Enzyme I from salmonella typhimurium.
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Amino Acids↗
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Reconstitution of TATA-binding protein-associated factor/TATA-binding protein complexes for in vitro transcription.
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Animals↗
Polymer-protein interactions in aqueous two phase systems: fluorescent studies of the partition behaviour of human serum albumin.
This study describes the partitioning of fluorescent macromolecules in aqueous two-phase systems (ATPS) comprising phosphate salt and poly(ethylene glycol) of three different molecular masses (i.e. 1000, 1450 and 2000 Da). The impact of system assembly was studied with fluorescent macromolecules introduced in contact with either (i) first salt, then polymer or (ii) first polymer, then salt, or (iii) with both salt and polymer simultaneously. Native human serum albumin (HSA) and derivatives labelled with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid (1,5-IAEDANS) were partitioned using selected ATPS. Partitioning behaviour was characterised by molecular rotational studies of recovered proteins based upon changes of depolarisation. Measurements were undertaken by steady-state fluorescence or time-decay fluorescence using a single-photon counting system. In addition, circular dichroism was used as a tool for the study of macromolecular secondary structure. Two discrete categories of stable molecular structure have been identified that exist irrespective of the phase environment. The findings form the basis for a discussion of polymer protein interactions and the molecular micro-environment of proteins in ATPS.
Chemistry Techniques, Analytical↗
Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group.
S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.
Amino Acid Sequence↗
Synthesis, characterization, and degradation behavior of amphiphilic poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(epsilon-caprolactone).
A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(epsilon-caprolactone) (PCL) sequences onto a water-soluble poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) backbone. The graft copolymers were prepared through the ring-opening polymerization of epsilon-caprolactone (CL) initiated by the macroinitiator PHEA with pendant hydroxyl groups without adding any catalyst. By controlling the feed ratio of the macroinitiator to the monomer, the copolymers with different branch lengths and properties can be obtained. The successful grafting of PCL sequences onto the PHEA backbone was verified by FTIR, 1H NMR, and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The hydrolytic degradation and enzymatic degradation of these graft copolymers were investigated. The results show the hydrolytic degradation rate increases with increasing content of hydrophilic PHEA backbone. While the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. In situ observation of the degradation under polarizing light microscope (PLM) demonstrates the different degradation rates of different regions in the polymer samples.
Aspartic Acid↗
PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome.
The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.
Adenosine Triphosphatases↗
Building doors into cells.
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Bacterial Toxins↗
An investigation of the membrane topology of the ionotropic glutamate receptor subunit GluR1 in a cell-free system.
We have utilized cell-free translation in rabbit-reticulocyte lysate supplemented with canine pancreatic microsomal membranes to study the processing and membrane topology of the rat ionotropic glutamate receptor subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translated to yield a primary translation product with an apparent molecular mass of 99 kDa. In the presence of microsomal membranes this protein was processed to give a band of 107 kDa. Treatment with peptide-N-glycosidase F showed that this increase in molecular mass was due to N-linked glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decreased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated across the microsomal membrane and protected from the action of the protease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following translocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR1 was abolished, but was retained for GluR1M1. These data allow identification of the orientation of the N- and C-termini of GluR1 within the microsome; results which are consistent with an extracellular N-terminal and intracellular C-terminal localization following incorporation into the plasma membrane.
Animals↗
Conditional regulation of adenylyl cyclases by G-protein beta gamma-subunits.
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Adenylyl Cyclases↗
Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli.
Type III secretion systems, designed to deliver effector proteins across the bacterial cell envelope and the plasma membrane of the target eukaryotic cell, are involved in subversion of eukaryotic cell functions in a variety of human, animal and plant pathogens. In enteropathogenic Escherichia coli (EPEC), several protein substrates for the secretion apparatus were identified, including EspA, EspB and EspD. EspA is a structural protein and the major component of a large transiently expressed filamentous surface organelle that forms a direct link between the bacterium and the host cell, whereas EspD and EspB seem to form the mature translocation pore. Recent studies of the type III secretion systems of Shigella and Salmonella pathogenicity island (SPI)-1 revealed the existence of a macromolecular complex that spans both bacterial membranes and consists of a basal structure with two upper and two lower rings and a needle-like projection that extends outwards from the bacterial surface. MxiH (Shigella) and PrgI (Salmonella) are the main components of the needle of the type III secretion complex. A needle-like complex has not yet been reported in EPEC. In this study, we investigated EscF, a protein sharing sequence similarity with MxiH and PrgI. We report that EscF is required for type III protein secretion and EspA filament assembly. Moreover, we show that EscF binds EspA, suggesting that EspA filaments are an extension of the type III secretion needle complexes in EPEC.
Amino Acid Sequence↗
Local drug and gene delivery through microbubbles.
Ultrasound contrast agents (microbubbles) lower the threshold for cavitation by ultrasound energy. Ultrasound microbubbles may be used as cavitation nuclei for drug and gene delivery. By tailoring the physical properties of microbubbles and coating materials, drugs and genetic drugs can be incorporated into ultrasound contrast agents. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing the therapeutic agents. Cavitation also causes a local shockwave that improves cellular uptake of the therapeutic agent. As a result of the human genome project and continuing advances in molecular biology, many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Transthoracic ultrasound can be focused on the heart so that an intravenous injection of gene-bearing microbubbles will deliver genes relatively selectively to the myocardium. Using this technique, we have produced high levels of transgene expression in the insonated region of the myocardium. This new technology, using microbubbles and ultrasound for drug and gene delivery, merits further study and development.
Animals↗
Inhibition of protein synthesis initiation by oxidized glutathione: activation of a protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2.
Oxidized glutathione (GSSG) (0.02-0.5 mM) inhibits reticulocyte lysates by a mechanism similar to that observed in heme deficiency. Incubation of hemin-supplemented postribosomal supernates with GSSG results in the activation of a translational inhibitor [I(GSSG)]. The activation of I(GSSG) is enhanced by the presence of an energy-regenerating system. The simultaneous addition of 1 mM dithiothreitol blocks the activation of the GSSG-induced inhibitor; however, once inhibitor is formed, its activity is not affected by 1 mM dithiothreitol. GSSG-treated postribosomal supernates and partially purified preparations of I(GSSG) inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. Inhibition by I(GSSG) is blocked by cyclic AMP (2-10 mM) and is potentiated by ATP (2 mM). The inhibition is also blocked or reversed by eukaryotic initiation factor eIF-2. The activation of I(GSSG) is accompanied by an increased cyclic AMP-independent protein kinase activity which phosphorylates the 38,000-dalton component (alpha subunit) of eIF-2; however, GSSG treatment of supernates does not alter the activity of the cyclic AMP-independent protein kinase activity that phosphorylates the 49,000-dalton polypeptide component (beta subunit) of eIF-2. These data indicate that GSSG treatment of reticulocyte lysates results in the activation of a protein kinase with inhibitory and phosphorylation properties similar to those of the heme-regulated cyclic AMP-independent protein kinase which is activated in heme deficiency.
Cell-Free System↗
Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids.
Purified acetylcholine receptor and total lipids, both extracted from Torpedo californica electroplax, were utilized to reconstitute chemically excitable membrane vesicles. Reconstitution was achieved by dialysis of the extraction detergent, octyl beta-D-glucoside from protein/lipid incubation mixtures. The reconstituted preparations could be fractionated by sucrose density gradient centrifugation and consisted of vesicular structures visible in electron micrographs. In addition, the reconstituted vesicles exhibited;the following properties characteristic of native receptor-enriched membranes: (i) an external distribution of alpha-bungarotoxin-binding sites, (ii) a time-dependent binding of alpha-bungarotoxin that is depressed by preincubation with the cholinergic agonist carbamoylcholine ("desensitization"), (iii) an ability to retain 22Na+ that is lost in the presence of detergents or gramicidin A, and (iv) a carbamoylcholine-induced acceleration of 22Na+ efflux that can be blocked by alpha-bungarotoxin. The purified acetylcholine receptor that was utilized in the reconstitution experiments apparently does not require other protein components for ligand recognition or ion translocation.
Animals↗
Camphor hydroxylase of Pseudomonas putida: vestiges of sequence homology in cytochrome P-450CAM, putidaredoxin, and related proteins.
The amino acid sequences of cytochrome P-450CAM and putidaredoxin of the camphor hydroxylase [camphor, reduced-putida-ferredoxin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.15.1] of Pseudomonas putida are compared to each other and then to the sequences of bovine adrenodoxin and cytochrome b5. The comparisons reveal areas of homology indicating that these four proteins may share a common evolutionary origin. Moreover, homologous segments can be recognized by proper alignment of the sequence of cytochrome P-450CAM to recently determined sequences of other P-450 hemeproteins. Further scrutiny indicates vestiges of internal sequence duplications that have been conserved in limited segments by the constraints of selection over a long time period, while other segments of the sequence diverged. It is predicted that the corresponding reductases will show a similar sequence pattern. The conserved regions of internal sequence repetition may serve a special purpose in protein-protein interactions within the multienzyme systems.
Amino Acid Sequence↗
Antigenic crossreactivity of the alpha subunit of complement component C8 with the cysteine-rich domain shared by complement component C9 and low density lipoprotein receptor.
Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.
Complement C8↗
Structural homology of complement protein C6 with other channel-forming proteins of complement.
The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.
Amino Acid Sequence↗
Platelet-derived growth factor (PDGF) and PDGF receptor are induced in mesangial proliferative nephritis in the rat.
We investigated whether platelet-derived growth factor (PDGF), or its receptor (PDGF-R), was upregulated in a rat model of mesangial proliferative glomerulonephritis. A marked increase in both PDGF A- and B-chain mRNA could be demonstrated in glomerular RNA by Northern blot analysis 3 and 5 days after disease induction, corresponding to the time of mesangial cell proliferation. PDGF-R beta-subunit mRNA and protein were also increased in glomeruli in mesangial proliferative nephritis, being maximal at day 5. The principal cells expressing PDGF B-chain appeared by immunostaining to be a subpopulation of mesangial cells; in contrast, the majority of the mesangial cells expressed the PDGF-R beta-subunit protein. Both complement depletion and platelet depletion significantly reduced cell proliferation and expression of both PDGF and PDGF-R. Thus, in mesangial proliferative nephritis there is a platelet- and complement-mediated induction of PDGF A and B chain and PDGF-R beta-subunit gene transcription and protein synthesis. The finding that the majority of PDGF is produced by the mesangial cell supports the role of PDGF as an autocrine growth factor in glomerulonephritis.
Animals↗
Supramolecular organization of the mammalian translation system.
Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.
Adenosine Triphosphate↗