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At least 811 records · Page 45Linked to original sources
PubMed1995

Intracellular stability of alpha fragments of beta-galactosidase: effects of amino-terminally fused polypeptides.

Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.

Amino Acid Sequence↗
PubMed1995

N-terminal and C-terminal fragments of the hormone stanniocalcin show differential effects in eels.

The effects of an N-terminal, a C-terminal, and a mid-fragment of stanniocalcin, the primary hypocalcemic hormone in fish, on plasma total and free (ionic) calcium levels and whole animal calcium influx were tested in eels. Both the N- and the C-terminal fragments were hypocalcemic, causing 18 and 12% reduction in plasma calcium in stanniectomized eels, respectively. With both fragments the hypocalcemic action is transient. The hypocalcemia caused by the C-terminal fragment, although more rapid, is not as pronounced as the hypocalcemic action of the N-terminal fragment. Only the C-terminal fragment reduced calcium influx. The hypocalcemic activity of the C-terminal fragment then can be explained by its effect on calcium influx. The N-terminal fragment appears to function in a different manner. The mid-fragment has no effect on plasma calcium or calcium influx. The different parts of the hormone are concluded to have different effects.

Anguilla↗
PubMed1998

Expression of an antibody fragment at high levels in the bacterial cytoplasm.

Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm.

Amino Acid Sequence↗
PubMed1999

Exploring the folding funnel of a polypeptide chain by biophysical studies on protein fragments.

We are examining possible roles of native and non-native interactions in early events in protein folding by a systematic analysis of the structures of fragments of proteins whose folding pathways are well characterised. Seven fragments of the 110-residue protein barnase, corresponding to the progressive elongation from its N terminus, have been characterised by a battery of biophysical and spectroscopic methods. Barnase is a multi-modular protein that folds via an intermediate in which the C-terminal region of its major alpha-helix (alpha-helix1, residues Thr6-His18) is substantially formed as is also its anti-parallel beta-sheet, centred around a beta-hairpin (residues Ser92-Leu95). Fragments up to, and including, residues 1-95 (fragment B95), appeared to be mainly disordered, although a small amount of helical secondary structure in each was inferred from far-UV CD experiments, and fluorescence studies indicated some native-like tertiary interactions in B95. The largest fragment (residues 1-105, B105) is compactly folded. The secondary structure in alpha-helix1 in the seven fragments was found by NMR to increase with increasing chain length faster than the build-up of tertiary interactions, indicating that alpha-helix1 is being stabilised by non-native interactions. This behaviour contrasts with that in fragments of the 64-residue chymotrypsin inhibitor 2 (CI2), in which tertiary and secondary structures build up in parallel with increasing length. CI2 consists of a single module of structure that folds without a detectable intermediate. The largest fragment of barnase, B105, has interactions that resemble its folding intermediate, whereas one of the largest fragments of CI2 (residues 1-60) resembles the folding transition state. The folding pathways of both proteins are consistent with a scheme in which there are low levels of native-like secondary structure in the denatured state that become stabilised by long-range interactions as folding proceeds. Neither protein forms a stable fold when lacking the last ten residues at the C terminus. Since at least 20 amino acid residues are bound to the ribosome during protein biosynthesis, these small proteins do not fold until they have left the ribosome, and so the studies of the folding of such proteins in vitro may be relevant to their folding in vivo, especially as the molecular chaperone GroEL binds only weakly to denatured CI2 and does not discernibly alter the folding mechanism of barnase.

Amino Acid Sequence↗
PubMed1985

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice.

In vivo localization of a mouse monoclonal antibody (F2-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab')2 fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radio-localization studies were performed by using 125I- or 131I-labeled IgM monoclonal antibody or its F(ab')2 fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab')2 fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of 125I-radiolabeled F(ab')2 fragments and 125I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab')2 fragments was lower than with IgM, and so gamma-camera imaging was workable with F(ab')2 fragments without background subtraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab')2 fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab')2 fragments make them hardly suitable as carriers of toxic drugs.

Animals↗
PubMed1993

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation.

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium dodecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65 degrees C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20-25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35-40% in the SDS denaturation.

Animals↗
PubMed1995

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles.

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.

Alleles↗
PubMed2001

Changes in low molecular weight DNA fragmentation in white blood cells of workers highly exposed to asbestos.

OBJECTIVE: The aim of this study was to determine low molecular weight (LMW)-DNA fragmentation changes after white blood cell (WBC) incubation in lysis buffer followed by constant-field gel electrophoresis (CFGE). WBCs were isolated from blood samples of workers highly exposed to asbestos fibres at the workplace in Germany, and were compared with those from healthy adults. This study was conducted parallel to the study presented in our preceding paper (Marczynski et al. 2000b) in which we described significant increases in the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts in the DNA of white blood cells from the same highly exposed workers relative to the levels found in the control group in all three study years (1994 to 1997). METHOD: We found that 15-h incubation in lysis buffer of WBCs embedded in agarose-plugs from healthy control donors with 2% SDS, proteinase K and Na2-EDTA at 42 degrees C followed by 0.5 h at 4 degrees C produced a characteristic DNA fragmentation pattern below 23 kbp using CFGE. RESULTS: In the 1st year of the study (1994-1995) changes were found in LMW-DNA fragmentation in 54.8% of the asbestos workers studied, compared with the DNA fragmentation pattern of controls. Interestingly, in the 2nd year of the study (1995 1996) changes in DNA fragmentation were found in only 39.9% of exposed subjects. In the 3rd year of the study (1996-1997) the highest number of workers exposed to asbestos (67.3%) with changes in the LMW-DNA fragmentation pattern was found. The Chi-square test for each year of the study revealed significant changes (P < 0.001). These changes may be due to the presence of hydrogen peroxide (H2O2), as has been shown in vitro. It is likely that a Fenton reaction involving the heterolytic reduction of H2O2 by traces of reduced transition metals such as Fe2+ and Cu+ is involved in the fragmentation of DNA. No difference was found in the changes in DNA fragmentation between asbestos-exposed subjects with and without benign asbestos-associated diseases (asbestosis, asbestos-associated pleural plaques). Significant correlations were not found after analysis of the changes in DNA fragmentation in relation to different possible occupational and non-occupational confounding factors, such as the duration of asbestos exposure, the latency period, estimated cumulative fibrous dust dose ("fibre-years"), and non-occupational confounding factors, such as age, smoking status, acute febrile infections, the intake of medicines, aspirin, Ca2+, Mg2+ and/or hormones, the intake of vitamins, and cases of cancer. CONCLUSIONS: Our data confirm that oxidative stress occurs in the WBCs of workers highly exposed to asbestos fibres, thus supporting the hypothesis that asbestos fibres damage cells through an oxidative mechanism. Oxidative stress and oxidative DNA damage may be induced by long-term exposure to asbestos. The new insights into the oxidative effects of asbestos fibres are of great importance because they provide a way forward for new preventive strategies. Preventive and therapeutic approaches using antioxidants should be taken into consideration.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Aging↗
PubMed1995

A novel strategy affords high-yield coupling of antibody Fab' fragments to liposomes.

A new assay for the production of reactive sulfhydryl-bearing antibody Fab' fragments has been utilized to develop conditions affording high efficiencies of coupling of mouse and rabbit IgG-derived Fab' fragments to lipid vesicles containing maleimidyl-functionalized phospholipids. Cysteine and mercaptoethylamine, but not dithiothreitol, reduce antibody F(ab')2 to Fab' fragments in very good yields under conditions where overreduction to heavy and light chains is minimized. Surprisingly, however, a large fraction of the Fab' fragments generated under these conditions can lack maleimide-reactive sulfhydryl groups, as demonstrated using a maleimidyl-poly(ethylene glycol) conjugate to shift selectively the electrophoretic mobility of the reactive sulfhydryl-bearing Fab' fragments. After modification of F(ab')2 reduction conditions specifically to maximize the yield of the latter fraction, it is possible to achieve high and very reproducible coupling of functional Fab' fragments to liposomes (equivalent to coupling of ca. 70% of total input protein and almost 100% of the reactive sulfhydryl-bearing Fab' fraction). A novel phospholipid-poly(ethylene glycol)-maleimide 'anchor' allows particularly efficient coupling of Fab' fragments to liposomes, even using relatively low liposome concentrations and molar percentages of the liposome-incorporated 'anchor' species. These results demonstrate that with appropriate optimization of the conditions for Fab' production and liposome coupling, Fab' fragments can be coupled to liposomes with efficiencies comparable to or exceeding those reported for coupling of intact antibodies. These results should facilitate the wider use of Fab' fragments as a potentially advantageous alternative to intact antibodies for liposomal targeting in various applications.

Animals↗
PubMed1981

Intraspinal distribution and reaction in the grey matter with tetanus toxin of intracisternally injected anti-tetanus toxoid F(ab')2 fragments.

Anti-tetanus toxoid F(ab')2 fragments were purified using immune-affinity chromatography on tetanus toxoid-Sepharose. Fragments were labeled with 125I. Labeled or non-labeled fragments were injected into the intrathecal space of rats. The labeled fragments were found in the spinal cord outside but not inside neurons. Tetanus toxin was injected into a muscle and 36 h later labeled fragments were injected intracisternally. After another 24 h the label was elevated in the spinal cord half segments giving neural supply to the injected muscle and in these half-segments the label was concentrated around some alpha-motoneurons. [125I]Tetanus toxin was injected into a muscle and at different times thereafter non-labeled fragments were injected intracisternally. The development of hindlimb rigidity but not the accumulation of [125I]tetanus toxin in alpha-motoneurons was prevented by early intracisternal injection of fragments. Injection of fragments after the appearance of hindlimb rigidity did not revert the rigidity but prevented the further development of symptoms. It is concluded that an action of tetanus toxin inside alpha-motoneurons is of no importance for the development of motor symptoms in clinical tetanus. The data suggest that in order to evoke spinal symptoms of toxicity tetanus toxin has to reach interneurons by transneuronal migration. In the very early stages of clinical tetanus the intrathecal injection of fragments may be useful.

Animals↗
PubMed1984

HN glycoprotein of HVJ (Sendai virus) enhances the selective cytotoxicity of diphtheria toxin fragment A-containing liposomes on subacute sclerosing panencephalitis virus-infected cells.

Fragment A of diphtheria toxin-containing liposomes (naked liposomes) selectively kill subacute sclerosing panencephalitis virus-infected cells (SSPE cells) (Exp cell res 132 (1981) 259) [10]. Fragment A-containing liposomes associated with either hemagglutinating and neuraminidase (HN) or fusion (F) glycoprotein of HVJ (Sendai virus) were prepared. These liposomes did not kill normal cultured cells. Fragment A-containing liposomes associated with HN protein were much more cytotoxic than naked liposomes containing fragment A to SSPE cells. Their cytotoxicity to the SSPE cells was influenced by the duration of incubation and the amount of HN protein. Fragment A-containing liposomes associated with F protein had about the same cytotoxicity on SSPE cells as had naked liposomes containing fragment A. Fragment A-containing liposomes associated with wheat germ agglutinin (WGA) were also prepared, but these also had the same toxicity as naked liposomes containing fragment A. The effects of monoclonal antibodies against HN protein on the cytotoxicity on SSPE cells of fragment A-containing liposomes associated with HN were studied. The significance of these results with regard to the actions of HN protein and possible reasons for the selective killing of SSPE cells are discussed.

Antibodies, Monoclonal↗
PubMed1994

Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies.

The thermal stability of Fv fragments was examined by circular dichroism (CD) spectrometry and high-performance liquid chromatography. We analyzed three Fv fragments: that of a monoclonal antibody D1.3 and two derivatives of it. After separation of wild-type VH and VL fragments, thermal denaturation of each fragment was monitored by CD spectrometry. The results indicated that the dissociation of Fv into VH and VL fragments seemed to be coupled with the denaturation of each fragment and that the thermal denaturation of VH and VL fragments was prevented when they were associated with one another. The analysis of the three Fv fragments also indicated that, in some cases, differences in amino acids even within the CDRs could have significant effects on the thermal stability of the complex between VH and VL fragments.

Amino Acid Sequence↗
PubMed1988

Preparation of F(ab')2 fragments from monoclonal mouse IgG1 suitable for use in radioimaging.

Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab')2 fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab')2 and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab')2 fragments was 24 +/- 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab')2 fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab')2 fragments from papain digestion was 50 +/- 5% of the theoretical maximum. Both the above procedures gave F(ab')2 fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab')2 preparations differed, suggesting different digestion sites.

Acid Phosphatase↗
PubMed1989

Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas.

Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.

Animals↗
PubMed1995

Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein.

The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass--i.e., approximate sequence positions--that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1-177 and 94-323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94-177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.

Amino Acid Sequence↗
PubMed1994

Inhibition of peptide binding to DR molecules by a leupeptin-induced invariant chain fragment.

Loading of peptides onto DR molecules was studied by characterizing precursors of the mature peptide-DR complexes expressed at the surface of B cells. Since invariant chain (Ii) prevents binding of peptides by DR molecules, it was speculated that analysis of complexes between DR heterodimers and proteolytic fragments of Ii offers the possibility to examine how DR molecules and peptides assemble. Using a procedure combining a two-step affinity chromatography and gel filtration, we isolated from leupeptin-treated B cells complexes between DR molecules and N-terminal Ii fragments previously called "leupeptin-induced polypeptides" (LIP; Blum and Cresswell, 1988, Proc. natn. Acad. Sci. U.S.A. 85, 3975-3979). It was observed that the most prominent LIP fragment has a relative molecular mass (M(r)) of 16 kDa. In addition, we show that this polypeptide species does not bear N-linked glycans, indicating that this fragment does not extend beyond residue 129 of Ii. Similarly to DR alpha beta heterodimers associated with the full length 33 and 35 kDa Ii forms, DR alpha beta heterodimers associated with LIP fragments are unstable in sodium dodecyl sulfate (SDS) at ambient temperature, whereas mature DR alpha beta heterodimers are resistant to dissociation with SDS. These results are indirect evidence that LIP-DR complexes are devoid of bound peptides. This possibility was supported by showing that LIP-DR complexes fail to bind a radioiodinated tetanus toxin peptide (125I-p2), while DR molecules, which are spontaneously released from complexes with LIP fragments, bind the labeled peptide. These results demonstrate that association with LIP fragments is sufficient to prevent binding of peptides by DR molecules. This notion was further documented by showing that binding of 125I-p2 on DR heterodimers is inhibited by preparations of LIP fragment. By contrast, a soluble recombinant fragment corresponding to the extracytoplasmic region of Ii did not block 125I-p2 binding. The results presented in this study indicate that the cytoplasmic and/or transmembrane region of Ii is required to prevent peptide binding by DR molecules, while the extracytoplasmic portion of Ii, though capable of associating with DR molecules, lacks the capacity to block peptide binding.

Amino Acid Sequence↗
PubMed1993

Purification of the antibacterial fragments of guinea-pig major basic protein.

In this study, we tried to purify the antibacterial fragments of guinea-pig major basic protein (MBP) using Staphylococcus aureus. The antibacterial activity of MBP was not affected by the pyridylethylation, suggesting that the disulfide bonds were not necessary for the antibacterial activity. When pyridylethylated-MBP was digested with alpha-chymotrypsin, the four potent antibacterial fragments (fragment V (Arg105-Tyr119), fragment IX (Thr1-Phe67), fragment X (Ile54-Leu97) and fragment XIII (Arg25-Phe67)) were isolated by reverse-phase high-performance liquid chromatography. Anti-MBP monoclonal antibody, BMK-13 neutralized the antibacterial activity of PE-MBP and fragments IX, X and XIII, but not the activity of fragment V, suggesting that Ile54-Phe67, the common amino-acid sequence of fragments IX, X and XIII, might be involved in the antibacterial activity of MBP. In fact, the synthetic peptide, Ile54-Phe67 exerted the antibacterial activity, and the activity was neutralized with BMK-13. The antibacterial activity of Ile54-Phe67 was lost by the modification with peptidylarginine deiminase which converted arginine residue to citrulline residue, suggesting that the arginine residues may be important for the antibacterial activity.

Amino Acid Sequence↗