Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A.

Three photosynthetic complexes, light-harvesting complex 2 (LH2), light-harvesting complex 1 (LH1), and the reaction centre-light-harvesting complex 1 photounit (RC-LH1), were purified from a single species of a purple bacterium, Rhodobacter sphaeroides, and reconstituted into two-dimensional (2-D) crystals. Vesicular 2-D crystals of LH1 and RC-LH1 were imaged in negative stain and projection maps at 25 A resolution were produced. The rings formed by LH1 have approximately the same mean diameter as the LH1 rings from Rhodospirillum rubrum ( approximately 90 A) and therefore are likely to be composed of 15 to 17 alphabeta subunits.

Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution.

Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens fibre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with N-octyl-beta-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-beta-D-maltoside.

Uptake of 125I-PDGF-AB to the blood after extravascular administration in mice.

The kinetics of exogenously given 125I-platelet-derived growth factor-AB (PDGF-AB) was studied in mice. 125I-PDGF-AB was injected either intraperitoneally, intramuscularly or subcutaneously and the resulting concentrations of 125I-radioactivity monitored in the blood at different times. The serum levels of 125I-radioactivity rose to a maximum 2-4 hours after injection, before decreasing.

Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.

Electron microscopy has become a powerful technique, along with X-ray crystallography and nuclear magnetic resonance spectroscopy, to study the three-dimensional structure of biological molecules. It has evolved into a number of methods dealing with a wide range of biological samples, with electron crystallography of two-dimensional crystals being so far the only method allowing data collection at near-atomic resolution. In this paper, we review the methodology of electron crystallography and its application to membrane proteins, starting with the pioneering work on bacteriorhodopsin, which led to the first visualization of the secondary structure of a membrane protein in 1975.

2D crystallization of membrane proteins: rationales and examples.

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy.

Localization of granule proteins in human eosinophil bone marrow progenitors.

Eosinophils have a characteristic content of cationic proteins, stored in core-containing specific granules and released at sites of inflammation; coreless granules (sometimes called primary) are present in eosinophil promyelocytes. In order to determine a possible relationship between the two granule subsets, immunoelectron-microscopic techniques were used to determine the presence and precise intragranular distribution of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and arylsulfatase B of eosinophil granules, as well as the Charcot-Leyden crystal (CLC) protein, in eosinophil progenitors of the bone marrow. MBP, ECP, EPO, and arylsulfatase B were observed in both coreless and core-containing (specific) granules.

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold.

The three-dimensional structure of aquaporin-1.

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1).

Two-dimensional crystallization of the light-harvesting I-reaction centre photounit from Rhodospirillum rubrum.

A stoichiometric unit of the light-harvesting complex I and the reaction centre (LHI-RC complex) has been isolated from a carotenoid-less mutant of the purple non-sulphur bacterium Rhodospirillum rubrum by mild solubilization of photosynthetic membranes with the phospholipid detergent diheptylphosphatidylcholine. Dialysis of the isolated LHI-RC complexes in the presence of added dioleoyl-sn-phosphatidylcholine produced ordered two-dimensional crystals. Digital image processing revealed that the LHI-RC are packed together in a square lattice (a = b = 16.

Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.

Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well-ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse.

Highly ordered two-dimensional crystals of photosystem I reaction center from Synechococcus sp.: functional and structural analyses.

The photosystem 1 reaction center complex from the thermophilic cyanobacterium Synechococcus sp. was isolated by Triton X-100 solubilization and fractional precipitation with polyethylene glycol. As shown by gel electrophoresis, the isolated complex was composed of the 83 kDa subunits A and B, and at least six other subunits with molecular mass below 20 kDa.

Tubular crystals of a photosystem II core complex.

An oxygen evolving photosystem II core complex containing all three extrinsic proteins (33, 23, 17 kDa) was isolated from spinach and reconstituted into tubular two-dimensional crystals of 72.9 nm diameter and 1-2 micrometers length. While the 17 and 23 kDa polypeptides were lost during crystallization, the extrinsic 33 kDa protein was retained.

Subcellular localization of transforming growth factor-alpha in human eosinophil granulocytes.

Eosinophils are involved in the inflammatory response seen in allergy and helminthic infestations. Eosinophils synthesize transforming growth factor-alpha (TGF-alpha), which may play a role in the development of the characteristic fibrosis seen in longstanding high eosinophilia. Using immunoelectron microscopic techniques, eosinophils from peripheral blood of healthy individuals and from one patient with high eosinophilia showed presence TGF-alpha in matrix of the specific crystalloid-containing granules.

Projection map of aquaporin-1 determined by electron crystallography.

Using cryo-electron microscopy we have determined a projection map of the structure of the water selective pore aquaporin-1.

Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure.

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm.

Transforming growth factor-alpha (TGF-alpha) in human bone marrow: demonstration of TGF-alpha in erythroblasts and eosinophilic precursor cells and of epidermal growth factor receptors in blastlike cells of myelomonocytic origin.

The expression of transforming growth factor-alpha (TGF-alpha) in human differentiating leukemic cell lines and in circulating human eosinophils prompted the search for an analogous function in normal human bone marrow (BM) cells. Immunohistochemistry, using a monoclonal antibody directed to the mature form of the TGF-alpha molecule, showed TGF-alpha on the erythroblasts of normal donors. This novel property of erythroid cells was found on cells at all stages of maturation, most clearly on nucleated forms but to some extent also on erythrocytes within the BM.

Production of transforming growth factor alpha by human leukemia cells (HL-60 and U-937) during monocytic differentiation.

We have previously demonstrated that human promyelocytic HL-60 cells express transforming growth factor-alpha (TGF-alpha) during granulocytic differentiation. The present experiments were carried out in order to determine whether cells differentiated towards monocytes/macrophages will analogously express the TGF-alpha proto-oncogene product. HL-60 cells were induced to differentiate with 1 microM 1,alpha 25-dihydroxycholecalciferol (vitamin D3), and the human monocytoid cell line, U-937, was induced with 1 microM retinoic acid (RA), 0.

The three-dimensional structure of human erythrocyte aquaporin CHIP.

Water-permeable membranes of several plant and mammalian tissues contain specific water channel proteins, the 'aquaporins'. The best characterized aquaporin is CHIP, a 28 kDa red blood cell channel-forming integral protein. Isolated CHIP and Escherichia coli lipids may be assembled into 2-D crystals for structural analyses.

Mammalian uroplakins. A group of highly conserved urothelial differentiation-related membrane proteins.

The asymmetric unit membrane (AUM) forms the apical plaques of mammalian urothelium and is believed to play a role in strengthening the urothelial apical surface thus preventing the cells from rupturing during bladder distention. We have shown previously that purified bovine AUMs contain four major integral membrane proteins: the uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa). This contradicts some previous reports indicating that some of these proteins are absent in AUMs of several species.

Transforming growth factor alpha expression in normal human blood eosinophils: differential regulation by granulocyte-macrophage colony-stimulating factor and interleukin-3.

We have previously demonstrated a constitutive expression of transforming growth factor alpha (TGF-alpha) in normal human blood eosinophils, both at the mRNA and protein level. This may indicate a novel function of the eosinophil, the regulation of which has not been clarified. Therefore human white blood cells (WBC) were treated with potential regulators of eosinophil function.

Biologically active two-dimensional crystals of aquaporin CHIP.

Plasma membranes of several mammalian tissues are highly permeable to water due to the presence of CHIP, the 28-kDa channel-forming integral protein which is the archetypal member of the aquaporin family of water channel proteins. To define its native structure, purified red cell CHIP protein was reconstituted into lipid bilayers at a high protein-to-lipid ratio, and the resulting 3-microns diameter membrane vesicles were examined by high resolution electron microscopy. The reconstituted membranes contained highly ordered two-dimensional crystalline lattices of p422(1) symmetry in which each CHIP tetramer contained a central depression extending from the outer and inner surfaces of the membrane into the transbilayer domain of the molecule.

Production of transforming growth factor alpha by normal human blood eosinophils.

Transforming growth factor alpha (TGF-alpha) is a pleiotropic factor mediating numerous cellular responses in normal and transformed cells. This includes differentiation, proliferation, migration, and formation of extracellular matrix. TGF-alpha has been demonstrated in circulating eosinophils from the idiopathic hypereosinophilic syndrome and in differentiating promyelocytic leukemia cells in vitro.

Human erythrocyte band 3. Solubilization and reconstitution into two-dimensional crystals.

Various polyoxyethylene alkylethers were used to extract integral proteins from human erythrocyte membranes. The solubilization power of these detergents and the oligomerization of solubilized band 3 were studied. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that short-chain detergents induced oligomers larger than the band 3 dimer.