Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of lauroyl sucrose capable of solubilizing 100% of beta-adrenergic receptors from bovine cerebellum membranes has been carried out. The preparative procedure for isolation of homogeneous beta-adrenergic receptors including affinity chromatography on the novel support, oxprenolol-Sepharose, is described. According to SDS-PAAG electrophoresis data, the Mr value for the beta-adrenergic receptor is 61 kD. The purified beta-adrenergic receptor can interact with the purified GTP-binding regulatory protein of adenylate cyclase (Gs) after their reconstitution into liposomes. Trypsin treatment of the purified receptor does not interfere with its functional properties, nor does it change the hydrodynamic parameters under non-denaturing conditions despite the fact that the polypeptide chain of the receptor is cleaved by trypsin.
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PMID:[Isolation of a homogeneous functionally active beta-adrenergic receptor from bovine cerebellum using lauroyl sucrose. Effect of trypsin on receptor activity]. 285 8

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.
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PMID:Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations. 294 92

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.
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PMID:Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen. 299 41

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta-mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39-kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39-kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.
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PMID:Properties and topography of the major integral plasma membrane protein of a unicellular organism. 313 63

L. donovani antigens were analyzed by a direct agglutination test (DAT), by ELISA using intact promastigotes and by the SDS-PAGE immunoperoxidase technique (SGIP). Sera of Chagas' disease patients cross-reacted in the ELISA and SGIP but not in the DAT. Trypsin treatment of the parasites removed concanavalin A-binding sites but not epitopes for antibodies present in Chagas' disease and in leishmaniasis sera, as seen in the SGIP. Eight bands were revealed after incubation of the gel sections with kala-azar or Chagas' disease sera, three of which were common to both sera. The major antigenic component recognized by leishmaniasis sera was a glycoprotein of 57 kD, and the major cross-reacting protein recognized by Chagas' disease sera was a glycoprotein doublet of 71.5-68 kD.
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PMID:Leishmania donovani donovani antigens recognized by kala-azar patient sera and identification of cross-reacting antigens to Chagas' disease. 314 97

Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca2+ channel.
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PMID:Localization by immunoelectron microscopy of spanning protein of triad junction in terminal cisternae/triad vesicles. 322 Sep 50

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.
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PMID:Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies. 328 Jun 2

A simple immunoaffinity column chromatographic procedure is described whereby recombinant human prorenin secreted from Chinese hamster ovary cells may be isolated in a high state of purity from serum-free culture medium. Prorenin thus purified has been characterized by SDS-polyacrylamide gel electrophoresis and by partial sequence analysis which has revealed the expected N-terminal sequence. Trypsin treatment gives rise to renin, and reversible acid activation has also been demonstrated for the recombinant zymogen.
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PMID:Immunoaffinity purification of human prorenin produced in Chinese hamster ovary cells. 328 27

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.
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PMID:Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis. 330 38

Electron microscopy and SDS-PAGE have been used to analyse the polypeptide and antigenic composition of the sheath and core components of the axial filament of Treponema pallidum. The sheath contains a major 37 kDa polypeptide which was solubilized by a combination of trypsin and urea treatments with concurrent loss of binding of anti-37 kDa monoclonal antibody. These studies also indicated some antigenic heterogeneity within the axial filament population. Trypsin treatment alone removed a number of antigenic determinants from the axial filament but left others intact, suggesting differences in their location within the sheath structure. A second 31.5 kDa polypeptide may also be associated with the sheath. The axial filament core comprises at least two components, an antigenically dominant 33.5 kDa polypeptide and a second of 34 kDa. The structure of the axial filament in T. pallidum and Treponema phagedenis biotype Reiterii was similar, but antigenic cross-reactivity of sheath and core components was incomplete.
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PMID:Analysis of sheath and core structures of the axial filament of Treponema pallidum. 331 73


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