scholarly journals Biosynthesis of rat MUC2 in colon and its analogy with human MUC2

1995 ◽  
Vol 309 (1) ◽  
pp. 221-229 ◽  
Author(s):  
K M A J Tytgat ◽  
F J Bovelander ◽  
F J M Opdam ◽  
A W C Einerhand ◽  
H A Büller ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Buoyant Density ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Rat Colon ◽  
Metabolic Labelling ◽  
Colonic Mucin ◽  
Sds Page ◽  
High Degree ◽  
Degree Of Similarity

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.

scholarly journals Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization

1994 ◽  
Vol 304 (3) ◽  
pp. 693-698 ◽  
Author(s):  
L W J Klomp ◽  
L van Rens ◽  
G J Strous
Keyword(s):  
Molecular Mass ◽  
Mechanical Damage ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Apparent Molecular Mass ◽  
Gastric Mucin ◽  
Gastric Mucus ◽  
Apical Side ◽  
Sds Page ◽  
Glycosylation Inhibitor

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.

scholarly journals Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the ‘26 S’ multienzyme complex

1991 ◽  
Vol 280 (1) ◽  
pp. 225-232 ◽  
Author(s):  
A Seelig ◽  
P M Kloetzel ◽  
L Kuehn ◽  
B Dahlmann
Keyword(s):  
Molecular Mass ◽  
Molecular Interaction ◽  
Gradient Centrifugation ◽  
Mass Range ◽  
High Molecular Mass ◽  
Multienzyme Complex ◽  
Multicatalytic Proteinase ◽  
Sds Page ◽  
Specific Antisera ◽  
Rabbit Reticulocytes

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a ‘26 S’ complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass ‘26 S’ multienzyme complex. In all instances proteasomes are identified in their ‘free’ 650 kDa ‘20 S’ form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described ‘26 S’ proteinase. This ‘26 S’ proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the ‘26 S’ proteinase complex.

scholarly journals Mucins secreted by a transformed cell line derived from human tracheal gland cells1

1997 ◽  
Vol 326 (2) ◽  
pp. 431-437 ◽  
Author(s):  
Jean-Marc LO-GUIDICE ◽  
Marc D. MERTEN ◽  
Geneviève LAMBLIN ◽  
Nicole PORCHET ◽  
Marie-Christine HOUVENAGHEL ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Cell Line ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Mass ◽  
Cscl Density Gradient

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose® CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to β-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc α2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520–528]; they should be considered as having a mixed, both serous and mucous, phenotype.

scholarly journals The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1

1991 ◽  
Vol 278 (1) ◽  
pp. 179-187 ◽  
Author(s):  
R V Ward ◽  
R M Hembry ◽  
J J Reynolds ◽  
G Murphy
Keyword(s):  
Molecular Mass ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
U937 Cells ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Gingival Fibroblasts ◽  
Tissue Inhibitor ◽  
Lower Molecular Mass ◽  
Inhibitory Activities

Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

scholarly journals Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences

1995 ◽  
Vol 307 (2) ◽  
pp. 341-346 ◽  
Author(s):  
S S Little ◽  
D A Johnson
Keyword(s):  
Mast Cell ◽  
Molecular Mass ◽  
Peptide Mapping ◽  
Proteinase Inhibitors ◽  
High Molecular Mass ◽  
Mass Separation ◽  
Human Mast Cell ◽  
Mast Cell Tryptase ◽  
Sds Page ◽  
Mass Form

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.

scholarly journals Lipopolysaccharides from Mesorhizobium huakuii and Mesorhizobium ciceri: chemical and immunological comparative data.

2002 ◽  
Vol 49 (4) ◽  
pp. 1043-1052 ◽  
Author(s):  
Adam Choma
Keyword(s):  
Fatty Acids ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Molecular Size ◽  
High Molecular Mass ◽  
Mesorhizobium Huakuii ◽  
Sds Page ◽  
Mass Fractions ◽  
Neutral Sugars ◽  
Molecular Size Distribution

Lipopolysaccharides of two Mesorhizobium species of different host specificity were compared: M. huakuii and M. ciceri. M. huakuii sp. was represented by five strains with special consideration of M. huakuii IFO 15243(T). SDS/PAGE profiles revealed that all M. huakuii LPS preparations contained low molecular mass fractions (LPS-II) of the same molecular size. All of lipopolysaccharides contained high molecular mass fractions (LPS-I). However, the high molecular mass fraction from each strain possessed an individual molecular size distribution pattern. The crossreactivity of blotted lipopolysaccharides with rabbit polyclonal antibodies against Mesorhizobium huakuii IFO 15243(T) whole bacteria indicated the presence of common epitope(s) within the investigated Mesorhizobium huakuii strains. Moreover, LPS from M. huakuii S52 also reacted with anti M. ciceri HAMBI 1750 serum showing that there are epitopes common for different mesorhizobial species. LPS isolated from Mesorhizobium huakuii strain IFO 15243(T) contained neutral sugars: L-6-deoxytalose, L-rhamnose, D-galactose and D-glucose, aminosugars:D-quinovosamine, D-glucosamine, D-2,3-diamino-2,3-dideoxyglucose and D-galacturonic and D-glucuronic acids. In the LPS preparation, fatty acids typical for Mesorhizobium strains were detected. 3-Hydroxydodecanoic, 3-hydroxy-iso-tridecanoic, 3-hydroxyeicosanoic, 3-hydroxyheneicosanoic and 3-hydroxydocosenoic acids were the major amide linked fatty acids, while iso -heptadecanoic, eicosanoic, docosenoic, as well as 27-hydroxyoctacosanoic and 27-oxooctacosanoic acids were the dominant ester linked fatty residues.

Purification and Characterization of a Novel Metalloproteinase, Acurhagin, from Agkistrodon acutus Venom

2002 ◽  
Vol 87 (04) ◽  
pp. 641-650 ◽  
Author(s):  
Wen-Jeng Wang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Molecular Mass ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Crude Venom ◽  
Reducing Conditions ◽  
Sds Page

SummaryAcurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anionexchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aα. chain of fibrinogen, followed by Bβ chain, while γ chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37° C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrinlike and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of plateletrich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

scholarly journals Analysis of purified human liver α-l-fucosidase by western-blotting with lectins and polyclonal and monoclonal antibodies

1992 ◽  
Vol 282 (3) ◽  
pp. 829-834 ◽  
Author(s):  
S W Johnson ◽  
S Piesecki ◽  
R F Wang ◽  
I Damjanov ◽  
J A Alhadeff
Keyword(s):  
Monoclonal Antibodies ◽  
Sialic Acid ◽  
Western Blot ◽  
Molecular Mass ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Liver ◽  
High Molecular Mass ◽  
Sds Page ◽  
Gel Isoelectric Focusing

Western-blot analysis [with lectins, polyclonal antibodies (pAbs) and four monoclonal antibodies (mAbs)] was employed to investigate the structural relationship between the separated isoforms and subunits of purified human liver alpha-L-fucosidase. SDS/PAGE and Western-blot analysis indicated the presence of two protein bands of 51 kDa and 56 kDa that were recognized by the pAbs. Polyacrylamide-gel isoelectric focusing (PAG-IEF) followed by blotting indicated that the pAbs and mAbs recognized at least five fucosidase isoforms (pI values 3.6-6.0). Lectin blotting indicated an enrichment of sialic acid residues in the more acidic isoforms. Western-blot analysis indicated that four mAbs recognized the 51 kDa subunit and at least two mAbs recognized the 56 kDa subunit. The subunit composition of the isoforms (separated by PAG-IEF) of human liver alpha-L-fucosidase was investigated by SDS/PAGE. One or two closely spaced bands were found for each isoform with a trend of increasing relative amounts of the high-molecular-mass band in the more acidic isoforms relative to the more neutral isoforms. Neuraminidase treatment of alpha-L-fucosidase resulted in a decrease in the amount of the high-molecular-mass subunit and an increase in the amount of the low-molecular-mass subunit, suggesting that these subunits are related at least in part by sialic acid residues. In addition, blotting with lectins indicated the presence of sialic acid residues only in the high-molecular-mass subunit. N-Glycanase treatment led to the disappearance of the glycosylated 56 kDa and 51 kDa protein bands and the appearance of non-glycosylated protein bands at 48 kDa and 45 kDa. The overall results indicate that (1) N-glycosylation contributes to, but does not account completely for, structural differences in the fucosidase subunits and (2) the more acidic isoforms of fucosidase contain enriched relative amounts of the sialylated high-molecular-mass subunit.

scholarly journals Bacillus galactosidilyticus sp. nov., an alkali-tolerant β-galactosidase producer

2004 ◽  
Vol 54 (2) ◽  
pp. 617-621 ◽  
Author(s):  
Marc Heyndrickx ◽  
Niall A. Logan ◽  
Liesbeth Lebbe ◽  
Marina Rodríguez-Díaz ◽  
Gillian Forsyth ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Raw Milk ◽  
Sequence Comparisons ◽  
16S Rdna Sequence ◽  
Bacillus Lentus ◽  
Sds Page ◽  
Phenotypic Tests ◽  
High Degree ◽  
Degree Of Similarity ◽  
Amplified Rdna Restriction Analysis

A novel Bacillus isolate from raw milk and four strains from diverse origins that were identified previously as Bacillus lentus, Bacillus firmus and Bacillus circulans showed a high degree of similarity in amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests, whilst 16S rDNA sequence comparisons and DNA relatedness data showed that this taxon was different from related Bacillus species. On the basis of these data, Bacillus galactosidilyticus sp. nov. is proposed, with the type strain LMG 17892T (=DSM 15595T=Logan B2188T=MB 800T).

Sign in / Sign up

Export Citation Format

Share Document