Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dermal keratin bodies, consisting mainly of keratin intermediate filament aggregates (KIFA) coated with IgM anti-KIF autoantibodies, are present in normal human skin and occur in increased quantities in certain skin diseases. Keratin bodies are normally rapidly removed, but in primary localized cutaneous amyloidosis (PLCA) they are converted by an unknown mechanism to amyloid. Amyloid P component (AP), a glycoprotein identical to, and derived from, the normal plasma protein serum amyloid P component (SAP), is present in all forms of amyloid including PLCA. We investigated the interaction between SAP, keratin bodies, and KIFA. Immunofluorescence staining of normal skin using fluoresceinated anti-SAP and rhodamine-conjugated anti-IgM, or AE-1/AE-3 anti-keratin antibodies followed by Texas Red-conjugated anti-mouse immunoglobulin, showed that 52% +/- 4 (mean +/- sem, n = 6) of keratin bodies bound anti-SAP. Similar findings were present in a biopsy from a patient with lichen planus. Isolated KIFA, prepared by 8M urea extraction of normal human epidermis or cultured keratinocytes, were preincubated with normal human serum as a source of SAP and then stained with fluoresceinated anti-SAP. Bright fluorescence seen when the incubation medium contained Ca++ was absent in the presence of ethylenediamine tetraacetic acid. Specific Ca++-dependent binding of SAP to KIFA was confirmed using immunoblotting. Binding of SAP to KIFA did not prevent their degradation following exposure to trypsin or alpha-chymotrypsin. Similarly, partial enzymatic digestion of KIFA did not abrogate their ability to bind SAP. Our findings, that SAP is associated with keratin bodies in skin and exhibits Ca++-dependent binding to KIFA in vitro, identify keratin filaments as a newly recognized ligand for SAP.
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PMID:Amyloid P component binds to keratin bodies in human skin and to isolated keratin filament aggregates in vitro. 245 1

In this study, we have labelled proteins on the surface of unfertilized, zona-free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow-bore glass pipette. Surface proteins were labelled using sulfo-NHS-biotin (sulfosuccinimidobiotin), a water-soluble, membrane-impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin-FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using streptavidin-HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of M(r) 82 and 69 kD, but other major proteins of M(r) 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of M(r) 18-100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos.
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PMID:Biotinylation of proteins on the surface of zona-free mouse oocytes. 835 34

Recently, we have observed a nuclear localization for human alpha(1)-antichymotrypsin (AACT) expressed in the cytosol of transgenic Bright Yellow-2 (BY-2) tobacco cultured cells (see accompanying paper: Benchabane, M., Saint-Jore-Dupas, C., Bardor, M., Faye, L., Michaud, D. and Gomord, V. (2008a) Targeting and post-translational processing of human alpha(1)-antichymotrypsin in BY-2 tobacco cultured cells. Plant Biotechnol. J. doi: 10.1111/j.1467-7652.2008.00382.x). In the present article, we assess whether the intrinsic DNA-binding activity of AACT can explain its nuclear localization, and whether this same activity has an impact on its protease inhibitory potency and stability in planta. An engineered form of AACT with no DNA-binding activity, rAACTDeltaK, was compared with the wild-type polypeptide, rAACT, in terms of chymotrypsin inhibitory potency, stability in planta and distribution in tobacco cells. In accordance with available data reporting distinct sites for protease inhibition and DNA binding, rAACT and rAACTDeltaK showed similar antichymotrypsin activity, similar to the activity of native AACT purified from human plasma. As observed for AACT in BY-2 tobacco cells, a green fluorescent protein (GFP)-AACT fusion transiently expressed in the cytosol of tobacco leaf epidermal cells was detected mainly in the nucleus by confocal laser microscopy. By contrast, rAACTDeltaK expressed as a GFP fusion showed a balanced distribution between the cytosol and the nucleus, similar to the distribution pattern of free GFP exhibiting no DNA-binding affinity. In line with immunodetection data showing higher accumulation levels for GFP-AACT in tobacco leaf cells, rAACTDeltaK was more susceptible than rAACT to tryptic digestion in the presence of DNA. Overall, these observations suggest the following: (i) a retention effect of DNA on AACT in the nucleus; and (ii) a stabilizing effect of the AACT-DNA interaction on rAACT challenged with non-target proteases, which, possibly, may be useful in protecting this protein in plant expression platforms.
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PMID:Nucleocytoplasmic transit of human alpha1-antichymotrypsin in tobacco leaf epidermal cells. 1905 6