Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli. Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P. falciparum blood stage parasite challenge. The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code). To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl). The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E. coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient. Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites. The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite.
Mol Biochem Parasitol 1991 Aug
PMID:A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1. 171 16

In order to study the antigenic structure of histone H1(0) the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1(0) antiserum. The C-terminal fragments 99-193 (obtained following acetic acid hydrolysis) and 107-193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1-30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1-22 and 1-28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1(0) are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.
Mol Cell Biochem 1991 Oct 16
PMID:Antigenic structure of histone H1(0). 172 85

Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria.
Mol Immunol 1992 Jan
PMID:Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E. coli: recovery of active FV fragments. 173 Nov 88

Histone H3 proteins were purified to near homogeneity from callus cultures of dicotyledonous plants alfalfa, soybean, Arabidopsis, carrot and tobacco to determine the number of histone H3 variants. In every species two histone H3 variants were identified by gradient gel electrophoresis and reversed-phase chromatography. They were named H3.1 and H3.2 in order of increasing mobility in acid-urea-Triton gels. Co-electrophoresis of histone H3.2 proteins of all species in this gel system and HPLC co-chromatography suggest that all histone H3.2 variants have a primary protein sequence identical to alfalfa H3.2. Two distinct H3.1 variant forms were identified, represented by alfalfa and Arabidopsis H3.1 proteins which differ only at residue 90. Soybean H3.1 resembles H3.1 of alfalfa. Carrot and tobacco H3.1 appear identical to the Arabidopsis H3.1 histone variant. All H3 proteins were acetylated to multiple levels and in each plant the histone H3.2 forms were more highly acetylated. An inverse relationship was observed between plant genome size and the relative abundance of histone variant H3.2 and also with the level of acetylation of both histone H3 variants. This correlation matches the general tendency that in plants with smaller genomes a larger fraction of the genome is transcriptionally active.
Plant Mol Biol 1992 Jan
PMID:Existence of two histone H3 variants in dicotyledonous plants and correlation between their acetylation and plant genome size. 173 82

The monomer-dimer association reaction of Arc repressor was studied by pressure-induced dissociation and by dilution. The dissociation was measured by the decrease (red shift) in the average energy of emission of the tryptophan fluorescence. Pressure dissociation also promoted a decrease in the excited-state lifetime of the single tryptophanyl residue, Trp14. These observations suggest that Trp14 becomes exposed to an aqueous environment following dissociation. The pressure-dissociation curves were concentration dependent, with p1/2 (half-dissociation pressure) shifting to higher pressures as the concentration increased. The dissociation constant (KdO) obtained by extrapolating the pressure-dissociation curves to atmospheric pressure was similar to that determined from the dilution curve (KdO = 30 nM). An anomalous steepness of dissociation in response to dilution was observed, suggesting that conformational changes occur as a result of dissociation of Arc repressor. Binding of bis(8-anilinonaphthalene-1-sulfonate) to Arc repressor was not significantly affected by pressure dissociation, whereas thermal or urea denaturation was accompanied by a dramatic decrease in binding. These results suggest that the conformational changes that follow dissociation induced by pressure are more limited than those following denaturation. The tryptophan anisotropy decreased by about one-half, suggesting the dissociation of a globular dimer to a compact monomer. On the other hand, denaturation by urea promoted an increase in anisotropy, as expected for a random-coil conformation. Dissociated Arc has the hydrodynamic properties of a folded monomer. On the other hand, dissociated Arc has a high degree of exposure of hydrophobic side-chains, and the distribution of conformations is much broader than that in the folded dimer. These features suggest that the dissociated subunit is a molten globule. The subunit interaction was substantially increased by a single amino acid substitution (Pro8----Leu), and the free energy of stabilization amounted to -2.9 kcal/mol. This increased stability suggests that residue 8 is located in the dimer interface and that part of the tertiary and most of the quaternary structure constraints result from the interaction between the intersubunit beta-strands.
J Mol Biol 1992 Jan 20
PMID:Dissociation of a native dimer to a molten globule monomer. Effects of pressure and dilution on the association equilibrium of arc repressor. 173 63

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1991 Dec 20
PMID:Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii. 176 47

Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c. 20 kDa, termed ADP-ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.
Mol Microbiol 1991 Nov
PMID:Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins. 177 53

Rapid, quantitative hybridization assays with good sensitivity are needed in many applications, for example, determining the amount of specific product from PCR. We have developed an assay which relies on the hybridization of a biotinylated oligomer and a fluoresceinated oligomer to a single-stranded target in solution. The hybridized complex is captured by streptavidin to a biotinylated membrane. After capture, the hybridization complex is detected by an antifluorescein-urease conjugate which binds to the fluoresceinated probe. The membrane-bound urease conjugate is exposed to urea and assayed with a pH-sensitive silicon sensor. The total assay time is less than 2 h and the sensitivity limit is 20 x 10(6) molecules with a coefficient of variation, CV, of less than 10%. The assay was applied to the analysis of a model target using PCR. We were able to measure the amount of specific product and the amplification factor during the exponential phase of PCR. Using extrapolation from the measured amounts of amplified product, the initial amounts of target molecules were calculated to be 1.2 x 10(6) and 4.0 x 10(2) when the added quantities were 3 x 10(6) and 3 x 10(3), as determined by serial dilution.
Mol Cell Probes 1991 Oct
PMID:Quantitation of DNA hybridization in a silicon sensor-based system: application to PCR. 179 56

Dehydroepiandrosterone (DHEA), administered per os, serves to prevent or retard the development of a variety of genetic and induced disorders in mice and rats. This treatment also results in the development of hepatomegaly, a change of liver color from pink to mahogany, peroxisome proliferation in hepatocytes and alterations in hepatocyte mitochondria morphology and respiration. We used one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to identify changes in the relative levels of liver proteins produced by DHEA treatment of rodents. In mouse liver, there were apparent increases in the levels of 26 proteins and decreases in the levels of 7 proteins. Of the induced proteins the most prominent had Mr approximately 72 K; this protein was identified in a previous study as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Another protein of Mr approximately 28 K, of unknown nature, also was induced markedly by DHEA treatment of mice and rats. A protein of Mr approximately 160 K, which was identified as carbamoyl phosphate synthetase-I (CPS-I), was decreased markedly by DHEA action. This enzyme, which comprises approx. 15-20% of mitochondrial matrix protein, is involved in the entry and rate-limiting step of the urea cycle. The specific activity of CPS-I also was significantly decreased by DHEA, but serum urea levels were normal. To determine whether steroids other than DHEA also induced similar changes, mice were treated with various steroids for 14 days and, thereafter, liver proteins were evaluated by SDS-PAGE: estradiol-17 beta and isoandrosterone induced both the approximately 72 and approximately 28 kDa proteins, testosterone and androsterone induced the 28 kDa protein only, but etiocholanolone, pregnenolone and progesterone were without effect. The findings of this study serve to demonstrate that: (i) hepatic protein levels are affected by DHEA treatment of mice and rats; (ii) liver CPS-I activity is decreased significantly by DHEA treatment, but serum urea levels remain within the normal range; and (iii) sex steroids and some of their precursors, when administered per os, also alter liver protein levels.
J Steroid Biochem Mol Biol 1991 May
PMID:Inhibition of carbamoyl phosphate synthetase-I by dietary dehydroepiandrosterone. 182 77

The effects of 40 days of treatment with Cyclosporine A (CSA) on plasma and urine free amino acids were investigated in sham-operated (C) and partially nephrectomized (Pnx) female Fischer 344 rats. High Dose CSA (30 mg/kg/day ip) was associated with reduced weight gain, increased plasma urea nitrogen, and hypoproteinemia in C and Pnx animals. These animals also demonstrated increased plasma levels of alanine, markedly reduced levels of tryptophan, and an increase in urinary excretion of methylhistidines. C but not Pnx animals also showed a significant increase in plasma serine and a decrease in plasma taurine. CSA treatment of group C resulted in a progressive aminoaciduria involving substrates of the neutral and acidic renal amino acid transport systems; however, the renal excretion of taurine and beta-alanine by these animals was markedly reduced as compared to vehicle treated controls. High dose CSA exacerbated aminoaciduria in Pnx animals, but in this group, the excretion of beta amino acids was also increased. Our findings demonstrate that chronic CSA toxicity in rodents with normal renal function is characterized by increased muscle protein catabolism, significant reductions in plasma tryptophan, and an apparent decrease in whole body taurine pools. With the exception of the taurine abnormalities. CSA treatment had similar effects on Pnx animals; however, in this group, CSA-induced pathological changes were superimposed on the changes due to renal insufficiency per se. CSA toxicity as identified by the parameters investigated in this study was no more severe in Pnx animals with moderate chronic renal insufficiency than in controls with intact renal function.
Exp Mol Pathol 1991 Aug
PMID:Free amino acids during chronic cyclosporine A toxicity in intact and partially nephrectomized rats. 188 71


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