Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fragmentation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources
PubMed2000

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis.

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.

Apoptosis↗
PubMed2002

High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm.

The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.

Animals↗
PubMed1999

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and H37Ra.

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra was performed by a number of restriction enzymes, including Nru I, EcoN I, Pst I, and Pvu II. No differences were found in the IS 6110-fingerprints of the study strains by Nru I. One differential IS6110-positive restriction fragment was detected by EcoN I in strain H37Ra, while analysis by Pst I revealed that two fragments of the strain H37Rv were replaced by four novel IS6110-positive fragments in the strain H37Ra. By using Pvu II, a restriction enzyme that cleaves IS 6110 once, and by probing for an IS6110 specific target sequence located to the right of the Pvu II site, we found that the strains H37Rv and H37Ra share 13 IS6110-positive restriction fragments and that one IS6110-positive restriction fragment of H37Rv is replaced by four novel fragments in H37Ra; by probing for an IS6110-specific target sequence to the left of the Pvu II site, 13 shared restriction fragments and 2 differential bands in strain H37Ra were detected. These findings demonstrate that novel insertions of the IS6110 element exist in the avirulent strain H37Ra and raise the question of the role, if any, of IS6110-insertional mutagenesis in the establishment of the avirulent M. tuberculosis H37Ra phenotype.

DNA Transposable Elements↗
PubMed2001

Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris.

The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments.

Amino Acid Sequence↗
PubMed1994

RFLP analysis of chromosomal fragments in genetic mosaic strains of Bombyx mori.

Mottled striped (pSm), a genetic mosaic strain for larval body marking of the silkworm, Bombyx mori, contains a small chromosomal fragment generated by breakage near the end of the 2nd chromosome. This fragment carries the striped marking (pS) gene and part of the chorion gene clusters. To determine the structural features of this fragmented chromosome, we studied the organization of the chorion genes on the fragment using restriction fragment length polymorphism (RFLP) analysis. Two of three classes of chorion gene family probes detected RFLPs in the two phenotypes, pSm (p/p/pS), and p (p/p), which had lost the fragment, segregated among the siblings of a mottled striped strain. Although hybridization patterns were basically identical between them, one or two additional bands were always observed associating with the mosaic phenotype (pSm). This suggests that the additional bands correspond to extra copies of chorion genes on the chromosomal fragment, which have a different structure from ones on the intrinsic 2nd chromosomes. Such heterogeneity of chorion genes may have been maintained since the beginning of mosaic induction, due to the absence of recombinational events between the two chromosomes. We are unable to detect any RFLPs by hybridization with the early class of chorion genes, implying that chromosomal breakage might have occurred between the two chorion clusters, Chl-2 and Ch3, which are located approximately 4 cM apart from each other. Based on RFLP analyses for two independent mosaic strains (788 and 872), we postulate a common chromosomal origin with independent breakpoints and construct structural models for the two kinds of chromosomal fragments.

Animals↗
PubMed1990

Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin.

A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized by E. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.

Actins↗
PubMed2003

Changes in low molecular weight DNA fragmentation in white blood cells after diisocyanate exposure of workers.

The pathogenesis of diisocyanate-induced asthma is still largely unknown. Recently, it has been shown that thiol-redox homeostasis of human airway epithelial cells may be altered after in vitro exposure to diisocyanates. In the present study, low molecular weight (LMW) DNA fragmentation patterns in white blood cells (WBCs) were assessed on 16 industrial workers with work-related asthma, before and after chamber challenges with one of three commonly used diisocyanates in concentrations up to 30 ppb. LMW-DNA fragmentation changes were evaluated after 15 h incubation of WBCs embedded in agarose plugs in lysis buffer with or without hydrogen peroxide (H2O2). Increased LMW-DNA fragmentation occurred in WBCs taken at 30 min or 19 h after the end of the chamber challenge in both subjects with positive and in 8 of 14 subjects with negative challenges. In contrast, no change in LMW-DNA fragmentation was seen in WBCs taken at the same time intervals from 11 non-exposed controls. There was no association between changes in DNA fragmentation patterns and possible confounding factors such as age, smoking status, atopy, medication, duration of occupational exposure and period since exposure cessation. These results indicate that diisocyanate exposure can induce DNA fragmentation. Similarities in the increased amounts of WBC LMW-DNA fragments following diisocyanate exposure with the DNA fragmentation after plugs lysis in buffer with H2O2 support the hypothesis that diisocyanates change the intracellular redox steady-state. Whether this effect plays any role in isocyanate-induced asthma has to be investigated in larger epidemiological studies.

Administration, Inhalation↗
PubMed2000

Neuronal nuclear DNA fragmentation in the aged canine brain: apoptosis or nuclear DNA fragility?

Neuronal DNA fragmentation, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation (TUNEL), has been reported in both the canine and human brains in normal ageing, and in some human age-related neurodegenerative diseases. These results have suggested that apoptosis plays an important role in age-related neuronal loss. It is not clear, however, whether the TUNEL method is highly specific for apoptosis, as DNA fragmentation also occurs in the late stages o necrosis. In this study we have examined 27 dogs aged from 8 to 18 years, to investigate the occurrence of nuclear DNA fragmentation. An autolysis index based on current histological criteria was assigned to each animal to evaluate the effects of autolysis on nuclear DNA integrity. Our results have shown that neuronal nuclear DNA fragmentation is frequent in aged dogs, although it is not accompanied by apoptotic morphology. Yet, a positive relation between TUNEL labelling and the degree of tissue autolysis was observed. In contrast, no TUNEL labelling was detected in young control dogs despite autolysis indices being similar to those in aged dogs. Taken together, these results suggest that neuronal nuclear DNA fragmentation is an age-related phenomenon, not due to apoptosis, whenever other factors render neuronal DNA more susceptible to autolytic fragmentation. We confirm the effect of autolysis in a subpopulation of neurons in the aged canine brain, inducing nuclear DNA fragmentation.

Aging↗
PubMed1999

Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA fragmentation.

Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very useful preparation. Enterocytes towards the apex of the villi become increasingly susceptible to apoptosis. Here, this "apoptotic gradient" is used to demonstrate the presence of BAX and BCL-2 proteins in the cytoplasm of cells at the onset of apoptosis. In semithin serial sections of the small intestine, BAX, BCL-2 and DNA fragmentation were demonstrated. BAX and BCL-2 are always colocalized and only in cells with fragmented DNA. The gradient of BAX or BCL-2 staining is similar to the gradient of DNA fragmentation. Immunoreactivity for BCL-2 or BAX is most intense in cells that are prone to become apoptotic next in the course of cellular turnover but not in cells in an advanced apoptotic state, showing strongly condensed chromatin. When using the same technique on semithin sections of kidney biopsies, containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Similar to the situation in the enterocytes, BAX staining was confined to the cytoplasm of epithelial cells with a moderate degree of DNA fragmentation and reduced in epithelial cells with a high degree of DNA fragmentation. In contrast to the situation in the small intestine, very low levels of BCL-2 were found. The results suggest that expression of BCL-2 and BAX is related to cell damage as indicated by DNA fragmentation but not to advanced stages of cellular death, as indicated by chromatin condensation and cellular shrinkage.

Animals↗
PubMed1987

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Animals↗
PubMed1987

Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change.

The human erythrocyte sugar transporter has been labelled at its internal site with cytochalasin B and at its outside site by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA). The cleavage of the transporter by various proteinases has been studied. Chymotrypsin, subtilisin and V8 proteinase give parallel fragmentation patterns for the two labels down to fragments as small as 7 kDa. Thus the binding sites for the two labels can only be separated by a small span of protein. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteines to give a 15 kDa fragment from the two labels. N-Bromosuccinimide (a reagent which preferentially cleaves at tryptophan residues) has revealed differences in fragmentation of the transporter labelled with either cytochalasin B or with ASA-BMPA. A major cleavage site is proposed to occur at tryptophan 186 which leaves a C-terminal fragment containing both labels. A tryptophan cleavage at residue 388 divides the cytochalasin B site and the ASA-BMPA site. A further tryptophan cleavage gives a cytochalasin B labelled 3 kDa fragment probably from residues 388-412. This gives an assignment of the cytochalasin B site as the inside of the hydrophobic span H 10. Since the ASA-BMPA site is probably only 7 kDa from residue 388 and is on the same 15 kDa NTCB fragment as cytochalasin B we assign this to the outside of hydrophobic span H 9. Thermolysin only cleaves the transporter labelled with cytochalasin B and not with ASA-BMPA. A 18 kDa cytochalasin B labelled fragment is formed. This is indicative of a change in conformation of the transporter when an outside ligand is bound such that the inside of the hydrogen bonding transmembrane segments H 7 and H 8 (and containing the proposed thermolysin cleavage site) are withdrawn from the cytosolic surface. Thus it appears that the core of the transporter (including the external and internal sites plus the transmembrane channel) is located between segments H 7 and H 10.

Affinity Labels↗
PubMed1989

Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity.

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.

Animals↗
PubMed1976

Preparation and properties of carp muscle parvalbumin fragments A (residues 1 leads to 75) and B (residues 76 leads to 108).

The calcium-binding protein (parvalbumin), isolated from carp (Cyprinus carpio) muscle, has been specifically fragmented into two polypeptides by tryptic hydrolysis at the single arginine residue at position 75. Fragment A contains residues 1 leads to 75 and fragment B is composed of residues 76 leads to 108. The fragments have been characterized according to size, amino acid composition, carboxyl- and aminoterminal analysis. Both fragments appear to be homogeneous by these criteria. The intact protein is known to bind 2 mol of calcium per mol of parvalbumin, and although each fragment alone contains all of the essential ligands for the coordination of one Ca2+, neither fragment displays calcium binding activity. Attempts to reconstitute the two fragments, under a variety of conditions, into a functional complex which can bind calcium have been unsuccessful. The side chain of Arg-75 is known to occupy an internal position in the crystalline structure of parvalbumin (Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313), where it is stabilized by an intricate network of hydrogen bonding involving the side chain of Glu-81. Although this internal salt bridge is approx. 20 A from either calcium binding site, it has been suggested that this structural feature of the molecule plays an essential role in the reversible binding of Ca2+. That the side chain of Arg-75 likewise occupies an internal position in the solution structure is indicated by its unavailability for reaction with 1,2-cyclohexanedione under conditions of physiological pH and temperature. However, in the presence of EDTA and at pH 8, it is readily modified by cyclohexanedione. This modification is accompanied by a concomitant loss in calcium binding activity. Reversal of the modification by treatment with hydroxylamine is accompanied by restoration of calcium binding activity. The serum of these data support the hypothesis that Arg-75 plays a critical role in the structural organization and calcium binding activity of the molecule, and in addition, suggests that the integrity of the peptide bond between Arg-75 and Ala-76 may be necessary for establishing the proper micro-environment required for formation of the internal salt bridge between Arg-75 and Glu-81.

Amino Acid Sequence↗
PubMed1979

Preparation of cyanogen bromide fragments of MM, NN, and MN glycoproteins (glycophorins) from human erythrocyte membranes of single donors.

A procedure is described for the preparation of three cyanogen bromide fragments of the MM, NN, or MN glycoprotein (glycophorin) of the human erythrocyte membranes, from erythrocytes of single donors. The fragments are obtained in pure form and excellent yields by employing procedures which include proteolytic inhibitors during membrane processing, thorough delipidation of the glycoprotein, and CNBr cleavage conditions which lead to quantitative fragmentation without loss of carbohydrates. A phenol-urea extraction resolves the two glycopeptide fragments from the carbohydrate-free fragment. The two glycopeptides are further purified by Bio-Gel P-6 and P-100 chromatography. The three fragments include the amino terminal 8 residue glycopeptide, a large glycopeptide form the middle of the molecule which bears the Asn-linked oligosaccharide and 8--9 O-glycosidically linked units, and a carboxyl terminal, carbohydrate-free, approx. 50 residue fragment. Their amino acid and carbohydrate composition, and size, are in close agreement with the sequence data of Tomita, M., Furthmayr, H. and Marchesi, V.T. (Biochemistry (1978), 17, 4756--4770). The fragments represent three well delineated portions of the glycoprotein molecule.

Amino Acids↗
PubMed1988

Location and characterization of the warfarin binding site of human serum albumin. A comparative study of two large fragments.

The warfarin binding behaviour of a large tryptic fragment (residues 198-585 which comprise domains two and three) and of a large peptic fragment (residues 1-387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate and characterize the primary warfarin binding site. The induced ellipticity of the warfarin-peptic fragment complex turned out to be pH dependent. This pH dependence occurs in the region of the neutral-to-base transition of the albumin molecule. The induced ellipticity of the warfarin-tryptic fragment complex is pH independent. Difference CD-spectra showed that the peptic fragment and albumin have similar warfarin binding properties whereas the tryptic fragment has deviant warfarin binding properties. The equilibrium dialysis experiments showed that the affinity of warfarin to the peptic fragment and to albumin is practically the same whereas the affinity of warfarin to the tryptic fragment is a factor 2-8 lower than the affinity of warfarin to albumin. Our results indicate that the main part of the primary warfarin binding site is located in domain two of the albumin structure and that domain one plays an important role in the N-B transition of albumin.

Binding Sites↗
PubMed1990

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains.

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.

Animals↗
PubMed1993

Characterization of the linker peptide of the single-chain Fv fragment of an antibody by NMR spectroscopy.

A comparison of the single-chain Fv fragment of the antibody McPC603 (scFv) with its corresponding unlinked Fv fragment has been carried out with 15N-edited NMR spectroscopy. The two Fv fragments adopt the same structure, indicating that the linker does not perturb the folding of the domains. This also directly demonstrates that folding in vivo (Fv fragment) and in vitro (scFv fragment) leads to the same structure. The main differences in the spectra of the uniformly 15N-labeled scFv and Fv fragments are due to signals of Gly and Ser from the linker peptide of the scFv fragment. The linker peptide has been mapped with NMR spectra of 15N-glycine- and 15N-glycine/15N-serine-labeled scFv fragments. The 15N T2 relaxation data indicate that the linker peptide is more flexible than the rest of the molecule.

Glycine↗