Sepharose 6B Gel Filtration Research Articles

The potentiating effect of platelet on plasminogen activation by tissue plasminogen activator

A new role for platelets in fibrinolysis is proposed. Platelets (euglobulin from platelet rich plasma and from human platelet extract) may potentiate plasminogen activation by tissue plasminogen activator(tPA). The potentiating activity was detected by both chromogenic substrate and fibrin plate analysis. The fibrinolysispotentiating substance in the platelets required the presence of both tPA and plasminogen, suggesting that it potentiates the activation of plasminogen by tPA. This substance was not related to fibrinogen degradation products because it was also present in platelets from two afibrinogenemic patients and did not lose its activity when separated from fibrinogen-related antigen by Sepharose 2B gel filtration. Since platelets contain both activator(s) and inhibitor(s) of plasminogen activation by tPA, a balance between activator(s) and inhibitor(s) in platelets may also be required for control of the fibrinolytic pathway.

Distinct hydrodynamic forms of the insulin receptor: electrophoretic analysis of the RI and RII species.

In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do represent larger (RI) and smaller (RII) oligomeric forms of the receptor, rather than representing receptor species separated from each other by differential adsorption to the Sepharose matrix. Receptor solubilized from isolated human placenta membranes was purified by lectin- and insulin-agarose chromatography and was radiolabeled with carrier-free 125I. The labeled receptor was separated by Sepharose 6B gel filtration into two fractions (peak I, KAV = 0.31; peak II, KAV = 0.53), was immunoprecipitated by anti-insulin receptor antibody, and was analysed by electrophoresis in nonreducing polyacrylamide slab gels. The autoradiograms of the gels indicated that peak I (KAV = 0.31, RI receptor form) contained a number of receptor species of 240 000 daltons or greater, whereas peak II (KAV = 0.53, RII receptor form) contained mainly receptor species of 210 000 daltons or smaller. In particular, large amounts of a 90 000 dalton species (presumably free receptor beta-subunit) were present in peak II. Incubation of the material obtained from peak I with insulin resulted in a change in the electrophoretic pattern, which became identical with that observed for material recovered from peak II.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet thrombospondin haemagglutinin activity is due to aggregate formation

Thrombospondin released from human blood platelets by thrombin activation formed high molecular weight aggregates which co-eluted with haemagglutinin activity on Sepharose 4B gel filtration. Thrombospondin aggregation was mediated by intermolecular disulphide bridges. The aggregates consisted of a series of oligomers ranging from a dimer to polymeric forms with Mr =40 x 10 6. Native monomeric thrombospondin obtained by a modified procedure was deficient in haemagglutination activity but inhibited haemagglutination induced by aggregated thrombospondin.

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid.

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain.

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.

Glycosaminoglycan profiles in cloned granulated lymphocytes with natural killer function and in cultured mast cells: their potential use as biochemical markers.

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.

Studies on protease of the rotifer Brachionus plicatilis - I. Purification of alkaline protease of the rotifer Brachionus plicatilis.

Alkaline proteases were isolated from the crude extract of the rotifer Brachionus plicatilis by the combination of gel filtration and ion-exchange chromatogaphy. Two-main active fractions, designated as F-I and F-II, were eluted on the second DEAE-cellulose column, and F-I was finally purified about 190-fold and F-II about 920-fold. F-II was homogeneous as judged from polyacrylamide disc gel electrophoresis. The molecular weihts of F-I and F-II were estimated to be about 800, 000 and 900, 000, respectively, by Sepharose 6B gel filtration, and their isoelectric points were found to be 9.7 and 5.6, respectively.

Cytosolic gangliosides of rat brain: their fractionation into protein-bound complexes of different ganglioside compositions.

The sialic acid moiety of rat brain cytosolic gangliosides was radiolabeled by intracranial injection of N-(3H)acetylmannosamine. Upon ammonium sulphate fractionation, Sepharose 6B gel filtration, and hydroxylapatite-cellulose chromatography, ganglioside-bound radioactivity of brain cytosolic extract followed the behavior of protein and not that of purified gangliosides. This indicates that cytosolic gangliosides occur as ganglioside-protein complexes. By application of hydroxylapatite-cellulose column chromatography, fractions were obtained having different ganglioside composition. In particular, one fraction contained GM1, one GD1a, and one GT1b with a ganglioside homogeneity better than 95% in each fraction. This indicates the occurrence in brain cytosol of a GM1-protein complex, a GD1a-protein complex, and a GT1b-protein complex.

Purification and properties of pyranose oxidase from Coriolus versicolor.

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag+ , Cu2+ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.

Purification and properties of a thiol protease from rat liver nuclei.

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K m = 0.1 mm and no change in V max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [ 3H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [ 3H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.

Partial purification and characterization of myosin light chain kinase from bovine thyroid gland.

Myosin light chain kinase has been purified approximately 1700-fold from bovine thyroid gland, using Affigel blue column chromatography, ammonium sulfate fractionation (0-60% saturation), Sepharose 6B gel filtration, and calmodulin-Sepharose affinity column chromatography. This partially purified kinase was Ca2+ and calmodulin dependent for its activity and specifically phosphorylated the 20,000-dalton light chain of chicken gizzard myosin. At higher CaCl2 concentration, thyroid myosin light chain kinase demonstrated a novel inhibition in the presence of calmodulin. The mechanism of this Ca2+-dependent inhibition is not clear at present. The existence of myosin light chain kinase in thyroid gland provides additional support for the role of the contractile system in the secretion of thyroid hormones.

Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors.

The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-C1) and mobile (MAT-B1) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-C1 cells with Triton X-100-containing buffers gave insoluble residues showing three major components by NaDodSO(4)/polyacrylamide gel electrophoresis: actin, a 58,000-dalton polypeptide, and a cell surface glycoprotein of 75,000-80,000 daltons. The ratio of these components in Triton X-100-insoluble residues, as determined by scintillation counting of bands from gels of [(3)H]leucine-labeled microvillar membranes, approached equimolar, suggesting a specific complex of the components. The three components of the putative complex cosedimented on sucrose density gradients of Triton X-100/buffer-treated membranes. Gel filtration on Sepharose 2B gave a peak included in the column that contained only the glycoprotein, actin, and 58,000-dalton polypeptide by one-dimensional NaDodSO(4) electrophoresis and by two-dimensional isoelectric focusing/NaDodSO(4) electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-B1 microvillar membranes by Sepharose 2B gel filtration in Triton X-100-containing buffers contained actin and the glycoprotein but no 58,000-dalton polypeptide. From these results we propose that the cell surface-cytoskeleton interactions in the 13762 tumor cell microvilli involve direct association of actin with the cell surface glycoprotein. We further suggest that the 58,000-dalton polypeptide stabilizes the association of this complex with the microfilaments in the MAT-C1 microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.

Enzyme replacement therapy in porphyrias—II: Entrapment of δ-aminolaevulinate dehydratase in liposomes

1. 1. Different types of neutral and negative, large, small and multilamellar liposomes were prepared and conditions for entrapping partially and highly purified preparations of blood and bovine ALA-dehydratase were studied. 2. 2. Gel filtration on Sepharose 4B, resolved liposomes formed in the absence of enzyme from free enzyme, when both were chromatographed 1–2 hr after mixing, showing that ALA-D did not become associated with liposomes. 3. 3. Preparation of liposomes in the presence of ALA-D resulted in partial entrapment of the enzyme and separation of the protein-containing liposomes from excess free protein was achieved by Sepharose 4B gel filtration. 4. 4. Part of the activity of ALA-D associated with liposomes was latent, and it could only be detected after treatment with Triton X-100, which, at the concentration used, disrupted the spherules and did not affect ALA-D activity. Some activity was also measured in intact ALA-D loaded liposomes. 5. 5. Of all preparations of liposomes, best entrapment values for ALA-D were consistently obtained with negatively charged large multilamellar vesicles, ( −LMV (ALA-D)). Considerable less yield was associated with negative small unilamellar vesicles ( −SUV) and neutral large unilamellar vesicles ( nLUV).

Comparison of biliproteins in the ovaries of marine snails.

Dark blue or violet chromoproteins were isolated from the ovaries of marine snails Haliotis discus hannai, Chlorostoma lischkei, Omphalius pfeifferi, and Turbo marmorata by ammonium sulfate fractionation and Sepharose 4B gel filtration. The chromoprotein was found to be the major pigment in each of these ovaries. These chromoproteins had similar u. v. and visible absorption spectra, their molecular weights were 500, 000-700, 000 and their sedimentatin coefficients were 16-18 S. Chromophores were readily liberated from the chromoproteins by the HCI-acetone treamtment and their methyl esters were then prepared. The chromophore of the chromoprotein of T. marmorata was identified as turboverdin on the basis of u. v. and visible spectra and TLC, while those of the other three species were violin-type bile pigments. In all cases, the linkage between chromophore and protein moiety was non-covalent.

Salt-dependent hemagglutination with Ibaraki virus and its inhibition by specific antisera.

Ibaraki virus grown in HmLu-1 cells, a stable cell line from hamster lung, agglutinated erythrocytes from cattle, sheep, goats, horses and rabbits, when concentrated by ultrafiltration from infectious culture fluid. The hemagglutination (HA) was dependent on not only the pH but also the NaCl concentration of the diluent; the HA titer significantly improved by increasing the NaCl molarity to 0.6 M and standardizing pH to 7.5. The incubation temperature did not affect the HA titer. In Sepharose 4B gel filtration the peak of the HA activity coincided in position with the infectivity peak. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual bovine sera showed a significant positive correlation with their neutralizing antibody titers.

F1-like ATPase from anaerobic bacterium Lactobacillus casei contains six similar subunits.

Membrane‐linked ATPase of an anaerobic bacterium Lactobacillus casei has been studied.It was found that L. casei membrane particles hydrolyzed 0.1–0.2 μmol of ATP × mg protein−1× min−1 in the presence of Mg2+ or Ca2+ ions at optimal pH 6.4. This activity was inhibited by N,N′‐dicyclohexylcarbodiimide. Oligomycin, ouabain, vanadate and hydroxylamine were ineffective.The addition of valinomycin to the intact L. casei cells in the absence of extracellular K+ and glucose resulted in an ATP synthesis which was abolished by the addition of K Cl, N,N′‐dicyclohexylcarbodiimide or carbonylcyanide m‐chlorophenylhydrazone. In the presence of glucose, L. casei cells showed an energy‐dependent accumulation of tetraphenylphosphonium cations, which was sensitive to N,N′‐dicyclohexylcarbodiimide and carbonylcyanide m‐chlorophenylhydrazone.A soluble ATPase was isolated from L. casei membranes by means of (a) washing with a low ionic strength buffer in the presence of EDTA, Mg2+ ions and a protease inhibitor ‐ diisopropylfluorophosphate or (b) a chloroform treatment. Crude ATPase extracts were purified using isotachophoresis and (or) Sepharose 6B gelfiltration. Preparations of purified ATPase had a specific activity 3.0–4.0 μmol ATP × mg protein−1× min−1 and were homogeneous when studied with analytical disc electrophoresis or analytical ultracentrifugation (s20,w= 12 ± 0.5 S). The molecular mass of the L. casei ATPase estimated with sieve chromatography on Sepharose 6B proved to be 270 kDa. Purified L. casei ATPase denaturated by 1% sodium dodecyl sulphate or 8 M urea and then studied with dodecyl sulphate or urea polyacrylamide gel electrophoresis showed a single 43‐kDa band. L. casei ATPase subunits could not be separated by isoelectrofocusing in 8 M urea/polyacrylamide gel (a pH gradient of 0.2–0.3 cm was used) and migrated as a single band of isoelectric point at pH 4.2.An electron microscopic study of the purified L. casei ATPase negatively stained with uranyl acetate showed particles of very regular structure. The predominant type of image was found to be a hexagonal particle 9.0–10.0 nm in size with 6 n point symmetry. An analysis of five different projections indicated that six similar subunits are arranged in two layers (three subunits in each layer) at vertices of two triangles rotated at 180°. This structure has a 32 point group symmetry.It is concluded that the detachable part of membrane N,N×‐dicyclohexylcarbodiimide‐sensitive ATPase of L. casei is composed of six similar subunits. Such a simplified version of factor F1 may be regarded as an evolutionary precursor of the catalytic part of the H+ ‐ATP synthase of aerobic and photosynthetic bacteria, mitochondria, and chloroplasts, containing two types of major and 2–3 types of minor subunits. The possibility is discussed that simplicity of L. casei ATPase is due to the fact that in the cell it operates as an ATP‐splitting, rather than a synthesizing, mechanism.

Purification and partial characterization of human placental particle-bound aminopeptidase

Particle-bound aminopeptidase was purified from the human term placenta by separation of the particles from the soluble fraction by centrifugation and solubilization of the particle fraction with 0.5 per cent (v/v) Triton X-100 followed by CM-cellulose chromatography, Sepharose 6B gel filtration, DE-cellulose and Con A-Sepharose 4B affinity chromatography. Polyacrylamide gel electrophoresis indicated enzymatic and protein homogeneity of the purified enzyme. The enzyme seemed to be a glycoprotein with a molecular weight of 320 000. The purified enzyme did not endure freezing but was fairly stable at 4 degrees C. At 60 degrees C more than half and at 65 degrees C all enzyme activity disappeared in 15 min. The pH dependence of the purified enzyme varied between 6.75 and 7.5, and 6.0 and 6.5, depending on the substrate used. The enzyme hydrolysed most rapidly LeuNA (Km 0.095 +/- 0.008 mmol) followed by beta-naphthylamide derivatives of alanine (Km 0.222 +/- 0.02 mmol), arginine (Km 0.041 +/- 0.002 mmol), lysine (Km 0.084 +/- 0.005 mmol), methionine (Km 0.085 +/- 0.004 mmol) and cystine (Km 0.048 +/- 0.004 mmol). Thus LeuNA was the most specific among the substrates for the enzyme. The purification process revealed, however, that CysNA was the most selective substrate for the particulate enzyme, which readily hydrolysed also Leu-p-na (Km 0.865 +/- 0.023 mmol) and Bz-Cys-p-na (Km 0.248 +/- 0.034 mmol).

Purification and substrate specificity of bovine liver-ferrochelatase.

Bovine ferrochelatase from liver mitochondria was purified 1434-fold with a 31% yield to apparent homogeneity by a procedure involving solubilization, ammonium sulfate fractionation and blue Sepharose CL-6B chromatography. The molecular weight of the homogeneous protein was 42 500 when measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 200 000 was obtained by Sepharose 6B gel filtration. The specific activity for mesoheme synthesis was 413 nmol x mg protein-1 x min-1 at 37 degrees C and for protoheme synthesis 88 nmol x mg-1 x min-1. The optimum pH was 8.0 and Km values for the substrates were: protoporphyrin IX, 54 microM; mesoporphyrin IX, 46 microM; iron with protoporphyrin IX, 46 microM, iron with mesoporphyrin IX, 44 microM. The purified enzyme inserted iron into the following dicarboxylic porphyrins in descending order: meso-, deutero-, 2,4-diacetyldeutero-, hemato-, and protoporphyrin IX. This did not take place in the case of 2,4-diformyldeuteroporphyrin IX. Porphyrin c was converted to only a negligible amount of heme c, and coproporphyrin III did not act as a substrate at all. When metal specificity was examined, the highest value was obtained with zinc, decreasing in order with iron, cobalt and nickel. The enzyme failed to catalyze the insertion of copper or manganese into porphyrin. An antibody specific for the purified bovine ferrochelatase was prepared, and studies confirmed that the synthetic activities of iron-porphyrin, zinc-porphyrin and cobalt-porphyrin are ascribable to ferrochelatase.

The composition and polymeric structure of mucus glycoprotein from human gallbladder bile

1. Human gallbladder bile was obtained from three patients with mixed-type gallstones undergoing cholecystectomy, and each patient sample was analysed individually. 2. Mucus glycoprotein was isolated by Sepharose 4B gel filtration followed by equilibrium density gradient centrifugation in CsCl. The purified glycoprotein was free from contaminant non-covalently bound protein as demonstrated by SDS-polyacrylamide gel electrophoresis. 3. The mucus glycoprotein was present in human bile at concentrations of 0.2–0.5 mg/ml. 4. Amino acid analysis of the purified glycoprotein was similar to that of other mucus glycoproteins in being rich in serine, threonine and proline, which accounted for 39% w/w of the total amino acids. 5. The hexosamine content of the glycoprotein depended on the blood group; patients with blood group O had a molar ratio of glucosamine:galactosamine of 4.0:1, and those with blood group A a molar ratio of 2.1:1. 6. Analysis of the mucus glycoprotein by gel filtration on Sepharose 2B showed that the major glycoprotein component was excluded. On treatment with 0.2 M mercaptoethanol or proteolytic enzymes, but not with 4 M guanidinium chloride, this excluded peak was broken down to a lower molecular weight glycoprotein component which eluted as a single included peak on gel filtration. 7. A protein of 70 000 molecular weight, identified by SDS-polyacrylanide gel electrophoresis, is released following reduction of the glycoprotein. 8. These results show (1) that human gallbladder mucus glycoproteins, in common with several other gastrointestinal mucus glycoproteins, consist of a polymeric structure of lower molecular weight glycoprotein units joined by disulphide bridges, and (2) that parts of the protein core are accessible to proteolysis, while other parts are glycosylated and protected from proteolysis, presumably by the carhohydrate side-chains. 9. Mucus glycoproteins from pig gallbladder bile were also shown to consist of large molecular weight polymers maintained by disulphide bridges and broken down by proteolysis.