Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “MACROMOLECULAR SYSTEMS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed1979

mRNA-directed biosynthesis of alpha subunit of thyrotropin: translation in cell-free and whole-cell systems.

mRNA from mouse thyrotropic pituitary tumors was translated in frog oocytes (a whole-cell system) and in wheat germ extract and reticulocyte lysate (cell-free systems) in the presence of [(35)S]methionine. Synthesized peptides related to thyrotropin were identified in the three systems by immunoprecipitation with subunit-specific antisera developed against the alpha subunit of ovine lutropin (luteinizing hormone) and the beta subunit of bovine thyrotropin. In wheat germ extract and reticulocyte lysate, a single immunoprecipitable form of the alpha subunit of thyrotropin was synthesized with an apparent molecular weight of 14,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the frog oocyte, three forms of immunoprecipitable alpha subunit of thyrotropin were synthesized with apparent molecular weights of 20,000, 14,000, and 10,000. The 20,000 form is similar to unlabeled rat pituitary standard alpha subunit and (35)S-labeled mouse tumor alpha subunit in cell cultures (20,000-21,000); thus, it may represent a precursor-cleaved and glycosylated form. The 14,000 form synthesized in all three systems probably represents the pre-alpha subunit of thyrotropin; the 10,000 form, synthesized only in the frog oocyte, could be a proteolytically cleaved but unglycosylated form. Because only the alpha subunit of thyrotropin was identified and no larger molecular weight immunoprecipitable form of either subunit was detected in any of the translation systems, alpha and beta subunits of thyrotropin appear to be translated from separate mRNAs.

Animals↗
PubMed1980

Structural details of membrane-bound acetylcholine receptor from Tropedo marmorata.

A projection, at 15- to 20-A resolution, is presented of the structure of the membrane-bound acetylcholine receptor protein from Torpedo marmorata. The projection has its axis perpendicular to the membrane plane; its main contribution originates from a hydrated portion of the protein, which extends from the membrane into the aqueous medium. The structure is distinctly asymmetric, with individual morphological subunits barely resolvable. These results have been obtained by noncrystallographic averaging, using correlation functions, applied to electron micrographs of receptor-rich membrane fragments. The micrographs had been taken with minimal beam exposure in a scanning transmission electron microscope.

Animals↗
PubMed1990

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

We have translated RNAs for the two rat asialoglycoprotein receptor polypeptides together in a cell-free system containing dog pancreatic microsomes and immunoprecipitated the products with antibodies that distinguish the two proteins. In this system the proteins oligomerize, as judged by their coprecipitation with either of the subunit-specific antisera. Oligomerization does not occur between subunits synthesized without microsomes or between subunits synthesized on separate microsomes mixed during detergent solubilization. Thus, oligomerization occurs within the microsomal membrane. We calculate that oligomerization proceeds with an efficiency of approximately 85%. The receptor complex appears to represent a specific oligomer because it excludes a third membrane glycoprotein synthesized in the same reaction. Oligomerization of the asialoglycoprotein receptor in vitro should provide a useful system to study the assembly of a membrane-protein complex.

Animals↗
PubMed1997

The alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor: analysis using a phage-epitope library.

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. alpha-Neurotoxins, in particular alpha-bungarotoxin (alpha-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with alpha-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to alpha-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the alpha-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind alpha-BTX. The library-derived peptide and the corresponding peptides containing residues 187-199 of the Torpedo AcChoR alpha-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human alpha7; ERFYECCKEPYPD) that binds alpha-BTX, inhibit the binding of alpha-BTX to the intact Torpedo AcChoR with IC50 values of 10(-6) M. A synthetic peptide from a neuronal AcChoR that does not bind alpha-BTX (e.g., human alpha2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the alpha-BTX-binding protein (alpha7)-i.e., Lys in alpha2 and Tyr in alpha7-does not inhibit the binding of alpha-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to alpha-BTX.

Amino Acid Sequence↗
PubMed1997

A region of vitamin K-dependent protein S that binds to C4b binding protein (C4BP) identified using bacteriophage peptide display libraries.

Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (KD = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer peptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity purification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to the identification of four regions in protein S that were similar to several of the selected peptides. These regions were synthesized as linear peptides and tested in inhibition experiments. Only one distinct peak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified peptides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, respectively) in a second set of synthetic peptides. Direct binding of the peptides to C4BP was inferred from titrations monitored by recording the near UV circular dichroism spectra or the polarization of tryptophan fluorescence. The results suggest that residues 447-460 constitute a portion of protein S that is important for the interaction with C4BP. These findings may have implications for patients suffering from thrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.

Amino Acid Sequence↗
PubMed2004

Preparation and evaluation of microcapsules using polymerized rosin as a novel wall forming material.

Sustained release diclofenac sodium microcapsules were prepared using polymerized rosin as a novel wall-forming material by a solvent evaporation technique. A novel method developed in our laboratory with the potential for scale-up and production of polymerized rosin microcapsules is detailed. These microcapsules might have application for development of implant/depot systems, primarily due to a sustained/controlled release capability and potential biocompatibility of polymerized rosin. The effect of variables like solvent systems, stirring speed and temperature were previously optimized. The solution system of drug and polymerized rosin dissolved in iso-propyl alcohol and acetone is sprayed with the help of a 0.5 mm nozzle spray gun in liquid paraffin maintained at 60 degrees C in the stirring condition. Varying drug:polymer ratios, namely 1:1, 1:2, 2:1, 1:3 and 3:1, were employed for microcapsule preparation. The prepared microcapsules were evaluated for size, shape, drug content and in vitro drug release. The morphology of microcapsules was characterized by scanning electron microscopy. The microcapsules show sustained release curves at pH 7.4 phosphate buffer for up to 10 h. The data obtained from the dissolution profiles were compared in the light of different kinetics models and the regression coefficients were compared. The in vitro dissolution study confirmed the Higuchi-order release pattern. Particle size and release data analysis from five consecutive batches prepared in the laboratory indicated suitable reproducibility of the proposed solvent evaporation process.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.

The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase. The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350). In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death. Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive. The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350. In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.

Antibodies, Monoclonal↗
PubMed2003

Metabolism of macromolecules in tissue.

Hyaluronan (HA) plays an important role in maintaining tissue integrity, as well as in facilitating the migration of cells during inflammation, wound repair, and embryonic development. By comparison with other macromolecules of the extracellular matrix, HA undergoes rapid turnover. Most of its degradation does not occur locally, but within distant lymph nodes. During this process, tissue HA enters the afferent lymphatics and is transported with the lymph to the draining lymph nodes. Because of its unique catabolic pathway, the fate of HA is an interesting subject for investigation, particularly in pathological states of lymphangiodysplasia or lymphatic damage.

Animals↗
PubMed1976

Studies on P-450. X. On the coordination structure of hemoprotein P-450.

The spectral properties of a P-450 preparation from adrenocortical mitochondria were compared with those of both other hemoproteins and model compounds to examine the possibility suggested by previous authors that the mercaptide anion (thiolate anion, RS-) of a cysteine residue might be one of the axial ligands of P-45O. 1. The following features were found in addition to the well known anomalous spectral characteristics of P-450. The Soret band of the ferrous form was at anomalously short wavelengths compared with those of other ferrous high-spin hemoproteins. The regularity in the shifts of the Soret maximum observed with various forms of usual hemoproteins was disrupted in the case of P-450 by the maximum of the ferrous form. 2. The complex of methemoglobin with 2-mercaptoethanol, which was the model for P-450 used by previous authors for spectral and ESR studies, was studied more precisely. The model showed an absorption spectrum very similar to that of the imidazole complex of ferric P-450, whereas the complex of methemoglobin with imidazole showed both Soret and visible absorption maxima at shorter wavelengths. The rapid rate of reduction of methemoglobin by dithionite decreased on binding to mercaptoethanol and the slow rate of reduction of P-450 increased on binding to imidazole. Thus the half-reduction times for both complexes were of the same order of magnitude. 3. An absorption spectrum which was very similar to the spectrum of ferric high-spin P-45O could be obtained by quick scanning immediately after ferric protoheme had been dissolved in a 33% solution of mercaptoethanol in water containing Tween 20 and NaOH. 4. Protoheme dissolved in an aqueous solution of Nikkol BL9EX (a non-ionic detergent) containing NaOH, dithionite, and 1 mM mercaptoethanol formed a transient intermediate complex having a Soret maximum at 470 nm. Treatment of this complex with carbon monoxide resulted in the formation of a CO adduct having a Soret maximum at 450 nm and a single band at about 555 nm in the visible region. These maxima were similar to those of the carbon monoxide complex of P-450. Many of the results could be explained readily by assuming that a mercaptide anion was one of the axial ligands, and also that heme was held in a hydrophobic crevice in the P-450 molecule. The coordination structure of P-450 seems to be more flexible than those of usual hemoproteins.

Adrenal Cortex↗
PubMed2006

Integrated software for macromolecular crystallography synchrotron beamlines II: revision, robots and a database.

This manuscript chronicles the evolution of software used originally to control a diffractometer at a macromolecular crystallography beamline. The system has been augmented and rewritten. A modular and carefully organized suite of programs now handles the whole experimental environment from a single vantage point. It provides automatic logging of the experiment and communication with the user, all the way from an initial proposal to perform the work to the end of data collection. This has included construction of a relational database to organize all details of the experiment and incorporation of a robotic specimen changer to provide automation for high-throughput applications.

Crystallography↗
PubMed2005

Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ.

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.

Amino Acid Sequence↗
PubMed1976

The action pattern of amylomaltase from Escherichia coli.

Amylomaltase, the inducible 4-alpha-glucanotransferase of Escherichia coli strain ML, has been purified to homogeneity. Its specific activity with a commercial maltose substrate was 500 mkat/kg protein (30 mumol glucose formed min-1 mg protein-1). The purified enzyme, dependent on buffer concentration, exists in interconvertible low-molecular-weight (apparent molecular weight 71000) and high-molecular-weight (apparent molecular weight 370000) forms. The specificity of amylomaltase has been redefined. Hitherto, the enzyme was thought to be a glucosyltransferase, catalysing the transfer of single glucosyl units, and maltose has been regarded as its most important substrate. Amylomaltase is now shown to exhibit both glucosyl-transfer and 4-alpha-glucanosyl-transfer specificity. 4-alpha-Glucanosyl chains containing up to at least nine glucosyl units can be transferred. However, it is concluded that the transfer reaction by which amylomaltase action was originally expressed, does not take place, i.e., Maltose + maltose in equilibrium Maltotriose + glucose and that maltose has a restricted role as a substrate. This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate. It is confirmed that when a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing-end-group. Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release. The lag pahse is overcome by addition of 'priming' (catalytic) concentrations of maltotriose or higher maltodextrins. An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose. The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous 1,4-alpha-glucans by E. coli.

Escherichia coli↗
PubMed1989

On the role of phospholipids in the reconstituted cytochrome P-450 system. A model study using dilauroyl and distearoyl glycerophosphocholine.

The difference in pentoxyresorufin O-dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P-450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P-450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P-450 for the NADPH-cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho-dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P-450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P-450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P-450 molecule, but also on the efficiency of electron transfer from NADPH-cytochrome reductase to cytochrome P-450.

Animals↗
PubMed1991

Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology.

An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.

Amino Acid Sequence↗
PubMed2005

Polarity of enteropathogenic Escherichia coli EspA filament assembly and protein secretion.

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic Escherichia coli, two important human enteric pathogens, is unique in that it has an approximately 12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).

Cell Line↗
PubMed1968

Screening of Escherichia coli temperature-sensitive mutants by pretreatment with glucose starvation.

A system for screening Escherichia coli temperature-sensitive mutants is described. The system involves glucose starvation and minimizes ambiguities introduced by the interdependencies of macromolecular synthesis during balanced growth. The system permits the quick recognition of protein synthesis mutants and their classification into two general catagories. Complete protein synthesis mutants are unable to make any polypeptide material, whereas partial protein synthesis mutants are able to produce inactive proteins. The phenotypes of several mutants are described.

Bacterial Proteins↗
PubMed1985

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.

Animals↗
PubMed1990

Characterization of a mammalian ribosomal protein gene promoter.

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.

Adenoviridae↗