Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.
(9/422)
Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins. (+info)
Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta.
(10/422)
The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1 beta (MIP-1 beta) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1 beta monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1 beta with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1 beta-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1 alpha-heparin interaction suggests a possible structural difference between the MIP-1 beta-heparin and MIP-1 alpha-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1 beta, the activities of wild-type MIP-1 beta and R46-substituted MIP-1 beta were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1 beta with GAGs is not intrinsically required for functional interaction of MIP-1 beta with its receptor. (+info)
Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A.
(11/422)
Proteins of approximately 35, 55 and 65kDa were purified from cauliflower extracts by microcystin-Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A-subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5,the casein phosphatase activity of plant PP5 was activated >10-fold by arachidonic acid, with half-maximal stimulation occurring at approximately 100 microM lipid. (+info)
Isolation and purification of a 316 Da preformed compound from strawberry (Fragaria ananassa) leaves active against plant pathogens.
(12/422)
An antibiotic called fragarin showing activities against bacterial and fungal plant pathogens was isolated and purified by FPLC chromatography from the soluble fraction of strawberry leaves. The molecular weight value determined by mass spectrometry is 316 Da. Fragarin remains fully active after protease treatment or alkaline hydrolysis at 100 degrees C for 20 min. Biological and chemical analyses suggest that fragarin may be a new type of an antimicrobial preformed compound--phytoanticipin--and would constitute a primary non-specific barrier of strawberry defense. (+info)
Carbohydrate analysis of bradyrhizobial (NC92) lipopolysaccharides by high performance-anion exchange chromatography with pulsed amperometric detection.
(13/422)
Composition analysis of monosaccharides of Sepharose 4B purified NC 92 LPS and the polysaccharides fractions from Sephadex G-50 chromatography was performed by high performance anion exchange chromatography using pulsed amperometric detection. Rhamnose, mannose, galactose and glucose are present in a substantial amount in the purified LPS (Pk I). High molecular weight purified polysaccharides (PS I) obtained after sephadex gel filtration of the purified LPS (Pk I) acid hydrolysate showed an increase in glucose:galactose ratio. This indicates the presence of the peanut root lectin (PRA II) specific sugar in higher proportion on the O-antigen part of the LPS molecule, which may aid in the critical recognition reaction. (+info)
Outer membrane lipoprotein e (P4) of Haemophilus influenzae is a novel phosphomonoesterase.
(14/422)
Haemophilus influenzae exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable H. influenzae strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein e (P4) within the H. influenzae sequence database. Escherichia coli DH5alpha transformed with plasmids containing the H. influenzae hel gene, which encodes lipoprotein e (P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated approximately 28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase. (+info)
A protein factor essential for microtubule assembly.
(15/422)
A heat stable protein essentail for microtubule assembly has been isolated. This protein, which we designate tau (tau), is present in association with tubulin purified from porcine brain by repeated cycles of polymerization. Tau is separated from tubulin by ion exchange chromatography on phosphocellulose. In the absence of tau, tubulin exists entirely as a 6S dimer of two polypeptide chains (alpha and beta tubulin) with a molecular weight of 120,000, which will not assemble into microtubules in vitro. Addition of tau completely restores tubule-forming capacity. Under nonpolymerizing conditions, tau converts 6S dimers to 36S rings-structures which have been implicated as intermediates in tubule formation. Hence, tau appears to act on the 6S tubulin dimer, activating it for polymerization. The unique ability of tau to restore the normal features of in vitro microtubule assembly makes it likely that tau is a major regulator of microtubule formation in cells. (+info)
Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.
(16/422)
Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA. (+info)