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At least 199 records · Page 11Linked to original sources
PubMed1992

Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.

Adult↗
PubMed1992

The roles of fibrinogen and fibrin in hemostasis and thrombosis.

Proteolytic conversion of fibrinogen to fibrin results in self-assembly to form a clot matrix that subsequently becomes cross-linked by fXIIIa to form the main structural element of the thrombus in vivo. Fibrin formation and assembly lead to new properties that regulate the rate and extent of clotting, cross-linking, and fibrinolysis. These are brought about by the ability of fibrin (1) to bind thrombin at a nonsubstrate site, thus limiting its diffusability but at the same time preserving its catalytic potential; (2) to bind fXIII, regulate its activation to fXIIIa, and limit further activation of fXIII once fibrin cross-linking has occurred; and (3) to bind alpha 2-PI, t-PA, and plasminogen and regulate the initiation and propagation of fibrinolysis. Fibrinogen and fibrin contain several potential platelet binding sites that interact with platelet GPIIb/IIIa receptors, and thus promote their participation in the hemostatic process. Additional, less well-defined interactions, not covered in detail here, such as those between fibrinogen or fibrin and other plasma proteins, cells, or tissue matrix components, suggest other functions that, along with those detailed above, will further define its multiple roles in modulating hemostasis, inflammation, and the wound healing process.

Amino Acid Sequence↗
PubMed1992

Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.

Adenosine Triphosphate↗
PubMedDate not supplied

[Role of phospholipids in the generation of membrane potentials by proteoliposomes].

Closed protein-phospholipid particles (proteoliposomes), obtained by self-assembly method, are capable to generate and to maintain the membrane potential in the case if their protein complex is represented by: a) a complex of mitochondrial ATPase; b) a complex of cytochrome oxidase and cytochrome c and c) bacteriorhodopsin from Halobacterium halobium; and their phospholipid component is represented by phosphatidylethanolamine or by a mixture of mitochondrial phospholipids. Only cytochromoxidase and bacteriorhodopsin (but not ATPase) proteoliposomes with phosphatidylserine are active. Cardiolipin also is not active in experiments with ATPase. Phosphatidylcholine produces in all the cases proteoliposomes incapable of maintaining the membrane potential. It is concluded that the inefficiency of phosphatidylcholine in the formation of proteoliposomes, generating the membrane potential, is due to the impossibility of obtaining closed membrane forms with a high electric resistance. The inefficiency of phosphatidylserine and cardiolipine, in the case of ATPase protein component of proteoliposomes, may be due to a specific requirement of this generator of the membrane potential in phosphatidylethanolamine.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Anticoagulants of the nondialyzed fractions of the ammonia extract of the herb Pulmonaria mollissima].

T-100 anticoagulants non-dialyzed through cellophane are isolated from ammonia extract of herb Pulmonaria mollissima. Their effect is mainly realized at the stage of coagulation conversions of fibrinogen, first of all at the stage of fibrin self-assembly. One of these anticoagulants is a peptide, another one is a glycopeptide which contains glucose residues like carbohydrate. Peptide components of the both anticoagulants are characterized by the high level of amino acids, their radicals are capable to ionization under physiological conditions. In contrast to animal-origin analogs, glycopeptide in nontoxic doses causes stable hypocoagulemia in animals. It is expedient to study Pulmonaria mollissima extracts as a source of direct anticoagulants.

Amino Acids↗
PubMed1991

Binding and calcium-induced aggregation of laminin onto lipid bilayers.

Direct binding of laminin in the form of its complex with nidogen to planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy. Binding occurred equally well to zwitterionic (phosphatidylcholine) and negatively charged (phosphatidylglycerol) lipids and was enhanced by sulfatides but only at nonphysiological molar ratios higher than 30 mol %. Strong interactions with lipid bilayers were also observed for bovine serum albumin. This explains a strong inhibition of laminin binding by this protein. However, binding of laminin to sulfatide-rich bilayers was not completely inhibited. Observable by the microscopic technique was the formation of laminin clusters on the surface of the bilayer which occurred concomitantly with binding. Both processes were strongly enhanced by the presence of calcium. These results show that calcium-induced laminin self-assembly is enhanced at lipid surfaces.

Calcium↗
PubMed1990

Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae.

Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pKa values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the reproducible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 A resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution.

Bacterial Outer Membrane Proteins↗
PubMed1991

Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein.

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

DNA-Binding Proteins↗
PubMed1991

Bovine glomerular basement membrane. Location and structure of the asparagine-linked oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer.

Collagen IV contains an amino-terminal tetramerization domain (7 S) that is involved in aggregation and cross-linking as part of the process of self-assembly of the collagen IV matrix of basement membranes. We determined the structure and location of the Asn-linked oligosaccharides of the 7 S tetramer. Two glycopeptides, GP-1 and GP-2, were isolated from tryptic digests of the 7 S tetramer and were characterized. GP-1 and GP-2 are derived from the alpha 1(IV) chain and the alpha 2(IV) chain, respectively. Each glycopeptide contained one sequence, -Asn-Xaa-Thr-, which was shown to be N-glycosylated at Asn, corresponding to position 126 of the alpha 1 chains and 138 of the alpha 2 chain. 1H NMR spectroscopic analysis of the oligosaccharide is a biantennary N-acetyllactosamine type of N-linked oligosaccharide with a broad heterogeneity in the presence of the sugar residues at their nonreducing termini as indicated. [formula: see text] The location of the Asn-linked oligosaccharide units and Hyl-linked disaccharide units and their orientation with respect to the surface of the triple helix were calculated using two models. We conclude that both units are important determinants in the assembly of the 7 S tetramer.

Amino Acid Sequence↗
PubMed1991

Human adenovirus serotype 3 fiber protein. Comparison of native and recombinant proteins.

We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.

Adenoviridae↗
PubMed1978

Protein-lipid interactions and the role of water.

The rigidity of the three-dimensional structure of a native protein is dependent on the network of hydrogen-bonded groups which provide the scaffolding for the other interactions. The structure is stabilized by the hydrophobic interactions of the nonpolar side chains. The latter are formed by the very unfavorable entropy change that occurs in water but not in less-polar solvents. It is unlikely that any solvent other than water can produce the same folding of a polypeptide chain to form the active native structure. Water plays a unique role, since it alone is responsible for the heat capacity changes observed when nonpolar groups are transferred from an aqueous to a nonaqueous environment, as exists in the interior of a protein. The need to juxtapose like groups and to avoid making contact among unlike groups imposes severe restrictions on the binding of small or large molecules to proteins. Consequently there must be proper pairing of polarities as well as close fitting of ligands for strong binding to occur. This is clearly evident from the x-ray studies of proteins containing subunits or prosthetic groups. The thermodynamic parameters observed in the most complex protein reactions--i.e., self-assembly systems--resemble rather well those observed in micelle association reactions or even in the solution of nonpolar gases in water. This interaction--hydrophobic--can be looked upon as the controlling reaction which stabilized the organized structures of most cellular entities aside from nucleic acids--i.e., membranes and organelles.

Butanes↗
PubMedDate not supplied

[Cooperative interaction of serum albumin with quaternized poly-4-vinyl pyridine and structure of the complexes].

Interaction of bovine serum albumin (BSA) with quaternized poly-4-vinyl pyridine (PE) in aqueous solutions at pH 7 was studied. It was shown that in a wide range of the ratios of the components (nBSA/nPE) soluble stable cooperative complexes were formed. At the same time a certain critical content of the protein exists at which the system loses its homogeneity. Complex formation is not accompanied by protein denaturation. At smaller nBSA/nPE ratios non-homogeneous distribution of protein globulas among polyelectrolite macromolecules was found; this corresponded to the "all or none" principle. Using ultracentrifugation technique viscosimetric measurements and electron microscopy it was shown that the soluble complexes exist in the form of rode-like particles consisting of protein globules stabilized by polycation chains. Such particle can be considered as a model of nucleoprotein complex. At certain crytical nBSA/nPE rations the rod-like particles aggregate with additional number of BSA-molecules and form more complicate soluble and insoluble cooperative complexes. Possible structural models of the complexes described were suggested and the thermodinamic and kinetic cryteria of their self-assembly were discussed.

Drug Stability↗
PubMed1977

[Quaternary structure of oligomeric immunoglobulin A forms from human blood serum].

A character of forces stabilyzing quaternary structure of dimer and more high molecular human immunoglobulin A oligomers is found to be different. Quaternary structure of IgA dimer is formed when joining subunits with disulfide bonds and is stabilized by non-covalent interactions between them. Disulfide bonds play a main part in the formation of trimers and tetramers. Dimer IgA reconstructs by 40% from subunits with intact interchain S--S bonds. The addition of exogenous J-chain does not significantly affect the process of dimer self-assembling from subunits with recovered and intact interchain disulfide bonds.

Chemical Phenomena↗
PubMed1979

The binding of ribosomal protein S4 does not change the gross conformation of the 16 S RNA.

The binding of ribosomal protein S4 to the 16 S RNA does not result in a large shape or conformational change in the 16 S RNA under the conditions of reconstitution. The sedimentation coefficient, frictional coefficient ratio, and effective hydrodynamic radius of the 16 S RNA.protein S4 complex are very similar to those obtained for the 16 S RNA free in solution. Only subtle conformational differences were obtained in the comparison of the complex and free 16 S RNA by circular dichroism. Thus, extensive organization of the 16 S RNA by ribosomal protein S4 is not a step in the process of self-assembly of the 30 S subunit.

Circular Dichroism↗
PubMedDate not supplied

[Spectrum differentiation as a method of studying protein preparations with a high turbidity level].

The studies on a model of a mixture of fibrin-monomer with glycogen and of fibrin gels of different turbidity show that in the first and second derivatives of the absorption spectra the contribution of turbidity is several orders lower. The derivative spectra are shown to be valuable technique to study the phenomena that are accompanied by substantial changes in turbidity: protein association and self-assembly of supermolecular structure.

Fibrin↗
PubMed1978

Monoclonal IgG anticoagulants delaying fibrin aggregation in two patients with systemic lupus erythematosus (SLE).

There is paucity of information regarding the prolonged plasma thrombin time known to occur in some patients with systemic lupus erythematosus. Detailed investigations of plasma from two such patients disclosed that IgG accounted for this defect in each case. IgG isolated from plasma of either patient possessed the property of delaying fibrin aggregation and prolonging the clotting times of fibrinogen. Preincubation of IgG from either patient with anti-IgG or anti-Fab (rabbit) serum abolished this anticoagulant property. Moreover, the anticoagulant IgG from the first patient was neutralized with anit-k chain and anti-IgG3, that from the second patient with anti-lambda chain and anti-IgG1 serum. These anticoagulants were also dissimilar with respect to their interactions with fibrin(ogen). IgG from the first patient had no anticoagulant activity against fibrin(ogen) species lacking intact Aalpha chains. IgG from the second patient displayed undiminished anticoagulant effect on such fibrin(ogen) species. We conclude that each anticoagulant interacted with a distinct region(s) on the fibrinogen molecule and that these interactions affect or involve sites that participate in the fibrin self-assembly process.

Adolescent↗
PubMed1976

Biogenesis of matrix vesicles in cartilage growth plates.

Although the structure and function of cartilage matrix vesicles have been comprehensively documented in the literature, the method of vesicle production and export to the extra-cellular matrix is less well understood. The existing data are grouped into four general hypotheses of matrix vesicle biogenesis, with a consideration of the relative merits and weaknesses of each postulate. The hypotheses are: a) budding from cellular processes; b) extrusion of preformed structures; c) cellular degeneration and disintegration; and c)subunit secretion and extracellular assembly. Each of these possibilities offers conceptual advantages and disadvantages, so that it is difficult to adopt a monistic stance. Previous notions of vesicle biogenesis have derived from statis morphologic and analytical data, which are necessarily limiting when attempting to draw kinetic conclusions. Radiotracer studies of lipid synthesis and transport in cartilage lead to the conclusion that matrix vesicles in the growth plate are derived from chondrocytes; the present data favor the hypotheses of budding from cellular processes and/or subunit secretion with extracellular self-assembly.

Cartilage↗
PubMed1977

Myogenesis and contraction in the early embryonic heart of the rainbow trout. An electron microscopic study.

Myogenesis in the embryonic heart of the rainbow trout, Salmo gairdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th osmite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.

Animals↗