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PubMed1991

Distinct pathways of CD4 and CD8 cells induce rapid target DNA fragmentation.

When activated with either Con A, a CD3-specific mAb, or Ag-pulsed B lymphoma (LK35.2) cells, CD4 (Th1) clones quickly induce DNA fragmentation in target cells followed by release of 51Cr-labeled intracellular materials. Both activated CD4 clones and CD8 (CTL) cells fragment target DNA into electrophoretically identical "ladder" pattern made of approximately 200 bp. The effect of various metabolic inhibitors on the ability of CD4 and CD8 cells to induce target DNA fragmentation was studied. Little effect was observed with the DNA synthesis inhibitor, mitomycin C. The RNA synthesis inhibitor, actinomycin D, and the protein synthesis inhibitor, cycloheximide, strongly inhibited the ability of CD4 cells, but not CD8 cells, to induce target DNA fragmentation. In contrast, target DNA fragmentation by CD8 cells, but not by CD4 cells, was inhibited by cholera toxin. Although cyclosporin A inhibited CD4 cells to fragment target DNA during the early phase (90 min) of E:T interaction, this inhibition was not sustained in the later phase (210 min) of the assay. Zinc ions inhibited the ability of both CD4 and CD8 cells to fragment target DNA. Treatment of effectors and targets with these inhibitors, followed by washings, demonstrated that the action of these inhibitors on effector cells alone is sufficient to inhibit target DNA fragmentation. The strong correlation among these parameters of DNA fragmentation and Cr-release assays supports the hypothesis of programed cell death. Although distinct cytolytic pathways are used by CD4 and CD8 cells to kill targets, both pathways deliver a signal that activates endonuclease(s), fragments target DNA, causes Cr-release, and lyses target cells. Taken together with our previous studies, the present findings demonstrate that activated cytolytic CD4 clones do not use perforin, serine proteases, and TNF as mediators for resistant target DNA fragmentation.

Animals↗
PubMed1984

Interference with tolerance induction of primed B cells by the Fc fragment of immunoglobulin.

The capacity to interfere with tolerance induction in primed B cells was examined. Previous work had shown that TNP-specific splenic B cells from mice primed and boosted with TNP-KLH are highly susceptible to in vitro tolerization upon a brief exposure to TNP on a carrier unrelated to KLH. In the present work it was found that tolerance induction in these primed B cells could be partially disrupted by addition of the Fc fragment of immunoglobulin, a B-cell mitogen, and adjuvant, during exposure of the B cells to tolerogen. Addition of Fc fragments prepared by papain digestion of human IgG interfered with tolerization routinely in approximately 30-60% of the spleen cells susceptible to tolerogen. Addition of whole IgG or Fab fragments had no effect on tolerance induction. As little as 5 micrograms/ml of the Fc fragment preparation significantly interfered with tolerization and 32-64 micrograms/ml was optimal. Disruption of tolerization was most effective when the Fc fragment was added to the spleen cells either 4 hr prior to tolerogen or simultaneously with tolerogen; addition of the Fc fragment 4 hr after exposure to tolerogen was significantly less effective. Disruption of tolerization by the Fc fragment was not through polyclonal activation of B cells, as antigen was required for generation of significant numbers of PFC to TNP. Also, disruption was not through expansion of low avidity clones of B cells insusceptible to tolerogen, as the avidity of the antibody produced with and without Fc fragments present was approximately the same. These results show that the Fc fragment of IgG can partially interfere with tolerization of primed B cells. The manner in which Fc fragments may function to prevent tolerization through its lymphoid cell stimulatory capacities is discussed.

Animals↗
PubMed1998

Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4.

Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast transcription factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

Amino Acid Sequence↗
PubMed1978

Isolation of enzymatically derived fragments of guinea pig IgG and an examination of their reactivity against Staphylococcal protein A.

Papain digest of guinea pig IgG2 was separated by ion-exchange chromatography into five fractions, one containing mainly Fab and four Fc fractions. Covalently linked Fc (cFc) and non-covalently linked Fc fragments (nFc) were present, both with a molecular weight (Mw) of about 56,000. nFc was split into half fragments by gel filtration under dissociating condition (Mw 25,000 to 27,000). An incomplete Fc fragment (iFc) was also isolated (Mw 36,000), which consisted of a Fc chain (Mw 26,000) non-covalently linked to a piece of another chain. These preparations all reacted with protein A. In addition, the Fc' fragment (Mw 22,000), which is the C-terminus of the Fc fragment, was isolated. This fragment did not react with protein A. The cFc was treated with acid and then digested with trypsin at pH 7.8 for 45 s. The digest were separated into four fractions (I-IV) by gel filtration on Sephadex G-100. Fraction I was indistinguishable from intact Fc, fraction II contained an incomplete covalent Fc fragment (Mw 34,000 to 38,000). Both these fragments reacted with protein A. Fraction IV consisted of fragments belonging to the C-terminus, and was protein A non-reactive. Fraction III was shown to contain a mixture of fragments belonging to fractions II and IV.

Animals↗
PubMedDate not supplied

[Immunochemical properties of tryptophanyl-tRNA synthetase and its fragments].

The interaction between beef pancreas tryptophanyl-tRNA synthetase and its fragments produced after limited proteolysis, with IgG fraction of antiserum and with Fab fragment of IgG has been studied. Both the intact antibodies and Fab fragments inhibit the enzyme activity in tRNA aminoacylation and tryptophan dependent ATP-32P pyrophosphate exchange reactions. However, the enzyme inhibited by antibodies is still able to form a complex with tryptophanyl-tRNA. The enzymatically active fragment obtained after endogenous proteolysis interacts only with 1/3 of the antibodies against native enzyme. The fragment produced by trypsinolysis possess similar immunochemical properties. This fragment has almost the same molecular weight but is enzymatically inactive. Pure antibodies against tryptic fragment isolated by means of specific immunoabsorbent inhibit the enzymatic activity. The antibodies which do not interact with this fragment (2/3 of the total amount of antibodies) have no influence on the enzymatic activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptis fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis. Probably the amino acid residues of this peptide participate in formation of the active centre of tryptophanyl-tRNA synthetase. A new procedure for determination of the number of antigenic determinants in proteins is developed. It is shown by this method that beef pancreas tryptophanyl-tRNA synthetase contains 9 +/- 1 antigenic determinants.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.

Proteins were digested in normal and highly 18O-enriched water using proteases commonly employed for protein sequencing. The extent of 18O incorporation into the resulting peptide fragments was characterized by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The endoproteinases trypsin, Lys-C and Glu-C incorporate two atoms of 18O, resulting in a mass shift of +4 D for the peptide fragments. This indicates that, following proteolytic cleavage, peptide products continue to interact with these proteases and undergo repeated binding/hydrolysis cycles, resulting in complete equilibration of both oxygens in the carboxy terminus of the fragments with oxygen from solvent water. In contrast, chymotrypsin and Asp-N incorporate only one atom of 18O, resulting in a mass shift of +2 D, indicating that after the cleavage step these proteases do not accept the peptides as substrates. In addition, it was found that the proteases trypsin, Glu-C, and Lys-C exhibit minor or nontypical sequence specificities, resulting in unexpected peptide fragments. These fragments incorporate only one 18O atom, indicating that they do not undergo further binding/hydrolysis cycles with the enzyme. Thus, it is possible to discriminate between enzyme-typical peptide fragments with mass shifts of +4 D and nontypical fragments with mass shifts of only +2 D. Based on these observations, protein digest strategies are described for the generation of 1:1 ion doublets spaced either by 2 or 4 D. In addition, the C-terminus of a protein can be identified by the absence of an ion doublet in the corresponding peptide fragment. In protein sequencing by mass spectrometry, digest protocols generating ion doublets provide the most clear-cut analytical results for the recognition of ion series in ESI-MS/MS and MALDI post-source decay (PSD) product ion spectra. Only the mass spectrometric fragment ions of a C-terminal series show ion doublets spaced either by 2 or 4 D, whereas the fragment ions belonging to an N-terminal series remain unshifted. This assignment unequivocally reveals the direction of the identified sequence.

Endopeptidases↗
PubMedDate not supplied

Cell adhesion and neurite extension in response to two proteolytic fragments of laminin.

Studies from several laboratories have suggested that laminin contains at least two domains that selectively mediate cell type-specific behavior. In this study, two proteolytic fragments of laminin are evaluated for their ability to interact with three different populations of embryonic chicken cells. A 600 kDa thrombin fragment, derived from the central portion of the laminin molecule, supports attachment of dorsal root ganglion (DRG), spinal cord (SC), and heart cells. Neurons from both DRGs and SCs extend neurites in response to this fragment. Quantitatively, both cell adhesion and neurite extension on the 600 kDa fragment are comparable to these responses to intact laminin. A 440 kDa chymotrypsin fragment, derived from either intact laminin or the 600 kDa fragment, does not support equivalent responses. Fewer DRG cells attach to this fragment and neurites are shorter than on the 600 kDa fragment. Heart and SC cell attachment is also reduced in comparison with activity of the 600 kDa fragment, and SC neurites do not form on the 440 kDa fragment. These results suggest that there are at least two cell binding domains in the laminin molecule, one with which a variety of cell types can interact and another that may mediate more restricted cellular responses. The latter site appears to be relatively inactive for SC and heart cell adhesion but supports limited attachment and neurite extension by DRG neurons.

Animals↗
PubMed2001

A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart.

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.

Amino Acid Sequence↗
PubMed1988

Heterogeneity of BamHi DNA fragments B and E in several HSV-1 strains and recombinants.

The restriction cleavage sites of the BamHI-B and BamHI-E DNA fragments of several Herpes simplex virus type 1 (HVS-1) strains were mapped. These fragments are situated at the ends of the long unique regions and share homologous sequences in the repeat components (TRL and IRL) of the genome. All the strains analyzed were found to have deletions in the Hpal-P fragment, situated in the BamHI-B fragment. Five strains were further analyzed and the deletions were located in the Smal-A fragment (within the Hpal-P fragment). The BamHI-E fragment of four recombinants (obtained by recombination between the HFEM genome and the BamHI-B fragment or part of it from the HSV-1 F strain) were almost identical but differed from another strain [NIH(LP)]. Comparison of the BamHI-B and the BamHI-E fragments of the same strain revealed that the fragments were not identical in all cases.

Cloning, Molecular↗
PubMed1991

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules.

Ca2+ titrations of the intrinsic fluorescence of a series of gamma-carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibit Tm (the (Ca2+)total concentration at which ln (B/F) = 0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase in Tm to 5.4 mM is observed for 8-GLA fragment 1. Tm decreases to 3.8 mM for the 7- and 6-GLA proteins. The value of h, about 2.8 +/- 0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2-1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca2+, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes in Tm and h response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.

1-Carboxyglutamic Acid↗
PubMed1978

Purification and characterization of the 45,000-Dalton fragment from tryptic digestion of (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum.

Tryptic digestion of (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaCl3, HgCl2, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane.

Animals↗
PubMed1998

Reducing the renal uptake of radiolabeled antibody fragments and peptides for diagnosis and therapy: present status, future prospects and limitations.

Elevated renal uptake and prolonged retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. Over recent years, one of the focuses of research has therefore been to develop suitable methods to reduce this renal uptake, and to evaluate whether the resulting methodology will benefit therapy with antibody fragments and peptides. In these studies it has been shown that the kidney uptake of antibody fragments in animals can be reduced in a dose-dependent manner by almost one order of magnitude by the systemic administration of cationic amino acids and their derivatives, whereas the uptake in all other organs, as well as the tumor, remains unaffected. A similar reduction in renal retention is achieved for all intracellularly retained radionuclides (e.g., radiometals) or radioiodinated immunoconjugates, as well as for smaller peptides. Lysine is usually the preferred agent, and its d- and l-isomers are equally effective whether given intraperitoneally or orally. Amino sugars are effective, but their N-acetyl derivatives, lacking the positive charge, are not. Basic polypeptides are also effective, and their potency increases with increasing molecular weight (i.e., the amount of positive charges per molecule). Urine analysis of treated individuals shows the excretion of unmetabolized, intact fragments or peptides, in contrast to mostly low-molecular-weight metabolites in untreated controls. In therapy studies using radiometal-conjugated Fab fragments, the kidney is the first dose-limiting organ. Administration of cationic amino acids results in a substantial increase in the maximum tolerated dose of such Fab fragments. No biochemical or histological evidence of renal damage has been observed under these conditions. As was the case in animal studies, in pilot clinical trials the renal uptake in patients injected with Fab' fragments and given amino acids could be decreased significantly, whereas the uptake by all other organs remained unaffected. These recent studies indicate that a variety of basic compounds are capable of inhibiting the tubular reabsorption of peptides and proteins, thus significantly lowering the renal uptake of antibody fragments or peptides in both animals and patients. On a molecular basis, the effect seems to rely essentially on the presence of a positively charged amino group. Thus, radiation nephrotoxicity of antibody fragments and peptides can be overcome successfully; this may provide new prospects for cancer therapy with radiolabeled antibody fragments and peptides.

Amino Acids↗
PubMed2004

Distribution of labelled anti-tenascin antibodies and fragments after injection into intact or partly resected C6-gliomas in rats.

INTRODUCTION: For treatment of malignant glioma, radioimmunotherapy has become a valuable alternative for more than 2 decades. Surprisingly, very little is known about the distribution of intralesionally administered labelled antibodies or fragments. We investigated the migration of labelled antibodies and antibody fragments injected into intact and partly resected C6-glioma in rats at different times after injection. MATERIALS AND METHODS: Nine days after induction of a C6-glioma, 5 microl of 125I-labelled murine anti-tenascin antibodies (n = 31) or 125I-labelled fragments of anti-tenascin antibodies (n = 32) was injected slowly into the tumour (group I). In group II the tumour was subtotally resected 9 days after induction of the C6-glioma, and 24 h later the labelled antibodies (n = 30) or fragments (n = 12) were injected into the resection cavity. At 6, 24 or 48 h after the injection, animals were sacrificed, and brains removed. Distribution of labelled antibodies and fragments was determined by superimposing autoradiographs onto frozen sections and HE-stained neighbouring sections using a digital image analysing system. RESULTS: After injection into intact C6-glioma, labelled antibodies covered a maximum distance of 3.2 +/- 1.0, 4.1 +/- 1.9 and 4.8 +/- 0.9 mm after 6, 24 and 48 h, respectively; while labelled fragments were found at a distance of 6.7 mm (+/-1.1) after 24 h and 5.8 mm (+/-0.9) after 48 h (significant in univariate analysis). Following partial tumour resection, the respective distances at 24 h were 3 +/- 0.4 mm for anti-tenascin antibodies and 3.4 +/- 0.3 mm for Fab fragments. CONCLUSION: After injection into C6-glioma, labelled fragments are able to cover a greater distance than labelled antibodies. Injection of antibodies and fragments 1 day after tumour resection results in reduced velocity of both antibodies and fragments.

Animals↗
PubMed1980

The interaction of bovine transferrin and monoferric transferrin fragments with rabbit reticulocytes.

1. The mechanism of interaction of transferrin with reticulocytes has been investigated using monoferric fragments derived by proteolysis from bovine Fe2-transferrin. 2. Rabbit reticulocytes readily took up iron from bovine transferrin, but only slight uptake occurred from the C-terminal fragment (S), and almost none from the N-terminal fragment (F). 3. The degree of binding of transferrin and fragments to the cells was in the order transferrin greater than fragment F greater than fragment S. 4. Binding of transferrin and fragment S, but not of fragment F, was reduced when incubation was performed at 4 degrees C instead of 37 degrees C, and all iron uptake was abolisehd. 5. Preincubation of reticulocytes with fragment S, but not with fragment F, somewhat reduced subsequent iron uptake from transferrin. 6. The presence of bovine serum albumin (40 mg/ml) in the incubation buffer inhibited iron uptake, but iron uptake nevertheless occurred from transferrin in bovine serum. 7. No differences were detected in the rate of 59Fe uptake from transferrin labelled asymmetrically by sequential additions of 59Fe and 56Fe to apotransferrin. 8. It is concluded that both halves of the transferrin molecule are involved, perhaps in different ways, in the interaction of transferrin with reticulocytes, and that rabbit reticulocytes do not take up iron preferentially from one of the binding sites of bovine transferrin.

Animals↗
PubMed1981

Purification of a trypsin-insensitive fragment of spectrin from human erythrocyte membranes.

When spectrin is treated with trypsin, a series of polypeptide fragments is generated, One particular fragment having an approximate molecular weight of 80 000 constitutes 18% of the trypsin-digested mixture and is trypsin-insensitive. This fragment has been isolated and purified by gel filtration followed by ion-exchange chromatography. The molecular weight of the fragment, as seen from sedimentation equilibrium measurements and from gel electrophoresis, both in the presence and absence of detergent, is close to 80 000. There was no evidence of self-association under the conditions used. Changes in the specific rotation at 365 nm were used to detect temperature-dependent conformation changes in the fragment and to compare these changes with those in the intact spectrin molecule. The fragment undergoes temperature-dependent transitions centered at 46 and 58 degrees C, similar to those in intact spectrin (49 and 55 degrees C). Although the thermal transitions exhibited by intact spectrin are markedly salt-dependent, those shown by the fragment are not. ORD (optical rotary dispersion) measurements indicate 53% apparent alpha-helix in the fragment, compared to 68% in intact spectrin. Antibodies raised against the fragment cross-react only with band 1, the largest polypeptide of spectrin, indicating that the fragment is derived from band 1.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Fragmented fibronectin and other synovial fluid proteins in chronic arthritis: their relation to immune complexes.

Fibronectin, an opsonic glycoprotein has been shown to exist in fragmented forms in serum and synovial fluid. Some fragments in synovial fluid appear to be polyethylene glycol (PEG) precipitable, suggesting incorporation into immune complexes (IC). PEG precipitation, SDS-PAGE and immunoblotting were used to determine whether PEG precipitable fragments are real or artefactual. Disease specificity of fragmentation and IC incorporation of fibronectin and other proteins were also studied using these techniques. PEG precipitable fragments do not appear to be artefactual, although some fibronectin fragments are cryoprecipitable. Protein fragments showed similar distributions in whole serum and synovial fluid, disease specific differences being confined to PEG precipitates. Rheumatoid arthritis (RA) synovial fluid PEG precipitates displayed the greatest array of fragmented immunoglobulins and fibronectin. No PEG precipitates contained albumin fragments. Protein fragments in IC may impair their effective removal from RA joints. Accumulated IC could lead to tissue damage via complement activation.

Adult↗
PubMed1986

Two plasma fibronectin fragments with different gelatin-binding properties.

Two subtilisin-generated fragments of human plasma fibronectin exhibit significantly different gelatin affinities. One fragment of 60 kdaltons elutes from gelatin-Sepharose, after intact fibronectin, with pH 5.5, 50 mM citrate, 0.1 M NaCl. A 40 kdalton fragment, which can be derived from the 60 kdalton fragment, remains bound to gelatin under the same conditions. Furthermore, upon urea gradient elution, the 60 kdalton fragment dissociates from gelatin before the smaller fragment. Of the two fragments, the 60 kdalton fragment also demonstrates affinity for heparin. Identical amino-terminal sequences for the two fragments indicate that the heparin-binding activity resides in the difference region at the carboxyl-terminal of the 60 kdalton fragment.

Amino Acid Sequence↗
PubMed1989

Binding of human IgA1 and IgA1 fragments to jacalin.

Interaction of jacalin, an N-terminal galactose specific lectin, with human IgA1 and IgA1 fragments was investigated. IgA1 and all galactose containing fragments bound to jacalin-Sepharose, including Fab fragments containing only the galNac linked to serine-224 and Fc fragments containing four gal-galNac sequences. These data indicate that both the galNac and gal-galNac sequences can interact with jacalin. Jacalin precipitated IgA1 and the fragments F(abc)2, F(ab')2 and Fc in agar gel and from solutions. It also precipitated Fab' fragments in agar gel. Jacalin did not precipitate Fab fragments significantly. This suggests that, except for the single binding site on the Fab fragments containing the galNac linked to serine-224, jacalin itself also has a limited number of sites to interact with N-terminal galactose residues. ELISA studies revealed that intact IgA1 had a lower jacalin binding capacity than F(abc)2 fragments which lack CH3 domains, than F(ab')2 which lack the CH2 and CH3 domains, and than Fc fragments containing four gal-galNac sequences. This led to the conclusion that part of the galNac or gal-galNac sequences in intact IgA1 molecules are inaccessible to interaction with jacalin. Cleaving the C-terminal domains off may have induced a reorientation of the hinge region structure, including the orientation of the carbohydrate units.

Animals↗