A cell membrane-associated lectin of the oyster hemocyte

The presence of a lectin in association with hemocytes of the American oyster, Crassostrea virginica, has been demonstrated by utilizing a microhemagglutination assay. The plasma membrane association of this lectin is shown by its copurification with the plasma membrane fraction of disrupted hemocytes, using sucrose density gradient centrifugation, and also by the binding of ¹²⁵I-labeled glycoproteins to intact hemocytes at 4°C. Based upon agglutinating spcificity for a range of vertebrate erythrocytes, both untreated and enzyme-treated, along with hemagglutination-inhibition assays and crossed-absorption tests, it is apparent that there are also two serum (soluble) lectins, each having a distinct serological agglutination specificity, and that the hemocyte membrane-associated lectin has a specificity that is identical with one of these two serum lectins. It is proposed that the hemocyte membrane-associated lectin may be a true integral membrane protein, and therefore may function as a membrane receptor in nonself recognition by molluscan hemocytes.     

The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat.

1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids.     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.     

Opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes fromMytilus edulis

Summary Mytilus hemolymph was found to contain an agglutinin which could be inhibited by mucin. The agglutinin was isolated by affinity chromatography using neuraminidase-treated mucin/Sepharose.In vitro phagocytosis experiments revealed that only about 5% of washed hemocytes phagocytosed yeast cells suspended in a Tris-buffered NaCl-solution, whereas yeast suspended in hemolymph was normally ingested by more than 50% of the hemocytes. This relatively high phagocytic activity was shown to depend on the presence of two serum factors: When purified agglutinin was added to saline-suspended yeast, phagocytosis rates returned to normal, demonstrating opsonizing properties of the purified agglutinin. — On the other hand, addition of Ca⁺⁺-ions to saline caused an increase of the phagocytic activity of hemocytes. This was interpreted to indicate the activation of divalent cation-dependent recognition molecules at the hemocyte surface. The function of these postulated recognition factors was demonstrated by phagocytosis inhibition tests. Their location at the hemocyte membrane became evident by binding of specific antiagglutinin IgG purified by help of an agglutinin/Sepharose column from an antiserum raised againstMytilus serum proteins. Consequently, humoral as well as cell bound agglutinin molecules are involved in the attachment of yeast cells toMytilus hemocytes which subsequently internalize foreign cells.Abbreviations DAB dimethylamino benzaldehyde - PO peroxidase - IgG immunoglobulin G     

Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors

Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells are larger, more granulated, and generally lack the BGH1 cell marker. Both hemocyte subpopulations bound fluorescent dye (Oregon Green)-conjugated excretory-secretory glycoproteins (fESPs), although the specific fESP binding signal (geometric mean value), after correction for cellular autofluorescence, was greater in the R1 hemocyte subpopulation compared to that of the R2 subset. Partial inhibition of fESP binding to hemocytes consistently was achieved using various glycoconjugates (mucin, asialo-mucin, asialo-fetuin, heparin) and polysaccharides (fucoidan, dextran sulfate 8000), suggesting the involvement of hemocyte carbohydrate-binding receptors (CBRs) in reactions with ESP-associated glycans. Although sulfation of carbohydrate ligands contributed significantly to ESP blocking activity of some inhibitory polysaccharides and heparin, other sulfated proteoglycans (chondroitins A and B, heparan sulfate) were noninhibitory, indicating that charge alone was not solely responsible for the observed inhibition of hemocyte binding by fESPs. A similar blocking effect by desialylated glycoproteins (asialo-mucin, asialo-fetuin) further supports the contention that ESP-hemocyte interactions are mediated primarily through CBRs. The glycoconjugate inhibitors of ESP binding were only partially effective over a range of concentrations and their glycan moieties (oligosaccharides or long-chain polymers) comprised a diversity of major sugar groups, suggesting that hemocyte CBRs and S. mansoni larval ESPs likely represent a multiple receptor-ligand system. Previously reported findings of differential effects of ESPs on a variety of in vitro hemocyte functions are consistent with such a hypothesis.     

Identification of gastric mucosal mucus glycoprotein sulfotransferase.

Sulfation of mucus glycoproteins, reaction catalyzed by Golgi resident sulfotransferase, is an important event in posttranslational processing of gastric mucins. Here we report the purification of mucus glycoprotein sulfotransferase enzyme from the microsomal fraction of rat gastric mucosa. The enzyme was released from the membrane with 0.5% Triton X-100 and precipitated from the 100,000xg supernatant with 90% ice-cold acetone. The enzyme activity (44.7 pmol/mg/45 min) in the precipitate was enriched nearly 10-fold compared to Triton X-100 extract of microsomal membrane (4.2 pmol/mg/45 min). On SDS-PAGE, the enzyme gave a single 43 kDa protein band, which was active towards mucin, but did not catalyze the sulfation of galactosylceramide. The study is the first to report the characteristics of a sulfotransferase enzyme specific for gastric mucin.     

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase and of acyl-CoA–cholesterol acyltransferase by the transfer of non-esterified cholesterol to rat liver microsomal vesicles

The incubation of rat liver microsomal fraction with a serum preparation followed by the re-isolation of the microsomal membranes has resulted in an increase in the concentration of non-esterified cholesterol, a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase and in an increase in the activity of acyl-CoA–cholesterol acyltransferase in the treated microsomal preparation. These effects were related to the concentration of serum in the incubation mixture and to the duration of the incubation. The transfer of non-esterified cholesterol was specific in that the content of protein and the total phospholipids were similar in the original microsomal fraction and the serum-treated microsomal preparation. The incubation of the microsomal fraction with lipoprotein-deficient serum or with no serum resulted in both cases in small changes in the non-esterified cholesterol, the esterified cholesterol and the total phospholipid content in the treated preparations compared with these concentrations in the original microsomal fraction, whereas the activity of acyl-CoA–cholesterol acyltransferase and of 3-hydroxy-3-methylglutaryl-CoA reductase was similar in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase was lower and the activity of acyl-CoA–cholesterol acyltransferase was higher in the lipoprotein-deficient-serum-treated and the buffer-treated microsomal preparations as compared with these activities in the original microsomal fraction. However, the serum-treated microsomal preparation had considerably lower activity of 3-hydroxy-3-methylglutaryl-CoA reductase and considerably higher activity of acyl-CoA–cholesterol acyltransferase than these activities in buffer-treated and in lipoprotein-deficient-serum-treated microsomal preparations.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Binding of human liver hydrolases by immobilized lectins.

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.     

Membrane-associated pyruvate kinase in developing guinea-pig liver.

During analysis of pyruvate kinase distribution in developing guinea-pig liver it was observed that a substantial proportion of the activity remained associated with the microsomal membrane fraction ('microsomes'). Although some of this could be removed by washing with sucrose, the majority required detergent treatment for liberation, and even then at least one-half remained attached to the microsomes. Estimates of the contribution of this fraction to total cell pyruvate kinase activity indicated that it was more than 50% of the total, and this is likely to be an underestimate because of the continued latency of the enzyme even in the presence of detergent. The susceptibility of the microsomal enzyme, whether released by detergent or sucrose washing, to inactivation by Triton X-100 suggested it to be different from the cytosolic enzyme, which was stable under such conditions. (The microsomal enzyme required the presence of additional protein, such as bovine serum albumin, to maintain stability.) This view was confirmed by DEAE-cellulose chromatography and particularly isoelectric focusing, where the microsomal enzyme was shown to consist of at least four forms, which were distinctly different from those in the cytosol. Those data and the kinetic properties of the four forms in the membrane fraction indicate that the microsomal pyruvate kinase could consist of four counterparts to the cytosolic isoenzyme forms. These results are discussed in relation to the two possible explanations for the phenomenon (not mutually exclusive): that the more hydrophobic membrane forms are precursors of the cytosolic enzyme and that they may be part of functional glycolytic pathway in the microsomes of developing liver.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

Characterization of rat colonic cell surface glycoconjugates by fluoresceinated lectins

Summary Cryostat and paraffin embedded sections from cecum, proximal and distal colonic segments of male Sherman rats were examined by fluorescence microscopy after labeling with six fluorescein-conjugated lectins. These FITC-conjugated lectins were used as specific probes to define the labeling pattern of carbohydrate containing components of the lumenal and basolateral surfaces of epithelial cells, goblet cell mucin and lumenal mucin at all three sites. Marked regional differences in labeling were detected, indicating that the various carbohydrate components of these cells differ significantly along the length of the colon. Furthermore, the patterns of labeling components with each lectin appeared to vary depending on the fixation technique employed. Cryostat preparations generally resulted in a broader distribution of label and more intense staining with these lectins than fixed paraffin sections. While the reason(s) for these variations remain unclear at this time and will require further studies, the present data emphasize the importance of the fixation method when interpreting results obtained utilizing FITC-conjugated lectins.     

Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification.

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.     

Detection of UDP-N-acetylgalactosamine-oligosaccharide N-acetylgalactosaminyltransferase activity in rat stomach and human serum by high-performance liquid chromatography

The enzymatic transfer of the sugar portion from UDP-N-acetylgalactosamine to pyridylamino (PA) lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-4Glc-PA) was detected by high-performance liquid chromatography. Separation of the fluorescent product from the fluorescence-labeled acceptor was achieved within 10 min by reversed-phase high-performance liquid chromatography. Rat stomach enzyme activity was detected in the microsomal fraction from antrum but not corpus. Ohara et al. (1986, Comp. Biochem. Physiol. 83B, 273-275) reported that the N-acetylgalactosamine content in antrum mucin was greater than that in corpus mucin and antrum mucin had strong blood group A activity. The prominent asymmetrical distribution of the enzyme detected here well supports these findings. The elution position of the fluorescent product was the same as that of the product formed by the action of type A human serum toward the acceptor. Its hydrolysis by alpha-N-acetylgalactosaminidase yielded the acceptor. It is thus evident that the detected enzyme is the same as that producing the blood group A structure.     

Screening for plant lectins by latex agglutination tests

The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.     

Dual localization of long-chain acyl-CoA hydrolase in rat liver: one in the microsomes and one in the mitochondrial matrix.

Subcellular fractionation studies of rat liver localized the activity of palmitoyl-L-carnitine hydrolase to the microsomal fraction whereas palmitoyl-CoA hydrolase activity was found both in the microsomal fraction and in mitochrondria. An unusual biphasic sataration curve for palmitoyl-CoA was observed when intact mitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl-CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin fractionation of rat liver mitochondria demonstrated that a palmitoyl-CoA hydrolase was associated with the matrix fraction. Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl-CoA were higher for the microsomal enzyme than for the matrix-associated enzyme.     

Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Mucin-specific bark lectin from elderberry Sambucus sieboldiana and its applications to the affinity chromatography of mucin

Three bark lectins were isolated from elderberry Sambucus sieboldiana using fetuin-Sepharose 4B and mucin-Sepharose 4B, and were studied comparatively for their binding to glycoprotein and to clarify various physicochemical features. For each, a unique pattern on isoelectric focusing was noted and their affinity toward various glycoproteins differed, indicating the structures of their carbohydrate binding sites possibly differ. One bark lectin showed specific binding toward porcine mucin. The purity of mucin from a crude porcine stomach mucin or an extract of porcine submaxillary glands could be improved by affinity chromatography on immobilized lectin having binding specificity toward mucin.