Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the invariant residue B26-tyrosine in determining the structural and biological properties of insulin has been extensively investigated by the use of semisynthetic des-(B27-B30)-insulins with modifications of position B26. Apart from the conventional trypsin-catalyzed peptide bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides we elaborated a new approach using des-(B26-B30)-insulin as substrate in alpha-chymotrypsin-mediated syntheses. Results obtained from bioassays and CD-spectroscopy underline the importance of position B26 to the association of the native molecule and to the modulation of structural and hormonal properties of shortened insulins.
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PMID:Enzyme-assisted semisynthesis of shortened B26-modified insulins. 182 45

Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.
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PMID:Semisynthetic des-(B27-B30)-insulins with modified B26-tyrosine. 193 Jul 32

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.
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PMID:Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain. 271 39

It has been shown that HLA-B27 lymphocytes from healthy individuals (B27+ ankylosing spondylitis [AS]-), which are not lysed by an antiserum against Klebsiella K43, can be rendered susceptible to lysis after incubation in the culture filtrate of Klebsiella K43. This finding is compatible with a specific modification by a Klebsiella K43-derived soluble factor of a B27-associated lymphoid cell component. Preliminary characterization of the factor has indicated that it is nondialyzable, but it is heat labile at 56 degrees C for 30 min and has a 35,000-50,000 mol wt. The modifying factor activity of the filtrate is destroyed by neuraminidase but not by trypsin and alpha-chymotrypsin. Furthermore, the ability of the factor to convert B27+AS- lymphocytes can be specifically absorbed by B27+AS- lymphocytes, but not by B27+AS+, B27-AS+, or by B27-AS- lymphocytes, which suggests that B27+AS- cells carry a hypothetical receptor which can specifically bind a Klebsiella K43 antigenic determinant. These results imply that the modification by environmental agents of specific major histocompatibility complex-associated gene products may be an important element in the pathogenesis of the HLA-B27-linked seronegative arthropathies.
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PMID:Characterization of a factor(s) present in Klebsiella culture filtrates that specifically modifies an HLA-B27-associated cell-surface component. 615 68

Although the association between human histocompatibility leukocyte antigen (HLA) B27 and ankylosing spondylitis is the prototype of HLA-disease association, the mechanism underlying these associations has not been determined. We have investigated the possibility that the B27 molecules from patients with ankylosing spondylitis are different from those of normals, and only the "different" molecules predispose the individual to disease. Biosynthetically radiolabeled HLA-B27 molecules from patients with ankylosing spondylitis and normal individuals were compared by two-dimensional gel electrophoresis and tryptic peptide mapping with high pressure liquid chromatography. Extensive charge heterogeneity in the 45,000-dalton heavy chain was detected when B27 molecules were analyzed by two-dimensional gel electrophoresis; the charge heterogeneity was reduced, but not eliminated, when the B27 molecules were treated with neuraminidase to remove sialic acid residues before analysis. No structural difference in the B27 molecules from an ankylosing spondylitis patient and a normal individual were detected by two-dimensional gel electrophoresis. Analysis of [(3)H]leucine-labeled and [(3)H]arginine-labeled tryptic peptides and chymotryptic peptides of the trypsin insoluble material by reverse-phase high pressure liquid chromatography revealed identity of the B27 molecules from ankylosing spondylitis patients and normal individuals. These studies indicate that development of akylosing spondylitis in only some B27 positive individuals is not attributable to those individuals possessing variant B27 molecules.
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PMID:Structural identity of human histocompatibility leukocyte antigen-B27 molecules from patients with ankylosing spondylitis and normal individuals. 705 55

Within the scope of structure-function studies on the proteohormone insulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26-B30 were synthesized by trypsin-catalyzed coupling reactions of des-(B23-B30)-insulin with synthetic peptides. The peptides were obtained by Fmoc solid-phase peptide synthesis. Insulins with multiple amino acid-->glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27-B30 on the self-association of the hormone. The analogues [GlyB27,B28,B29,B30]insulin and [GlyB27,B28,B30]insulin exhibit relative receptor affinities of 80% and self-associate. The successive extension of [AlaB26]des-(B27-B30)-insulin-B26-amide (relative receptor binding 273%) with amino acids corresponding to the native sequence B27-B30 showed the influence of the length of the B-chain on receptor affinity: the extension by B27-threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28-side chain on main-chain conformation, self-association and receptor binding was examined with [XB28]des-(B29-B30)-insulin-B28-amides (X = Phe, Gly, D-Pro). While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [PheB28]des-(B29-B30)-insulin-B28-amide (relative binding 50%) shows diminished self-association. The backbone-modified insulin derivative [SarB26]des-(B27-B30)-insulin-B26-amide (sarcosine = N-methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding.
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PMID:The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone. 856 84

The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines. Immunometric assays using all possible antibody combinations were used to study the topography of antibody binding sites on the antigen. We conclude that the CA 125 antigen carries only two major antigenic domains, which classifies the antibodies as OC125-like (group A) or M11-like (group B). One antibody, OV 197, showed binding specificity related to some of the OC125-like antibodies, but was classified into a separate group C. The OC125-like group of antibodies has four subgroups with different binding specificities. These are A1 = OC 125 and K 95, A2 = K 93, A3 = B43.13, and A4 = ZS 33, B27.1 and CCD 247. Binding of nonlabelled OC 125 or K 95 to CA 125 caused a marked increase in binding of labelled OV 197 to the complex. This conformational change was not observed with any other antibody combinations. Antibody B43.13 could form immunometric assay combinations particularly with antibodies of subgroup A4, indicating that the B43.13 epitope is in the periphery of the binding area of OC125-like antibodies. The M11-like group of antibodies is more homogenous with strong cross-inhibition between most antibodies. Only one antibody, ZR 38, would form an immunoassay combination with other M11-like antibodies and thus represents a distinct subgroup. The main group of M11-like antibodies are M 11, ZR 45, MA602-6, K 91, OV 185, K 101, K 90, K 96, K 97, K 102, CCD 242, 145-9, and 130-22. Antibody OV 197 binds to a domain designated C and is unique, as stated above. Antibody pairs from any two of the three groups may be used in immunometric assays. Three antibodies were not studied by complete cross-inhibition due to low affinity (OV 198 and K 100) or lack of material (MA602-1). OV 198 and K 100 are most likely OC125-like and MA602-1 is M11-like. Antibody affinity was estimated with labelled antigen in solution or with antigen absorbed on microtiter wells. Western blot analysis showed staining both in the stacking gel and corresponding to a molecule of 200 kDa. There was a marked difference between the antibodies in their ability to bind to CA 125 immobilized on a membrane. Strongest binding was observed with the M11-like antibodies, particularly M 11, K 96, K 97, MA602-6, 145-9. Antibodies belonging to the subgroup A4 were the only OC 125-like antibodies which reacted well with CA 125 in Western analysis. Digestion of CA 125 with proteolytic enzymes showed it to be particularly sensitive to trypsin cleavage. However, no low molecular weight fragments with preserved immunoreactivity were found.
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PMID:Specificity and affinity of 26 monoclonal antibodies against the CA 125 antigen: first report from the ISOBM TD-1 workshop. International Society for Oncodevelopmental Biology and Medicine. 868 1

Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.
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PMID:High efficacy of clonal growth and expansion of adult neural stem cells. 1286 Oct 35

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.
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PMID:Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain. 1497 29

Refolding of the heavy chain of the Class I HLA molecule, HLA-B27, in the absence of beta(2)m, yields soluble high molecular weight (HMW) oligomers reminiscent of the oligomeric forms of beta(2)m-free heavy chains (FHCs) of class I HLA antigens observed on cell surfaces. Here we examine the structural characteristics of HMW B27 in respect of features potentially relevant to autoimmunity, such as: (a) retention of native-like structure, since this could facilitate non-canonical interactions with T-cell receptors even in the absence of bound beta(2)m and peptide, or (b) presence of non-native structure, since this could yield novel (non-self) antigenic conformational epitopes that could elicit immune attack. We report that HMW B27 is characterized by high secondary structural content, structural stability, stability to proteolysis by trypsin, and structural features that are both partly native-like, and partly non-native-like, as assessed through the binding of conformationally-distinguishing and cross-reacting scFv antibodies specifically selected against HMW B27. We also present cell ELISA data with conformation-specific scFv antibodies that distinguish between lymphocytes from individuals who are healthy and B27 positive, and those who are B27 positive but suffering from ankylosing spondylitis.
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PMID:Refolding of HLA-B27 heavy chains in the absence of beta2m yields stable high molecular weight (HMW) protein forms displaying native-like as well as non-native-like conformational features: implications for autoimmune disease. 1803 53


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