Comparison of glycopeptides from control and virus-transformed baby hamster kidney fibroblasts

Glucosamine-labeled glycopeptides from control and virus-transformed BHK fibroblasts were characterized by size, lectin affinity, charge, and composition. As already demonstrated, on the basis of elution position on a column of Sephadex G-50, transformed cells contained a greater proportion of large glycopeptides than did control cells. Transformed cells also contained a larger proportion of glycopeptides which do not bind to Con A-Sepharose. By sequential chromatography on Sephadex G-50, Con A-Sepharose, and DEAE-Sephadex, approximately 40 individual peaks were partially or completely resolved. If sialic acid was removed from the glycopeptides prior to analysis by ion-exchange chromatography, 95% of the glycopeptides from control cells and 85% of the glycopeptides from transformed cells were no longer bound by DEAE-Sephadex. It was concluded that the DEAE-Sephadex elution properties of the glycopeptides are determined almost entirely by the sialic acid content of the molecules. A comparison of the profiles of control and transformed cell glycopeptides simultaneously eluting from columns of DEAE-Sephadex revealed that the differences between the two cells were largely quantitative; however, the possibility of the existence of qualitative differences as well cannot be excluded. In particular, there was one component present on the surface of transformed cells that was virtually absent in control cells. It was degraded by nitrous acid hydrolysis and heparinase and appeared to be heparan sulfate like material. After fractionation, each isolated glycopeptide population was analyzed for carbohydrate and, in some cases, amino acid content. The apparently larger glycopeptides, group A, the dominant population in transformed cells, were found to contain 3 to 4 mannose residues/glycopeptide when the sugars were normalized to sialic acid content. On the basis of the same criteria, group B glycopeptides contained 4-6 mannose residues/glycopeptide. The carbohydrate and amino acid compositions of the glycopeptides from transformed cells were, with a few exceptions, similar to those from control cells. Some isolated glycopeptides appeared to contain both O-glycosidic anad N-glycosidic linkages on the same oligopeptide.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

In vitro isolation of Plasmodium chabaudi merozoites by continuous flow ultrasound, cell sieving, concanavalin A-affinity chromatography and poly-L-lysine coated bead support columns

Techniques of merozoite isolation based on continuous flow ultrasound, cell sieving, Concanavalin A-affinity chromatography and cationically charged bead support columns were compared using the rodent malaria parasite, Plasmodium chabaudi. While each technique proved useful in isolating merozoites in reasonable numbers, the use of Con A-Sepharose 4B columns consistently provided the greatest numbers which were free of host cell contaminating membrane material and which were invasive to cells both in vivo and in vitro. In addition, Con A-Sepharose columns could be regenerated using alpha methyl-D mannoside. These results, when considered in light of merozoite isolation procedures used for other malarial species, indicated that the host-parasite model system had a bearing on which merozoite isolation technique was most likely to be successful.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, JM, Warth, R. and Mu?oz, E. (1978) FEBS Letter, 86, 1-5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (alpha, beta, gamma, delta and epsilon) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

Interaction of carbohydrate binding sites on concanavalin A-agarose with receptors on adipocytes studied by buoyant density method.

The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads.     

Properties and separation of T lymphocyte growth stimulatory activity (TL-GSA) and of granulocyte-macrophage colony stimulatory activity (GM-CSA) produced separately from two human T lymphocyte subpopulations.

We have previously identified two stimulatory activities affecting blood cell maturation in PHA-stimulated human lymphocytes conditioned medium (PHA-LyCM). One was granulocyte-macrophage colony stimulatory activity (GM-CSA), and the other was T lymphocyte growth stimulatory activity (TL-GSA) in suspension culture. In this paper we have shown that although both activities can be produced from purified non-adherent human T lymphocytes, they are produced from two distinct subpopulations. The production of these activities was greatly enhanced by T cell mitogens. Both protein factors were relatively heat stable (56 degrees, 30 minutes), were sensitive to trypsin treatment and were specific for primate blood cells. These two activities were fractionated by means of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE cellulose and Con A-Sepharose column chromatographies. MW of the major peak estimated from the elution volume of gel filtration in the presence of 0.5 M NaCl was 40,000 for GM-CSA and 13,000 for TL-GSA. Results from Con A-Sepharose column showed that while about 70% of TL-GSA was bound to Con A, less than 25% of GM-CSA was bound. These observations show that the majority of TL-GSA and GM-CSA were separable by these two conventional column chromatographic methods.     

Leaching of concanavalin A during affinity chromatographic isolation of cell surface glycoproteins from human fetal neurons and glial cells.

Preparation of surface glycoproteins from human fetal brain cells by affinity chromatography on Con A-Sepharose 4B was a problematic endeavor due to leaching of Con A from the matrix. Dissociation of Con A from the matrix took place irrespective of the presence of lipid and/or detergent and the buffer composition during chromatography and was apparently related to the nature of the protein under study. Pretreatment of Con A-Sepharose with 6 M guanidine or 8 M urea reduced Con A leaching. The Con A eluate also contained noncovalently associated glycolipid. Elution at 25 degrees C rendered fractions containing a higher degree of Con A and glycolipid contamination compared to the negligible contamination by these two components when elution was carried out at 4 degrees C. This phenomenon was attributed to the formation of heterogeneous mixed micelles of glycoprotein.     

Production and properties of histamine-releasing factor of guinea pigs

The production of histamine-releasing factor (HRF) by human mononuclear cells has previously been reported. In this paper we describe the production of HRF by guinea pig spleen cells, thymocytes, and PBMC. Guinea pig lymphoid cells were cultured either alone or in the presence of mitogens (PHA and Con A) or specific Ag(OVA and keyhole limpet hemocyanin) and the dialyzed cell-free supernatant was tested for histamine-releasing activity on guinea pig lung mast cells and blood basophils. Lung mast cells were isolated by enzymatic digestion and partially purified by countercurrent elutriation and discontinuous Percoll gradient centrifugation. Guinea pig spleen cells, thymocytes, and PBMC spontaneously produced significant amounts of HRF. The production was enhanced upon stimulation with PHA or specific Ag in animals immunized with Ag in CFA. Two distinct species of HRF were identified with m.w. of 50,000 to 70,000 and 5000 to 8000 by gel chromatography. HRF is a trypsin- and chymotrypsin-sensitive heat-stable protein. It does not bind to Con A-Sepharose and its production is not inhibited by tunicamycin. HRF-induced histamine release from lung mast cells is a temperature-dependent process and is complete in 10 min at 37 degrees C. Intradermal injection of HRF caused an immediate ear-swelling reaction in guinea pigs. The most severe ear-swelling reactions did not resolve within 1 h, but instead evolved over a period of 12 to 24 h.     

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3H-Water and 3H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3H-F and 3H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.     

Affinity chromatographic isolation of cell surface glycoproteins from human foetal brains and their interaction with lectins.

The cell surface glycoproteins of foetal human neurons and glial cells were isolated by affinity chromatography on Con A-Sepharose 4B. Dissociation of Con A from the matrix took place independent of buffer composition and the absence of lipids and/or detergents during chromatography. It was apparently related to the nature of glyco proteins. Pretreatment of Con A-Sepharose 4B with urea or guanidine minimized this problem. The elution of glycoproteins from the affinity matrix at 4 degrees C, instead of the usual 25 degrees C, reduced both Con A and glycolipid contamination in the eluate. Dot-enzyme-linked-lectin assay was carried out with horse radish peroxidase conjugated lectins and serotonin. It was observed that total glycoproteins contained high mannose, hybrid and a limited quantity of biantennary complex type oligosaccharide chains. O-linked oligosaccharides were also present. Desialylation and sodium chloride inhibited binding to serotonin and wheat germ agglutinin indicating the presence of sialic acid residues. Fucose was attached to the innermost core GlcNAc residue, as revealed by affinity towards pea lectin.     

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.     

Eosinophil-particle interactions: a model system for study of cellular adherence and activation

In its role as an effector capable of killing large multicellular parasites, the eosinophil must be especially adapted for dealing with noningestible surfaces. A model system of Sepharose beads coated with serum protein or concanavalin A (Con A) has been used to study interactions between guinea pig peritoneal exudate eosinophils and noningestible particles. A small percentage of eosinophils were adherent to serum treated Sepharose; however, many cells were adherent to Con A-Sepharose. Adherence to Con A-Sepharose was decreased by pretreatment with 1 mM alpha-methylmannoside (alpha-MM). As compared to resting eosinophils, incubation of eosinophils with serum-treated Sepharose led to activation of oxidative metabolism as indicated by an eight-fold increase in superoxide anion production and an approximately threefold increase in quantitative leukocyte iodination. Eosinophils which were adherent to Con A beads could not be activated by either phorbol myristate acetate (PMA) or preopsonized zymosan. However, if adherence was reduced by preincubation with alpha-MM, PMA was able to activate the eosinophils. Neither soluble Con A nor Sepharose beads interfered with the assay of superoxide anion. These studies demonstrate the utility of Sepharose beads for studying interactions between eosinophils and noningestible particles.     

Interaction of concanavalin A with lactogenic receptors in isolated membranes

Specific binding of lactogenic hormones to their receptors in membranes from lactating mouse liver is inhibited by concanavalin A (Con A). Binding to solubilized receptors is not affected by the presence of Con A in the binding reaction. However, these solubilized binding proteins are retained on Con A-Sepharose columns. Prebound hormone-receptor complexes are also retained on Con A-Sepharose. These data indicate that lactogenic receptors have a Con A binding site distinct from the hormone binding site. Moreover, once bound, the hormone is not released by the action of Con A. Somatogenic receptors do not have a Con A binding site and the binding of bovine growth hormone to hepatic membranes is not affected by the presence of Con A in the binding reaction. Inhibition of binding to lactogenic receptors by Con A occurs independently from other membrane perturbing events such as phospholipid methylation.     

Binding of bovine brain tissue factor to concanavalin A-Sepharose. Partial purification of coagulant, arylamidase, and alkaline phosphatase activities.

Tissue factor coagulant activity is adsorbed onto concanavalin A-Sepharose from sodium deoxycholate extracts of delipidated bovine brain powders. Coagulant activity is eluted with alpha-methyl-D-glucoside in sodium deoxycholate with 2--25-fold purification. This material has the same coagulant specific activity as that previously prepared in this laboratory. Alkaline phosphatase and alanyl-beta-naphthylamidase activities in the detergent extract also bind to concanavalin A-Sepharose and elute under the same conditions with 4- and 7-fold purification. In addition to these biological activities, the eluate was composed of protein (67.7%), neutral and amino sugars and sialic acid (22.3%), phospholipid (4.5%), uronic acid (3.8%) and nucleic acid (1.7%). This preparation is slightly enriched in carbohydrates compared to previous preparations. Concanavalian A-Sepharose therefore appears to be useful material for partial purification of several mammalian plasma membrane components with retention of biological function.