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PubMed1992

Isolation and characterization of genes encoding chaperonin 60 beta from Arabidopsis thaliana.

Chaperonins (Cpn) are implicated in the folding and assembly of multimeric proteins in plastids and mitochondria of eukaryotes and in prokaryotes. Plastid Cpn is composed of two different polypeptides termed Cpn60 alpha and Cpn60 beta. We have isolated cDNA and genomic clones encoding Cpn60 beta from Arabidopsis thaliana. The steady-state level of the cpn60 beta mRNAs is higher in etiolated leaves and sucrose-treated plants as compared to control leaves. The A. thaliana cpn60 beta gene family consists of at least three different coding units. It was confirmed that Cpn beta-encoding genes have a high level of conservation among plants.

Amino Acid Sequence↗

PubMed1991

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.

Acetylcholinesterase↗

PubMed2010

Sites in the AAV5 capsid tolerant to deletions and tandem duplications.

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10(5) members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.

Amino Acid Sequence↗

PubMed1990

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora.

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1986

Circadian rhythms in Drosophila melanogaster: analysis of period as a function of gene dosage at the per (period) locus.

Mutations at the per (period) locus of Drosophila melanogaster affect the period of its circadian rhythms. An analysis of published data on strains having duplications and deletions of the per locus indicates that the period is a logarithmic function of the level of the per gene product. The analysis also indicates that period is relatively insensitive to the level of that gene product; a presumed 300% increase in the gene product level produces only a 4.6% decrease in the period. The period of a strain transformed with per+ DNA conforms to the same logarithmic relationship if the level of mRNA in the transformant, which is one-tenth that in the wild type, is considered equivalent to a gene dosage one-tenth the wild type dose of 2, or 0.2. The periods of strains having various doses of mutant per alleles which shorten (pers) or lengthen (per1) the period can be fitted to the same logarithmic function. The analysis may provide an explanation for the partial dominance of pers over per+ and the dominance of per+ over per1, since it suggests that the per1 gene product is nearly inactive while the pers gene product is more than 34 times as active as the wild type product. Analysis of periods of strains heterozygous at the per locus suggests that the per gene product may be a multimeric protein. Three possible roles for the per gene product in circadian rhythmicity are discussed, including a role in synchronizing rhythm-producing cells.

Alleles↗

PubMed1975

Evidence for an active dimer of Escherichia coli beta-galactosidase.

BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.

Dithiothreitol↗

PubMed1985

Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli.

Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin [Nagai, K. & Thøgersen, H. C. (1984) Nature (London) 309, 810-812]. The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated. Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers. The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb. Two mutant Hbs (Hb Nymphéas [Cys-93 beta----Ser] and Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg]) were constructed by site-directed mutagenesis using synthetic oligonucleotides. Hb Nymphéas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects. Hb Daphne showed low cooperativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation. As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.

Amino Acid Sequence↗

PubMed1987

Predominant role of amino-terminal sequences in dictating efficiency of class II major histocompatibility complex alpha beta dimer expression.

Cell surface expression of class II major histocompatibility complex-encoded (Ia) molecules depends on association of the component alpha and beta chains into a stable heterodimer. In the mouse, two isotypes of class II molecules have been identified, A beta A alpha and E beta E alpha. However, experiments from this laboratory have shown that, following DNA-mediated gene transfer into murine L cells, an A beta E alpha-mixed-isotype molecule can be assembled and expressed at the cell surface. In the present study, we have investigated the structural features of the beta chain that control the extent of association and level of membrane expression of A beta E alpha interisotypic pairs. The use of intact allelic A beta genes demonstrated that only A beta d chains, but not A beta b or A beta k chains, can be coexpressed on the surface membrane with E alpha chains. Transfection of recombinant A beta genes that encode all or half of the beta 1 domain from one allele and the rest of the chain from another allele revealed that the 5-7 polymorphic residues in the amino-terminal 50 residues of the A beta chain completely controlled this variation in expression with E alpha. Isotypically mixed beta genes encoding the A beta 1 domain of either A beta d or A beta k chains and the beta 2, transmembrane, and intracytoplasmic portions of E beta chains were used to assess the role of isotypically conserved structures in alpha beta pairing and expression. In marked contrast to the major alterations in expression accompanying changes in the amino-terminal polymorphic residues, exchange of these carboxyl-terminal isotypic segments had no detectable influence on the efficiency of expression with either A alpha or E alpha chains. These results argue strongly that variations in the efficiency with which distinct Ia alpha beta dimers assemble and are transported to the membrane is determined almost exclusively by a critical chain interaction involving the amino-terminal domains of the molecules.

Alleles↗

PubMed1991

Role of a carboxyl-terminal helix in the assembly, interchain interactions, and stability of aspartate transcarbamoylase.

The six individual catalytic polypeptide chains within the two catalytic trimers of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) are folded into two discrete structural domains interconnected in part by helix 12, which comprises residues 285-305 and is located near the carboxyl terminus of the chain. The essential role of this helix in folding of the chains and their assembly into ATCase was demonstrated by introducing a stop codon at the position corresponding to amino acid 284, 291, or 299. Cells containing these mutations are pyrimidine auxotrophs lacking ATCase-like protein in cell extracts. In contrast, stable active enzyme is formed from chains truncated at position 306 or 307, showing that all 310 amino acids are not required for assembly. Replacements of Gln-288, Asn-291, Arg-296, and Ala-298 were introduced to assess the effect of alterations within helix 12 on protein stability. Stability of the trimers was measured both by differential scanning microcalorimetry and by the rate of exchange of chains at 4 degrees C when mutant trimers were incubated with succinylated wild-type trimers. Melting temperatures of the mutant trimers spanned a range of more than 20 degrees C, with a few higher and others lower than that of wild-type trimers. Large changes in interchain interaction energies were observed for the trimers, but there was no direct correlation between the ease of dissociation of the trimers and their thermal stability. Calorimetry on the mutant holoenzymes revealed alterations in the interactions between trimers and regulatory subunits within the intact enzymes. The striking changes in stability of both trimers and holoenzymes demonstrated that effects of relatively localized amino acid replacements in helix 12 are manifested by indirect global alterations propagated throughout the structure.

Amino Acid Sequence↗

PubMed1975

Genetic and physiological characterization of met15 mutants of Saccharomyces cerevisiae: a selective system for forward and reverse mutations.

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus. -- It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.

Alleles↗

PubMed1988

Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space.

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.

ATP-Binding Cassette Transporters↗

PubMed1992

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation.

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose-6-phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6-phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Amino Acid Sequence↗

PubMed1992

Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli.

The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component.

Animals↗

PubMed1990

A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.

Amino Acid Sequence↗

PubMed1992

Characterization of apolipoprotein B mRNA editing from rabbit intestine.

Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally converted to a uridine by an RNA editing mechanism that transforms the codon for glutamine 2,153 to a termination codon. The editing reaction can be duplicated in a cell-free extract. In this study, the apo B-48 mRNA editing activity derived from partially purified extracts of rabbit enterocytes was characterized. The optimum conditions for the editing reaction were determined to be a salt concentration of 0.125-0.150 M NaCl or KCl, a pH of 8-8.5, and a temperature of 30 degrees C. The reaction rate was linear up to 45 minutes and was proportional to the editing extract concentration. No metal ion cofactors, DNA or RNA cofactors, or energy requirements were identified. At optimum conditions, the reaction followed Michaelis-Menten kinetics, with a Km of 0.4 nM for the rabbit RNA substrate. In addition, the reaction rate was enhanced by the addition of 25 micrograms/ml heparin or 40% glycerol. The characteristics of the editing reaction suggest that it is catalyzed by a nucleotide sequence-specific cytidine deaminase that is either a single enzyme or a multimeric protein.

Animals↗

PubMed1985

Effects of plasmin on von Willebrand factor multimers. Degradation in vitro and stimulation of release in vivo.

von Willebrand factor (vWF), a multimeric protein that mediates platelet adhesion, circulates in association with the procoagulant Factor VIII (FVIII). In previous reports, plasmin was shown in vitro to inactivate FVIII and cleave the vWF subunit extensively, but to cause only a modest decrease in vWF platelet-agglutinating activity. In the present study, the digestion of vWF multimers by plasmin was analyzed by sodium dodecyl sulfate-agarose gel electrophoresis and radioimmunoblotting. In vitro, plasmin degraded the large vWF multimers to smaller forms that could be distinguished from the small multimers present before digestion only by a slightly increased electrophoretic mobility. These plasmin-cleaved "multimers" were composed of disulfide-linked fragments with no intact vWF subunits. Thus, many plasmin cleavages occur within disulfide loops. The slight increase in mobility of plasmin-digested vWF is in part explained by the early cleavage from the multimers of a 34,000-mol wt peptide, which was purified and partially sequenced. The amino-terminal sequence (33 residues) agrees with the previously reported sequence (15 residues) for the amino terminus of the intact vWF subunit. Analysis of plasmin-digested vWF allowed deduction of a model for the native vWF structure, including the approximate location of the interprotomer disulfide bond(s). To determine whether plasmin would digest vWF in vivo, plasmas from 12 patients and 2 normal volunteers who received intravenous streptokinase (SK) were analyzed. Rather than vWF digestion, a two- to threefold rise in vWF antigen and platelet-agglutinating activity occurred within 2 h after a single SK dose, and the increase was greatest among the largest multimers. In contrast, FVIII clotting activity dropped to 10-20% of pre-SK levels. Thus, although plasmin destroys FVIII, a pharmacologically induced fibrinolytic state is associated with significant release of vWF from endothelial cells, platelets, or some other storage pool.

Blood Coagulation Factors↗

PubMed1992

Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.

Crotalid Venoms↗

PubMed1991

[Overview of erythropoietin].

Erythropoietin (Epo) is a glycoprotein that promotes the proliferation and differentiation of erythrocyte precursors. The major site of Epo production is the kidney and the liver is the main extra renal site of Epo production. Epo producing cells were identified by in situ hybridization, in the kidney, they are peritubular cells, most likely endothelial cells of the cortex and outer medulla; in the liver, they are mainly hepatocytes. The Epo secretion is stimulated by hypoxia, which is detected by an oxygen sensor. The Epo receptor is a multimeric protein, one chain which binds Epo has been cloned. However the structure of the Epo receptor is still puzzling, and one or more accessory chains remain to be identified. Since the clonage of the Epo gene, recombinant Epo has been available and allowed the treatment of patients with renal diseases with a constant efficacy.

Erythropoietin↗