ABSTRACT
Rat liver beta-glucuronidase was studied by sequential lectin affinity chromatography. beta-Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography. Ulex europaeus agglutinin-agarose chromatography revealed the presence of alpha(1-3)linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin and Phaseolus vulgaris erythroagglutinin.
Subject(s)
Glucuronidase/chemistry , Polysaccharides/analysis , Animals , Chromatography, Affinity , Lectins , Liver/enzymology , Molecular Structure , RatsABSTRACT
1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.
Subject(s)
Chondro-4-Sulfatase/isolation & purification , Liver/enzymology , Lysosomes/enzymology , Animals , Chondro-4-Sulfatase/chemistry , Chondro-4-Sulfatase/metabolism , Electrophoresis, Polyacrylamide Gel , Immunoelectrophoresis/methods , Isoelectric Focusing , Lectins , RatsABSTRACT
Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A.
Subject(s)
Cerebroside-Sulfatase/isolation & purification , Isoenzymes/isolation & purification , Liver/enzymology , Oligosaccharides/analysis , Animals , Carbohydrate Sequence , Cerebroside-Sulfatase/chemistry , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Immunoelectrophoresis, Two-Dimensional , Isoelectric Focusing , Isoenzymes/chemistry , Lectins , Lysosomes/enzymology , Molecular Sequence Data , Molecular Weight , Oligosaccharides/chemistry , Rats , UltracentrifugationABSTRACT
The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I.
Subject(s)
Carbohydrates/chemistry , Chromatography, Affinity , Glucuronidase/isolation & purification , Immunoelectrophoresis , Liver/enzymology , Animals , Concanavalin A , Electrophoresis, Polyacrylamide Gel , Glucuronidase/chemistry , Hydrogen-Ion Concentration , Isoelectric Focusing , Isoelectric Point , Liver/ultrastructure , Lysosomes/enzymology , Male , Molecular Weight , Rats , Rats, WistarABSTRACT
In human lens, cataract development causes an increase in the amount of protein aggregates. Their specific density of 1.25 g/ml is much higher than that of 1.14 g/ml and 1.06 g/ml obtained for water-soluble proteins. During the formation of water-insoluble aggregates their size varies up to some micrometers. Infrared spectra of water-soluble protein and in the aggregates confirm that the content of bound water in aggregates decreases.
