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At least 199 records · Page 11Linked to original sources
PubMed1991

[Study of the interaction of the monoclonal antibody to human interleukin-2 and its Fab-fragment with synthetic peptides --interleukin-2 fragments by (1)H-NMR].

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

Amino Acid Sequence↗
PubMed1988

Activation of B cells in vivo by a Fab/Fc fragment of a monoclonal anti-IgD antibody requires an interaction between the antibody fragment and a cellular IgG Fc receptor.

To investigate activation of B lymphocytes in vivo by an interaction between B cell surface Ig (sIg) and an anti-Ig antibody, we compared the abilities of a divalent IgG2b anti-IgD mAb, H delta a/1, and its univalent Fab/Fc fragment to enhance B cell sIa expression in vivo. The Fab/Fc fragment consists of a single Fab linked to Fc, and can interact with C and cellular Fc receptors. Although injection of BALB/c mice with either intact H delta a/1 or H delta a/1 Fab/Fc enhanced splenic B cell sIa expression, Ia expression was enhanced more by intact H delta a/1. Furthermore, injection of mice with 24G2, a mAb to the B cell and macrophage IgG2b Fc receptor, completely blocked the ability of 20 to 500 micrograms of H delta a/1 Fab/Fc to enhance B cell sIa expression, but had no effect on enhancement of B cell sIa expression by 100 micrograms of intact H delta a/1. This effect of 24G2 was mediated by its blocking of IgG2b receptor function rather than simply by its binding to B lymphocytes, since a mAb to the B cell IgE receptor did not interfere with the ability of H delta a/1 Fab/Fc to enhance B cell sIa expression. The different effects of 24G2 on B cell activation by intact H delta a/1 and H delta a/1 Fab/Fc were not a result of differences in the abilities of the intact antibody and its Fab/Fc fragment to activate B cells, since 24G2 did not interfere with the ability of AMS-15, a IgG2a anti-IgD mAb, to slightly increase B cell sIa expression. These observations indicate that a univalent ligand for B cell sIg can activate B lymphocytes in vivo through an IgGFc-IgGFc receptor-dependent interaction.

Animals↗
PubMed1994

C-terminal fragments of parathyroid hormone-related protein, PTHrP-(107-111) and (107-139), and the N-terminal PTHrP-(1-40) fragment stimulate membrane-associated protein kinase C activity in rat spleen lymphocytes.

Membrane-associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C-terminal parathyroid hormone-related protein fragments, PTHrP-(107-111) and PTHrP-(107-139), at concentrations from 10(-3) to 10(4) pM. By contrast, the same concentrations of PTHrP-(120-139), without the 107-111 TRSAW (-Thr-Arg-Ser-Ala-Trp-) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N-terminal PTHrP-(1-40) fragment also stimulated membrane-associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response.

Animals↗
PubMed1993

Dissection of the DNA-binding domain of Xenopus laevis TFIIIA. Quantitative DNase I footprinting analysis of specific complexes between a 5 S RNA gene fragment and N-terminal fragments of TFIIIA containing three, four or five zinc-finger domains.

Recombinant zinc finger proteins corresponding to N-terminal fragments of Xenopus laevis transcription factor IIIA (TFIIIA) comprising three, four and five fingers produced in Escherichia coli as cleavable hybrid proteins were shown to form specific stoichiometric complexes with DNA fragments containing the internal control region (ICR) of a 5 S RNA gene. The ordered set of DNase I footprints of each of the three proteins on the ICR comprise a nested set of footprints extending upstream from its 3' end (position +96 relative to start of the mature transcript) 20 bp, 20 bp or 34 bp into the ICR, respectively. Quantitative analysis of the footprinting data provided firm evidence that the DNase I footprint, and hence the structure, of the authentic TFIIIA:ICR complex in this region is fully and precisely accounted for by the N-terminal three fingers binding within the +77 to +96 region plus the pair of fingers 4 and 5, both required to extend the footprint upwards from the +77 to the +63 position. A structural interpretation of this set of new footprinting data in view of previous results and data is presented and discussed in terms of a refined model in which the protein-DNA interaction between the ICR and the three N-terminal fingers corresponds closely to that observed in the homologous three-finger zif268:DNA complex, whereas the basic mode of protein-DNA interaction, in which the pair of fingers 4 and 5 is engaged in forming the TFIIIA:ICR complex is of an entirely different, albeit not yet understood nature. To allow assessment of our model in terms of potential specificity-determining H-bonding patterns, a molecular model of the complex between the three-finger TFIIIA fragment and the ICR was constructed, using the zif268:DNA co-ordinates. Eight out of the nine amino acid residues, which according to our model are suitably located for forming hydrogen bonds with the bases, are potential H-bond acceptors or donors.

Amino Acid Sequence↗
PubMed1982

Isolation of a cationic fragment of high molecular mass kininogen and effect of this fragment on human plasma kallikrein activity.

The effect of a cationic fragment of high molecular mass (HMM) kininogen on kallikrein activity and the autocatalytic process of prekallikrein-to-kallikrein conversion were studied. Prekallikrein and kallikrein were obtained by ion exchange and affinity chromatography; the purification of HMM kininogen was achieved using QAE- and DEAE-Sephadex, and separation of the cationic fragment was carried out by gel filtration. It was shown that the fragment of HMM kininogen (mol. mass. 7000, high lysine content) suppressed the activity of a labile form of kallikrein and prevented activation of prekallikrein. A model of the prekallikrein-kallikrein system is proposed. The model is based on the recently established fact of occurrence of two kallikreins and two kallikrein precursors. It involves formation from kininogen, besides kinins, of a kallikrein inhibitor.

Cations↗
PubMed1994

Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.

Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

Amino Acid Sequence↗
PubMed1996

Direct excision of 50 kb pair DNA fragments from megabase-sized fragments produced during apoptotic cleavage of genomic DNA.

The DNA of U937 cells exposed to two different apoptotic stimuli, namely the cocktail H2O2/3-aminobenzamide (3AB) and etoposide, was analyzed using field inversion gel electrophoresis (FIGE) as well as programmable, autonomously controlled electrode electrophoresis (PACE). The results obtained indicate that FIGE is not appropriate for sizing apoptotic DNA fragments. PACE appears to be more accurate and reliable and the results obtained with this technique strongly suggest that the 50 kb DNA fragments are directly excised from Mb-sized DNA fragments without the intermediate cleavage of 200-300 kb products.

Apoptosis↗
PubMed1994

Monoclonal antibody F(ab')2 fragment labeled with N-succinimidyl 2,4-dimethoxy-3-halobenzoates: in vivo comparison of iodinated and astatinated fragments.

N-Succinimidyl 3-[211At]astato-2, 4-dimethoxybenzoate (SADMB) was prepared from a trialkylstannyl precursor in about 70-75% yield. With either trimethyl or tri-n-butylstannyl precursor, no temporal effect was found in the astatodestannylation yield. However, the methyl analog gave slightly better yield which was found to be not statistically significant. A monoclonal antibody (MAb) fragment, Mel-14 F(ab')2, could be labeled using SADMB in 28% coupling efficiency. The specific binding of this labeled fragment to tumor homogenates in vitro was 61.0 +/- 0.5% (62.8 +/- 0.9% for the 131I labeled fragment). Paired-label tissue distribution in normal mice showed similar uptake of 131I and 211 At in many tissues. However, by 14.5 h selectivity of spleen, lungs and stomach for Mel-14 F(ab')2 labeled with 211At compared to 131I was 4.1, 3.8 and 6.4, respectively.

Animals↗
PubMed1998

Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent--application to a human single-chain Fv fragment.

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.

Animals↗
PubMed2000

Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments.

Polypeptide release factor one from Thermus thermophilus, ttRF1, was purified and subjected to crystallization. Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction. Stable fragments of ttRF1 suitable for crystallization were screened by limited proteolysis. Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry. They were N-terminal fragments generated by proteolysis at amino acid positions 211, 231 and 292. The corresponding recombinant polypeptides, ttRF1(211), ttRF1(231) and ttRF1(292), were overproduced and subjected to crystallization. Of these polypeptides, ttRF1(292) gave rise to crystals that belong to P3(1) (or P3(2)) space group with unit cell parameters a = b = 64. 5 A, c = 86.6 A and diffract up to 7 A resolution.

Amino Acid Sequence↗
PubMed1990

Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A.

Examination of a selection of serum samples from adults from two regions of England showed that 50% of men in the 16-24 years and over 55 years age groups had high titres of antibody to diphtheria toxin (DT). In contrast, only 11% of women aged 16 to over 55 years had high titres of antibody to DT. All human antisera with high anti-DT titres reacted with a synthetic peptide (SP) corresponding to the amino acids 141-157 of DT fragment A, with sera from men aged 35 to over 55 years showing the highest titres. High antibody titres to fragment A paralleled those to SP in both sexes. Titres of antibody to DT fragment B were highest in individuals with high titres to DT. In sera from both sexes immunoglobulin G1 was the predominant subclass reactive with all three antigens. However, both IgG1 and IgG4 and to a lesser extent IgG2 and IgG3 were present in immunoglobulin concentrates.

Adolescent↗
PubMed1994

Generation of a family of protein fragments for structure-folding studies. 1. Folding complementation of two fragments of chymotrypsin inhibitor-2 formed by cleavage at its unique methionine residue.

The suitability of the barley chymotrypsin inhibitor-2 for study by fragmentation and complementation has been analyzed. The primary residue for binding to proteases, Met-59 (the unique methionine in the sequence), lies in a broad, solvent-exposed loop. The bond between Met-59 and Glu-60 was cleaved by cyanogen bromide. The two fragments thus obtained, i.e., CI-2(20-59) and CI-2(60-83), associate (KD = 42 nM) to yield a complex that has fluorescence and circular dichroism spectra identical to those of uncleaved chymotrypsin inhibitor-2. Recovery of native-like structure is further indicated by the ability of the complex to inhibit chymotrypsin, although the [I]50% is 140-fold higher than for the uncleaved inhibitor. CI-2(60-83) appears to be highly disordered in water, but fragment CI(20-59) forms significative structure, as judged by its circular dichroism spectra and evidence from one-dimensional NMR. The circular dichroism spectra of CI-2(20-59) approach the baseline in 4 M guanidinium chloride but display characteristics of an alpha-helix in the presence of trifluoroethanol. Analytical ultracentrifugation shows no concentration-dependent change in the molecular weight of the monomer of CI-2(20-59). Both one- and two-dimensional NMR of the complex [CI-2(20-59).(60-83)] show unequivocally the presence of a folded structure, which appears to be slightly different from the uncleaved native protein.

Guanidine↗
PubMed1999

Fragment reconstitution of a small protein: disulfide mutant of a short C-terminal fragment derived from streptococcal protein G.

Hierarchical studies on the folding of protein G B1 domain have shown that the C-terminal fragment (C16) has a considerable amount of beta-hairpin structure that exchanges between the folded and unfolded states at room temperature, and that the C16 fragment binds noncovalently to an N-terminal fragment (N40) under physiological conditions. Those studies have led us to the hypothesis that the amphipathic beta-hairpin structure of C16 initiates folding of the domain. To obtain a more detailed understanding of the folding mechanism of the domain, we designed a mutant of C16 (SS16ox) with a disulfide bond between residues 41 and 56, and then examined the interaction of the mutant with N40 by surface plasmon resonance (SPR) and by thermal denaturation studies using circular dichroism. SS16ox strongly interacted with N40, with an equilibrium constant, KD, that was 7-fold higher than wild-type. The association rate constant, kon, of SS16ox was 8.7-fold higher than that of wild-type. This strong interaction can be explained by the entropic effect of the disulfide bond. The introduction of the disulfide bond into C16 stabilizes the beta-hairpin structure of C16, accelerates the association rate with N40, and then stabilizes the whole complex. These results support a hypothetical folding mechanism of protein G where the amphipathic beta-hairpin structure of C16 acts as a nucleus and accelerates folding of the whole molecule.

Amino Acid Sequence↗
PubMed1989

Protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein.

We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein. The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates. Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity. By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20%. None of these three peptides has any significant effect on the activity of the folded enzyme. Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition. We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms.

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis.

We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the DFF40, the active component of DFF. Purified DFF40 exhibited an intrinsic DNase activity that was markedly stimulated by chromatin-associated proteins histone H1 and high mobility group proteins. DFF40 also triggered chromatin condensation when incubated with nuclei. These data suggest that DFF40 is sufficient to trigger both DNA fragmentation and chromatin condensation during apoptosis.

Amino Acid Sequence↗
PubMed2000

Resistance to paclitaxel induces time-delayed multinucleation and DNA fragmentation into large fragments in MCF-7 human breast adenocarcinoma cells.

DNA fragmentation was investigated in MCF7 and the MCF7TAX19 paclitaxel-resistant subline exposed to paclitaxel for 24 h. No nucleosome-sized DNA fragmentation was observed by DNA agarose gel electrophoresis in both cell lines. However, DNA fragmentation was detected by flow cytometry sub-G1 peak analysis in both cell lines immediately after paclitaxel exposure. Nuclear abnormalities were observed in both cell lines in the range of 35-40% of the total cell population. This value was reached immediately in MCF7 cells but was time-delayed in MCF7TAX19 cells. Significant morphologic differences were observed between sensitive and resistant cell lines, 24 h after exposure to 50 nmol/l paclitaxel. Although no difference in the sub-G1 cell population was observed between sensitive and resistant cells, a significantly higher rate of multinucleated cell features was observed in resistant cells.

Adenocarcinoma↗
PubMed1997

Comparative evaluation of the synthesis and purification of transmembrane peptide fragments. Rat bradykinin receptor fragment 64-97 as model.

The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin, receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of alpha-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor.

Amino Acid Sequence↗
PubMed1995

The enhancement of the human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells by trypsin treatment is ascribed to the binding of trypsin to the Fc fragment.

Human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells was remarkably enhanced by trypsin treatment of the cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of proteins of the cells treated with trypsin was the same as that of the cells treated with pronase, although pronase treatment had been shown to reduce the activity in our previous study (FEMS Microbiol. Lett. 123, 305-310, 1994). This contradiction was clarified by the finding that trypsin bound the Fc fragment more strongly than the cells, and a small amount of trypsin remained in the cells treated with trypsin and washed well. On the basis of these results, it was concluded that the enhancement of cell activity by trypsin treatment was ascribed to binding of the Fc fragment to trypsin remaining in the trypsin-treated cells.

Immunoglobulin Fc Fragments↗