Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The lectin-like activity of the Lactobacillus acidophilus strain JCM 1026 was studied by hemagglutination and hemagglutination-inhibition assays. L. acidophilus strain JCM 1026 was found to hemagglutinate human and animal erythrocytes. Neuraminidase-treatment of human-type O erythrocytes enhanced the activity. Treatment of the bacterial cells with proteinase K reduced hemagglutinating activity significantly. Although several mono- and disaccharides did not inhibit hemagglutination, several different glycoproteins did. These data indicate that a proteinaceous lectin-like component(s) recognizing carbohydrate-containing molecules is located on the cell surface of L. acidophilus.
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PMID:Lectin-like activity of Lactobacillus acidophilus strain JCM 1026. 145 21

The structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation. Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.
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PMID:Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties. 160 63

The orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.
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PMID:Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes. 166 Apr 76

The adherence of Treponema denticola GM-1, TD-4, and MS25 to human gingival fibroblasts (HGFs) was studied to serve as an introduction to investigations into the interactions of these oral bacteria with human host cells. Under both aerobic (5% CO2) and anaerobic (85% N2 plus 10% H2 plus 5% CO2) environments, the interactions with the HGFs were such that strains GM-1 and MS25 were consistently more adherent than strain TD-4. Polyclonal antibodies to GM-1 inhibited GM-1 adherence by 70%, while MS25 and TD-4 showed differing degrees of cross-reactive inhibition, indicative of common but not identical epitopes on the surface of the three T. denticola strains. Pretreatment of the three strains with trypsin did not inhibit adherence; proteinase K did, however, inhibit this interaction by 80%. Trypsin pretreatment of the HGFs resulted in increases in adherence of 50 and 86% for GM-1 and MS25, respectively, while a decrease of 41% was noted for TD-4. Exposure of the T. denticola strains to sugars and lectin pretreatment of the HGFs implicated adherence mediation by mannose and galactose residues on the HGF surface. Periodate treatment of HGFs resulted in a 50% drop in adherence for GM-1 and MS25, but did not decrease that of TD-4. Addition of fetal bovine serum inhibited adherence of the three strains to differing degrees, with TD-4 being the most susceptible. Addition of purified fibronectin (100 micrograms/ml) resulted in greater than 50% inhibition in GM-1 and MS25 adherence, while a 25% increase occurred with TD-4. While strain differences were noted in some of the parameters studied, the results indicate two possibilities for T. denticola-HGF adherence: a lectinlike adhesin(s) on the T. denticola surface with affinity for galactose and mannose on the HGF surface, and a serum host factor(s) bridging T. denticola and HGFs.
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PMID:Interaction of Treponema denticola TD-4, GM-1, and MS25 with human gingival fibroblasts. 216 Apr 30

Frozen sections of human gingiva and skin, fixed in acetone, were subjected to limited enzyme digestion (neuraminidase, proteinase K, trypsin) or, respectively, the application of solvents (chloroform/methanol, triton X-100) to allow a partial characterization of epithelial lectin binding sites. Gingiva differs from normal skin in that more conA-binding glycolipids are present in the lower cell layers. In the upper layers conA-fixing glycoproteins are prevailing. Psoriatic foci regularly exhibit an increased presence of conA-binding glycolipids. Gingiva and normal skin have some common features in the behavior of the lectin binding sites of HPA, WGA and UEA I. Analogies in the binding pattern of conA and UEA I in gingival tissue and in psoriatic foci are thus due to different lectin receptors.
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PMID:[Partial characterization of the epithelial lectin binding sites of human gingiva and skin]. 259 11

Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.
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PMID:Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli. 267 61

The functional domains of the glycoproteins of the pig zona pellucida have been analysed using lectin binding, peptide mapping, and immunoblotting in conjunction with analysis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and protein detection with the silver-based colour stain. Two of the pig zona pellucida glycoproteins identified in 2D-PAGE were differentially proteolysed within the intact matrix by a variety of enzymes. This proteolysis of specific proteins, however, did not affect the suprastructure of the matrix, or inhibit spermatozoa from adhering to the surface of the zona pellucida. The major glycoprotein appears to be involved in the structural maintenance of the zona pellucida because dissolution of the matrix correlated with proteolysis of this glycoprotein by proteinase K. These glycoproteins were further evaluated by lectin blotting with Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) before and after proteolysis of zona pellucida with trypsin. The lectins bound to all charge species of the three major zona pellucida glycoproteins. Only the most acidic components of the major glycoprotein family, which are not extensively digested, were recognized by these lectins after proteolysis. These studies provide evidence that the major glycoprotein family I of the pig zona pellucida is primarily responsible for maintaining the integrity of the matrix.
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PMID:Evidence for a role of the major glycoprotein in the structural maintenance of the pig zona pellucida. 343 Apr 58

Complexes of HLA class II alpha- and beta-chains with invariant chain were proteolytically digested to study domain interactions between these molecules. Detergent extracts of metabolically labeled monensin-treated B lymphoblastoid cells (B-LCL) were digested with proteinase K and immunoprecipitated with anti-HLA-DR or anti-invariant chain antibodies. Subsequent two-dimensional polyacrylamide amide gel electrophoresis showed that proteinase K treatment results in the sequential generation of three polypeptides of approximately 21,500, 19,500, and 18,000 daltons respectively. All are proteolytic fragments derived from invariant chain, and all remain associated with class II antigens. Two-dimensional gels of endoglycosidase H-treated immunoprecipitates showed that all three fragments contain two N-linked oligosaccharides. Neuraminidase treatment of immunoprecipitates and Bandeiraea simplicifolia lectin binding of cell extracts showed that the largest fragment, but not the smallest fragment, also contains O-linked oligosaccharides. None of the fragments possess the transmembrane region; fragments were released in soluble form when biosynthetically labeled B-LCL were ruptured by freezing and thawing and intact membranes were separated from aqueous components by ultracentrifugation. Lack of the transmembrane sequence was confirmed on the 18,000 dalton fragment by demonstrating through specific peptide cleavage at tryptophanyl residues that this fragment retains a substantial portion of the C-terminal region of I chain beyond trp162. Retention of the C-terminal region excludes the presence of the transmembrane region when m.w. are considered. Our data, taken in context of the amino acid sequence of the invariant chain predicted by the cDNA clone, demonstrate that invariant chain interacts with class II antigens via its extracytoplasmic region.
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PMID:Invariant chain associates with HLA class II antigens via its extracytoplasmic region. 351 15

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.
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PMID:The cell surface of a restrictive fenestrated endothelium. I. Distribution of lectin-receptor monosaccharides on the choriocapillaris. 394 19

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.
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PMID:Changes in cell surface antigens during in vitro lizard myogenesis. 634 59


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