Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Variation in endoplasmic-reticulum-associated glycoproteins of carrot cells cultured in vitro

Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with M_r 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with M_r 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A concanavalin A - 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GP 31, GP 32 a glycoprotein with an apparent molecular mass of 31 or 32 kdalton - kDa kilodalton - MS Murashige and Skoog - M_r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point ($\text{p}\text{I}$), apparent molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP_132–135^4–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ but different $\text{p}\text{I}$ were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Extracellular folate deaminase of Dictyostelium discoideum

Folate deaminase released from cells of Dictyostelium discoideum is heterogenous with respect to molecular weight and stability at 60°C. The most heat-stable component isoelectrofocuses in a broad band at approx. pH 6. The K_m value of this component for folate is approx. 7 · 10^?7 M and M_r approx. 40 000. The major portion if not all of the deaminase binds to immobilized concanavalin A and lentil lectin. Extracellular folate deaminase has a pH-optimum of approx. pH 6.0. This is higher than that of lysosomal enzymes, which are also glycoproteins released into the extracellular medium.     

Accessibility of plasma membrane sialoglycoconjugates of novikoff tumor cells to exogenous neuraminidase and proteases

Plasma membrane glycoconjugates of Novikoff tumor cells were radioactively labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄ Submission of the radioactively labeled glycoconjugates to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by fluorography revealed the presence of at least ten major glycoproteins and a glycolipid fraction. The glycolipid fraction contained 34% of the cell-surface radioactive label. Pretreatment of cells with neuraminidase from Vibrio cholerae reduced radioactive labeling of the glycoproteins by 71% and that of the glycolipids by 39%. Sequential treatment of cells with papain and neuraminidase further reduced radioactive labeling of the glycolipid fraction, indicating that resistance of this fraction to the hydrolytic action of neuraminidase was determined, at least in part, by steric factors. Incubation of cells with papain resulted in extensive degradation of most of the radioactively labeled glycoproteins with the exception of a subset of glycoproteins having apparent molecular weights of $\text{48 000 ± 5000}$. Trypsin was more selective, degrading three glycoproteins having apparent molecular weights of 200 000, 140 000 and 37 000.     

Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells

Glycoproteins released from carrot cells into culture media were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue or with the periodic-acid Schiff procedure. The appearance or disappearance of two glycoproteins of M_r 65,000 (GP65) and M_r 57,000 (GP57) was closely related to the formation of somatic embroys. GP65 was released specifically from embryogenic cells cultured in a medium without 2,4-dichlorophenoxyacetic acid, in which they can form somatic embryos. GP57 was released from the same embryogenic cells, if they were cultured in a medium with 2,4-dichlorophenoxyacetic acid, in which they cannot form somatic embryos. Nonembryogenic cells which cannot form somatic embryos, released only GP57.     

Concanavalin A is synthesized as a glycoprotein precursor

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (M_r) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with M_r 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-M_r polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 M_r. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [³H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, -mannosidase or -N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (M_r=30000). The 4000 decrease in M_r is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.Abbreviations Con A concanavalin A - Glc glucose - GlcNAc N-acetylglucosamine - IgG immunoglobulin G - Man mannose - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Summary Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with ³⁵S-methionine-labeled cells showed that HPA recognized two glycoproteins of M_r 170000 and 400000 among the secreted products of fibroblasts and two major cellular glycoproteins of M_r 40000 and M_r 180000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulselabeled shortly with ³⁵S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

Selective suppression of the murine autologous mixed lymphocyte reaction by physiological concentrations of hydrocortisone: effects on cell-surface Ia antigens

Strong, adult (Type II) autologous mixed lymphocyte reactions (AMLR) were observed in cultures of lymphoid cells from both A.TH and A.TL mice. These were suppressed by more than 90% in the continuous presence of 7.5 × 10^?8M hydrocortisone-21-sodium succinate. This concentration of hormone had minimal effects on the allogeneic mixed lymphocyte response (MLR) and the mitogenic response to concanavalin A (Con A). Higher concentrations suppressed all three responses. Treatment of autologous cell mixtures for the first 30 hr with 7.5 × 10^?8M hydrocortisone resulted in a 78% suppression of the AMLR. This was not associated with a detectable decrease in the quantity of Ia antigens on the stimulator-cell surface, as evaluated by the susceptibility of treated cells to antibody dependent, complement-mediated lysis, using [A.TH × B.10M]F₁ anti-A.TL antiserum. Hence, this suppression did not appear to result from an alteration of the antigens putatively associated with stimulation of the AMLR. Separate pretreatment of stimulator and responder cells with 7.5 × 10^?8M hydrocortisone followed by culturing with appropriate companion cells had no major effect on the AMLR. Therefore, low-dose hydrocortisone did not appear to selectively eliminate or permanently inactivate subpopulations of responder or stimulator cells. Rather, it appeared to regulate active cellular processes that are initiated by the coculturing of these cells and are required for the early stages of autologous lymphocyte activation.     

Lymphocyte receptors for polyclonal T mitogens: II. High-molecular-weight glycoproteins are the best candidate sites for mitogenic action by concanavalin A and galactose oxidase

Murine lymphocytes oxidized by galactose oxidase were radiolabeled by reduction with NaB³H₄. The labeled cells were incubated with Con A and the Con A-Con A receptor complexes formed in situ on the viable cells were isolated by immuno-precipitation with anti-Con A serum and fixed Staphylococcus aureus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography analysis of the precipitates demonstrated four high-molecular-weight glycoproteins which were oxidized by GO and which bound Con A. These same four glycoproteins were also oxidized and labeled by $\text{IO}{}_4\text{NaB}{}^3\text{H}{}_4$. [³H]Tyrosine biosynthetic labeling identified these four plus several other Con A receptors. Because Con A sterically inhibits GO mitogenic stimulation, these four glycoproteins are likely to represent the necessary sites of oxidative mitogenic action and are good candidates for the targets of Con A mitogenesis.     

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Polyadenylated RNA prepared from first trimester human placenta was translated in a membrane-free cell-free system derived from wheat germ. Analysis of the [³⁵S]methionine-labeled products by SDS-polyacrylamide electrophoresis demonstrated two proteins with apparent M_rs of 14,500 and 16,000 that were specifically immunoprecipitated by antiserum to reduced and carboxylated bovine LH_α, and two different proteins with apparent M_rs of 18,500 and 21,000 that were specifically immunoprecipitated by antiserum to hCG_β. None of these products was sensitive to cleavage by endoglycosidase H, whereas the M_r 21,000 product precipitated by antisera to bovine LH_α and to hCG_α from translations supplemented by canine pancreatic microsomes was processed to a product with M_r 13,000 by endoglycosidase H. We suggest that the two forms of the α and β subunit precursors could arise from the translation of two distinct mRNAs encoding each subunit.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.     

Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons

-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (M_r) 66000 and M_r 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. -Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an M_r of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the M_r-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that -mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [³H]glucosamine shows that after processing only the M_r-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to -mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of -mannosidase out of the ER-Golgi system.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - H L heavy, light subunit - IgG Immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Lectin receptors in Trypanosoma cruzi an N-acetyl-d-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the ¹³¹I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent M_r 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}$ inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}\text{-}\text{specific}$ receptor activity.     

The positively cooperative binding of concanavalin a to corn starch: The isotherm of binding and a measure of the cooperativity

The binding of concanavalin A to corn starch was investigated by fluorimetric assay. The extent of binding varied linearly with the mass of ligand, and followed a hyperbolic law with respect to the mass of starch. This led to an isotherm of binding: r = 0.33A_oME_o^?0.88, where r is the extent of binding, A_o is the mass of concanavalin A present (both bound and unbound), and M_o is the mass of starch. These results, and Scatchard plots of the data, showed the binding to be positively cooperative. The exponent of the M_o term was shown to be a measure of cooperativity. The binding was dependent on the ionic strength of the dispersion medium, and this indicated that the binding may have an electrostatic component.