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Results for “Protein Multimerization”

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PubMed1988

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution.

The effect of alkaline pH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in the pH range 7-12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates at pH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S in equilibrium 7S in equilibrium 4S----2S. Above pH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at approximately pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.

Hydrogen-Ion Concentration↗

PubMed1989

The transition state transcription regulator AbrB of Bacillus subtilis is autoregulated during vegetative growth.

The DNA-binding AbrB protein of Bacillus subtilis is an ambiactive transcriptional regulator of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Studies on the transcriptional control of AbrB synthesis using abrB-lacZ fusions indicated that the abrB gene was autoregulated. This was consistent with the observation that purified AbrB protein bound specifically to the promoter region of its own gene in DNase I protection experiments. The structural gene mutation abrB4 abolished the autoregulation and purified AbrB4 protein did not have the promoter binding properties associated with the wild-type protein. Both AbrB and AbrB4 proteins were shown to be hexamers of 10,500 Dalton subunits and subunit exchange occurred between the proteins in vitro. However, the presence of only one or two mutant subunits dramaticaly altered the DNA-binding ability of the multimeric protein. The results support a model in which autoregulation of the abrB gene is an important factor in preventing sporulation-associated genes from being expressed during vegetative growth.

Bacillus subtilis↗

PubMed1990

The gene coding for the yeast oligomycin sensitivity-conferring protein.

The gene coding for the yeast Saccharomyces cerevisiae mitochondrial oligomycin sensitivity-conferring protein (OSCP) has been sequenced, and the gene products have been characterized. The OSCP is subunit 5 of the mitochondrial ATP synthase, a multimeric protein complex. As such, the gene coding for the yeast OSCP is referred to here as the ATP5 gene. From the predicted primary sequence, the calculated molecular weight of the immature yeast OSCP is 22,813 and the amino acid sequence is 35% identical and 65% homologous to bovine OSCP. A null mutant has been constructed. This mutant strain is unable to grow on glycerol medium, has no detectable oligomycin-sensitive ATPase activity, and has no detectable immune reactive proteins with the corresponding molecular weight of the OSCP (using antibodies reactive to the yeast OSCP). The transcription products of the yeast gene have been characterized. There is a single major transcript from the ATP5 gene of 1.05 kilobases. The level of the transcription product is increased from 3-5-fold after growth in galactose medium as compared to cells grown in glucose medium. The transcriptional initiation sites were determined to occur at +68(G) and +69(T) at comparable frequency and were not dependent on the growth medium. These results suggest that transcription of the ATP5 gene is catabolite-repressed.

Adenosine Triphosphatases↗

PubMed1986

Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake).

The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.

Animals↗

PubMed1989

Structural characterization of alpha-bungarotoxin-binding proteins from Aplysia californica.

Structural features of alpha-bungarotoxin-binding proteins from the marine mollusc Aplysia californica have been examined as a first step toward delineating their potential role in cholinergic neurotransmission. Protein blotting with 125I-alpha-bungarotoxin was used to identify binding proteins in membranes prepared from Aplysia muscle and nervous tissue. Binding proteins from both tissues exhibited similar physical characteristics, which distinguish them from the prototypical alpha-bungarotoxin-binding protein, the nicotinic acetylcholine receptor obtained from Torpedo californica electric organ membranes. Aplysia binding activities migrate with an apparent molecular weight of 250 kDa on sodium dodecyl sulfate (SDS) gels in the presence of reducing agents. Binding of alpha-bungarotoxin to blots of Aplysia membranes is abolished by exposure of samples to heat or to low pH but is unaffected by reduction-alkylation treatment. In contrast, the alpha-bungarotoxin-binding subunit of the acetylcholine receptor from Torpedo membranes migrates on SDS gels at 40 kDa. It retains binding activity following exposure to heat or to low pH, but binding is substantially diminished by reduction-alkylation treatments. Another distinguishing characteristic of the Aplysia binding activities is revealed by examining recovery of membrane alpha-bungarotoxin-binding on protein blots; the high recovery of Aplysia binding contrasts sharply with the low recovery of Torpedo binding activity. The high apparent molecular weight of the Aplysia alpha-bungarotoxin-binding activities, their most distinguishing feature, is similar to an alpha-bungarotoxin-binding activity recently identified in lower vertebrate brain. Covalent cross-linking with 125I-alpha-bungarotoxin demonstrates, however, that the mobility of both Aplysia binding activities is due to a multimeric protein that is unusually resistant to dissociation in SDS. The covalently radiolabeled Aplysia alpha-bungarotoxin-binding activity migrates at approximately 260 kDa on SDS gels when solubilized at room temperature. When it was boiled before electrophoresis, the mobility of the radiolabeled protein shifts to approximately 70 kDa. Resistance to dissociation in the absence of boiling may explain both the high recovery of activity on blots and the insensitivity to reductive alkylation. Conversely, dissociation of the multimeric complex upon boiling may explain the observed loss of binding activity. Our results demonstrate structural similarities and differences between Aplysia alpha-bungarotoxin-binding proteins and the Torpedo acetylcholine receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis.

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.

Animals↗

PubMed1990

The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis.

We have cloned and characterized the mtr operon of Bacillus subtilis. This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon. We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively. MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4. The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter. Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein. The in vivo action of the mtrA and mtrB gene products was analyzed in an E. coli strain containing a trpE-lacZ gene fusion under control of the B. subtilis trp promoter/attenuator region. Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.

Amino Acid Sequence↗

PubMed1988

Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro.

The efficient export of a subset of Escherichia coli envelope proteins is dependent upon the product of the secB gene. Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form. In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits. SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles. The addition of purified SecB to an in vitro system prepared from SecB- cells significantly enhanced MBP translocation. The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes. Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.

ATP-Binding Cassette Transporters↗

PubMed1991

Protein kinase activity required for an early step in interferon-alpha signaling.

Interferon-alpha (IFN alpha) induces an immediate transcriptional response of a restricted set of genes in target cells. Specific transcription is mediated by the cytoplasmic activation of a transcription factor complex termed ISGF3. ISGF3 is a multimeric protein complex composed of a regulatory component (ISGF3 alpha), which is activated following IFN alpha treatment, and a DNA-binding component (ISGF3 gamma), which recognizes the IFN alpha-stimulated response element (ISRE). Following activation, ISGF3 alpha translocates to the nucleus where ISGF3 assembles as a high affinity complex on the ISRE. The biochemical basis for receptor-mediated activation of ISGF3 is unknown. We report that two potent protein kinase inhibitors, staurosporine and K-252a, ablated the transcriptional response to IFN alpha treatment. These inhibitors prevented the activation of the ISGF3 alpha component without affecting the ISGF3 gamma component, resulting in no accumulation of mature ISGF3 in nuclei of treated cells. Although these agents are potent inhibitors of protein kinase C (PKC), PKC does not mediate ISGF3 alpha activation. Down-regulation of PKC by chronic exposure of cells to 12-O-tetradecanoylphorbol-13-acetate, which led to complete loss of PKC-immunoreactive material, failed to ablate the transcriptional response to IFN alpha or the activation of ISGF3 alpha. The PKC-specific inhibitor calphostin C did not perturb activation or nuclear accumulation of ISGF3. We conclude that a novel, staurosporine/K-252a-sensitive kinase is required for ISGF3 activity and may participate in receptor-mediated signal transduction.

Alkaloids↗

PubMed1990

Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein.

A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.

Amino Acid Sequence↗

PubMed1988

Stability and self-organization of proteins.

X-ray crystallography and NMR provide insight into the three-dimensional structure of proteins at high resolution yielding a relatively small number of stable "topologies". Their free energies of stabilization are minute compared to the total molecular energy. Molecular adaptation to extremes of physical conditions at the protein level refers to marginal alterations of the intramolecular interactions; clearcut predictions with respect to the correlation of protein structure and protein stability are at present not possible. The acquisition of the native structure of proteins is determined by the kinetically accessible minimum of potential energy. The overall mechanism may be described as a hierarchical condensation with elements of secondary/super-secondary structure and subdomains/domains as intermediate stages. In the case of oligomeric and multimeric proteins, folding and association of subunits follow a consecutive uni-bimolecular mechanism, the kinetics of which can be simulated with high precision. The "code of protein folding" is still unknown.

Drug Stability↗

PubMed1990

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures.

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH. In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of diassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles. Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups. The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride. Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides. With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride. Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride.

Amino Acids↗

PubMed1992

Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly.

In the endoplasmic reticulum (ER), newly synthesized subunits of the T-cell antigen receptor (TCR), membrane-bound immunoglobulin (mIg), and major histocompatibility complex (MHC) class I antigens must fold correctly and assemble completely into multimeric protein complexes prior to transport to the cell surface. Although folding and assembly may occur spontaneously, the concept that molecular chaperones facilitate these events is emerging. Here, an intracellular protein of 90-kDa apparent molecular mass, denoted IP90, was shown to be an ER resident protein that associated with partial complexes of the TCR, mIg, and MHC class I proteins but was absent from fully assembled complexes. We speculate that IP90 might participate in folding and assembly processes of these and other multisubunit protein complexes during their transit through the ER.

Animals↗

PubMed1992

Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity.

Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for phosphorylation by protein kinases. Neurotransmitter receptors that mediate rapid synaptic transmission in the nervous system are the ligand-gated ion channels and include the nicotinic acetylcholine receptors of muscle and nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligand-gated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity.

Amino Acid Sequence↗

PubMed1985

Mannan-binding protein and conglutinin in bovine serum.

Conglutinin is a bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b complex (conglutination). The serum mannan-binding protein (MBP) is a lectin specific for mannose and N-acetylglucosamine. Since conglutination was shown to be inhibited specifically by N-acetylglucosamine [Leon, M.A. & Yokohari, R. (1964) Science 143, 1327-1328], the possibility was raised that conglutinin might be a bovine serum MBP. The present study, undertaken to solve this problem, revealed that bovine plasma contained an MBP besides conglutinin. These two proteins were very similar in their chemical and physicochemical properties as well as binding specificity. Both bound with high affinity (Kd = 10(-8) M) to glycoproteins terminated with mannose and/or N-acetylglucosamine residues in the presence of calcium, although conglutinin preferred N-acetylglucosamine rather than mannose. They were multimeric proteins of large molecular size (over 1,000,000 daltons, and approximately 600,000 daltons for conglutinin and MBP, respectively) and consisted of a single kind of subunit with molecular weight of around 45,000. The MBP was shown to have a collagen-like structure in the molecule, as was recently reported for conglutinin [Davis, A.E., III & Lachmann, P.J. (1984) Biochemistry 23, 2139-2144]. Despite these similarities, the MBP and conglutinin were immunochemically distinct, and the MBP did not show any conglutination activity.

Amino Acids↗

PubMed1991

Assessing the multimeric states of proteins: studies using laser desorption mass spectrometry.

We have developed a technique which utilizes matrix-assisted laser desorption mass spectrometry to study the subunit association of proteins. Aqueous protein samples are treated with a dilute solution of glutaraldehyde, a cross-linking agent which reacts with free amino groups on proteins. This agent effectively traps the multimeric form, preventing it from dissociating in the sample preparation and desorption process. Proteins measured include lysozyme, carbonic anhydrase, apomyoglobin, glucose 6-phosphate dehydrogenase, ovine lutropin, yeast alcohol dehydrogenase, avidin and pyruvate kinase. Dimeric and tetrameric complexes up to 250,000 Da have been measured in this manner.

Alcohol Dehydrogenase↗

PubMed1988

Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.

Antibodies, Monoclonal↗

PubMed1990

DNA-binding activity of Jun is increased through its interaction with Fos.

Transcription factor AP-1 mediates induction of a set of genes in response to the phorbol ester tumor promoter TPA. Recently, AP-1 preparations from HeLa cells were shown to contain a product of the c-JUN protooncogene (Jun/AP-1) which forms a tight complex with the Fos protein. In this paper, we examine the role of the Fos protein in the DNA-binding activity of the AP-1 complex. We show that the DNA-binding activity of bacterially expressed trpE-Jun fusion proteins is increased many-fold upon their interaction with Fos (or a Fos-related antigen) expressed from a baculovirus vector. The site of Fos interaction is within the DNA-binding domain of Jun/AP-1, and anti-Fos antibodies interfere with the binding of affinity purified AP-1 to DNA. These results suggest that, by associating with Jun/AP-1, Fos is responsible for the formation of a multimeric protein complex that has greater affinity for the target sequence than does Jun/AP-1 alone.

DNA↗