolobophora caliginosa (Oligochaeta,Annelida)

• 1.1. In Allolobophora caliginosa, a Cd-binding protein distinct in charge from Cd-BP 14, a Cd-binding protein previously isolated from the same oligochaete species [Nejmeddine et al. (1992) Comp. Biochem. Physiol.101C, 601–605], has been purified by a three-step chromatographic procedure including gel permeation and cation-exchange chromatography.
• 2.2. This Cd-binding protein exists in a monomeric form with a molecular weight of 14 kDa and does not contain carbohydrate.
• 3.3. The purified protein significantly absorbed at 280 nm and its amino acid composition revealed the presence of a high level of aromatic amino acids and a lack of cysteine, indicating that the molecule is distinct from metallothioneins.
• 4.4. By contrast, except for its chromatographic behavior on an ion-exchange chromatography column, the metalloprotein was found to be similar to Cd-BP 14. We thus conclude that it represents a charge-variant of Cd-BP 14.

     

Immunogenicities of haemocyanins from the giant African snail (Achatina fulica) and the keyhole limpet (Megathura crenulata).

1. The immunogenicities of the giant African snail (Achatina fulica) haemocyanin (AFH) and the keyhole limpet (Megathura crenulata) haemocyanin (KLH) were compared by determining their capacities to induce humoral and cell-mediated immune response in rats. 2. KLH was found to be more immunogenic than AFH, and this was attributed to the fact that KLH contains more moieties, and probably more antigenic determinants than AFH. 3. Since AFH was found capable of stimulating both humoral and cell-mediated immunity, it was suggested that it could be used as an antigen for the investigation of immune responses.