Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino acid sequence of the light chain of bovine protein C was determined by sequenator analysis of the carboxymethylated light chain and fragments obtained by cyanogen bromide treatment, tryptic digestion after blocking of lysine residues, and cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole). The sequence was (in the standard one-letter code) A-N-S-F-L-X-X-L-R-P-G-N-V-X-R-X-C-S-X-X-V-C-X-F-X-X-A-R-X-I-F-Q-N-T-X-D-T-M-A-F-W-S-K-Y-S-D-G-D-Q-C-E-D-R-P-S-G-S-P-C-D-L-P-C-C-G-R-G-K-C-I-H-G-L-G-G-F-R-C-D-C-A-E-G-W-E-G-R-F-C-L-H-E-V-R-F-S-N-C-S-A-E-B-G-G-C-A-H-Y-C-M-E-E-E-G-R-R-H-C-S-C-A-P-G-Y-R-L-E-D-D-H-Q-L-C-V-S-K-V-T-F-P-C-G-R-L-G-K-R-M-E-K-K-R-K-T-L. The first eleven glutamic acid residues were carboxylated to gamma-carboxyglutamic acid (X). The NH2-terminal, vitamin K-dependent part showed an extensive homology to both prothrombin and factor X, whereas the rest of the chain showed a strong homology to factor X but little similarity to prothrombin.
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PMID:Bovine protein C: amino acid sequence of the light chain. 28 10

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.
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PMID:Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement. 171 May 19

The majority of methods used to prepare immunosorbents immobilize antibodies through their reactive amino acid residues. The bound antibody activity of these immunosorbents is low. Hydrazide-based matrices couple antibodies through carbohydrate chains frequently located in the Fc region. This paper reports a comparative study of the performance of immunosorbents prepared by cyanogen bromide or hydrazide immobilization methods. The experiments utilized murine monoclonal antibodies to the human plasma proteins Factor IX or Protein C. The antibodies were immobilized at low densities to beaded agarose matrices which had similar properties. The hydrazide immunosorbents had binding efficiencies which were lower (anti-Factor IX) or up to 1.6-fold higher (anti-Protein C) than comparable cyanogen bromide coupled gels. However, there was no improvement in performance due to lower recoveries of bound protein from the hydrazide gels. Control experiments demonstrated that oxidation of antibody which is required for its coupling to hydrazide gels had no effect on antibody binding to antigen. Our results indicate that, as with cyanogen bromide coupling methods, site-directed immobilization through carbohydrate residues results in a restricted ability to bind to antigen. Both monoclonals were found to contain carbohydrate in their Fab' regions through which coupling may have occurred. The frequency of carbohydrate in the Fab region and the ability to control glycosylation at these sites are factors which may impact the utility of carbohydrate-directed immobilization of antibodies.
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PMID:Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies. 174 16

Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Influences of antigen processing on the expression of the T cell repertoire. Evidence for MHC-specific hindering structures on the products of processing. 245 73

We have isolated a fragment (approximately equal to 10 kDa) of thrombomodulin containing the fifth and sixth epidermal growth factor (EGF)-like regions which retains thrombin binding capacity. The amino-terminal sequence of a 50-kDa active fragment of thrombomodulin derived from elastase proteolysis begins 11 residues before the first EGF-like structure of native thrombomodulin. Subsequent digestion with cyanogen bromide yields a 10-kDa thrombin binding fragment. The amino-terminal sequence of this fragment starts at the fifth EGF-like structure (Phe407). The amino acid composition suggests that this fragment contains the fifth and sixth EGF-like structures with a total of approximately 77 residues. This fragment lacks cofactor activity, but acts as a competitive inhibitor for protein C activation (Ki = 8.6 +/- 1.4 nM). We propose that the fifth and sixth EGF-like structures contain the thrombin binding site of thrombomodulin.
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PMID:A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site. 283 58

An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.
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PMID:Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells. 307 Mar 57

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.
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PMID:Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores. 677 41

A panel of five multiallelic and highly informative dinucleotide CA repeat markers flanking the APC gene was used for presymptomatic diagnosis of familial adenomatous polyposis coli (FAP). Marker regions were amplified by PCR. DNA fragments were separated by electrophoresis in denaturing polyacrylamide gels and visualised by ethidium bromide staining. Two or more markers were found to be informative in all nine families tested, and all 23 persons at risk could be diagnosed as affected or unaffected by the disease gene, the probability being > 99.9% in 14 cases from six families in which flanking markers were informative. We found no indication for locus heterogeneity of the disease in our sample. The polyposis phenotype and its extracolonic manifestations co-segregated with a distinct haplotype determined by the markers flanking the APC gene. In one family with no remaining living affected members, we could infer the high risk haplotype from genotyping of first degree relatives. The segregation of this haplotype is consistent with the occurrence of CHRPEs in the progeny. In a sporadic case we made use of the typical early extracolonic manifestations of the disease (osteomas, desmoids) to identify the high risk haplotype. We conclude from our experience that indirect genotyping of FAP with this particular panel of closely linked and highly polymorphic microsatellite markers is a rapid, efficient, and highly reliable method for presymptomatic diagnosis of FAP.
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PMID:Presymptomatic diagnosis in families with adenomatous polyposis using highly polymorphic dinucleotide CA repeat markers flanking the APC gene. 791 30

During a systematic search for germ-line APC mutations causative of familial adenomatous polyposis, we discovered what appeared to be an insertion mutation while simply checking exon 14PCR products by agarose gel electrophoresis (AGE). On AGE, exon 14PCR product from the known affected member of this family gave two bands: one of normal length, the other retarded on the gel equivalent to an increase in length of some 20-25 bp. Direct sequencing of DNA purified from the two bands gave identical results, and was consistent with amplification from the same two alleles: one wild-type, and the other having an 1893del4 mutation. This suggested that the normal length band on AGE consisted of DNA homoduplexes (normal:normal and mutant:mutant) and the retarded band consisted of DNA heteroduplexes (normal:mutant and mutant:normal). This hypothesis was tested by subjecting purified material from each of the two bands alone to a single cycle of heat denaturation and annealing, which showed that either band was equally capable of regenerating both bands. Because the anomalous migration of the heteroduplexes is observed in the presence of ethidium bromide, it implies that they have a cruciform of cruciform-like structure. This case illustrates the necessity to be aware of anomalous DNA migration and always sequence all putative mutations.
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PMID:Appearances can be deceptive: an APC 1893del4 mutation with unusual properities. Mutations in brief no. 171. Online. 1065 83

Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA approximately 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4-6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).
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PMID:Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator. 1168 79


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