Linkage regions between dermatan polysulfates and peptides.

Three different types of dermatan polysulfate peptides I, II and III, isolated from hagfish notochord, hagfish skin and shark skin, all contained serine, xylose and galactose in a molar ratio of about 1.2 : 1 : 2. After beta-elimination-reduction, dermatan polysulfate peptides II and III produced alanine and xylitol in amounts equivalent to the amount of the serine decrease. Accordingly, it was shown that these dermatan polysulfates were linked to peptides by O-glycosidic bond between xylose and serine, as in chondroitin sulfates and dermatan sulfate. However, dermatan polysulfate peptide I produced N-acetylgalactosaminitol, in addition to alanine and xylitol, in a molar ratio of about 2 : 3 : 1, although the increase of alanine was equivalent to the serine decrease. Consequently, it was concluded that the linkage region of dermatan polysulfate peptide I has two types of O-glycosidic bond: one between xylose and serine and the other between N-acetylgalactosamine and serine. This is the first finding of an N-acetylgalactosamine involved in the linkage region of glycosaminoglycans consisting of uronic acid as repeating constituents.

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines.

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.

Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains.

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.

Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide.

We isolated vitronectins from the plasma or sera of 14 animal species including mouse and rat by heparin affinity chromatography. They cross-reacted with anti-vitronectin antibody and their amino terminal sequences showed strong homology. They also promoted spreading of BHK cells and were bound to heparin and collagen in the same way. Therefore, these properties appear to be essential for vitronectin function. However, the apparent molecular weights of these vitronectins varied considerable from 59 to 78 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1 to 3. To search for the uniformity of vitronectin polypeptide, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptides were speculated to be synthesized de novo in the size range of 50-57 kDa. Proteins reacting with anti-vitronectin antibody were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almost all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.

Capsaicin-induced activation of fine afferent fibres from rat skin in vitro.

A preparation of the hindpaw-skin together with the saphenous nerve from the adult rat was maintained in vitro. This was used to characterize the properties of sensory receptors with slowly conducting nerve fibres (C- and A delta) and to evaluate the effects of capsaicin and the capsaicin antagonist, capsazepine. Mechano-heat sensitive C-fibres were the most sensitive to capsaicin (threshold < 0.3 microM) applied to the receptive field. Other types of C-fibres were less sensitive (mechano-cold sensitive fibres threshold 1 microM) or insensitive (high- and low-threshold mechano-sensitive fibres). Mechano-heat and mechano-cold sensitive A delta-receptors were also activated by capsaicin but high- and low-threshold mechano-sensitive A delta-fibres were insensitive to capsaicin (maximum concentration 3 microM). The capsaicin-induced activation of mechano-heat sensitive C-fibres was concentration dependent with an EC50 = 350 nM. Responses to capsaicin, administered at submaximal concentrations were highly reproducible when administrations were separated by 30 min. Administrations at greater frequency reduced responsiveness to capsaicin. This was accompanied by a slowing of conduction velocity or production of a conduction blockade which was reversible after a few minutes. The activation of mechano-heat sensitive C-fibres by capsaicin could be prevented by capsazepine, indicating the involvement of specific capsaicin receptor-sites. These data show that fine afferents in the rat hindpaw-skin retain receptive properties when maintained in vitro. These fibres exhibit differential sensitivity to capsaicin; mechano-heat sensitive C-fibres being the most sensitive. The activation of this class of fibre was mediated via a specific capsaicin-receptor.

Thalamocortical and corticocortical excitatory postsynaptic potentials mediated by excitatory amino acid receptors in the cat motor cortex in vivo.

Intracellular recordings were made from neurons in the motor cortex of an anaesthetized cat, together with iontophoretic application of excitatory amino acid receptor agonists and antagonists, in order to evaluate the role of such receptors in excitatory postsynaptic potentials evoked from stimulation of afferent and recurrent pathways in vivo. Excitatory postsynaptic potentials which were evoked by stimulation of the ventrolateral thalamus were found to be largely insensitive to antagonism by N-methyl-D-aspartate receptor antagonists, although they were susceptible to blockade by the non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Increasing the ventrolateral thalamus stimulation frequency from 0.5 or 1 to 5 Hz caused an increase of evoked excitatory postsynaptic potential amplitudes and number of action potentials. These augmented excitatory postsynaptic potentials remained insensitive to application of N-methyl-D-aspartate antagonists. In contrast, recurrent excitatory postsynaptic potentials evoked by stimulation of the pyramidal tract were found to be sensitive to N-methyl-D-aspartate receptor antagonists and/or non-N-methyl-D-aspartate receptor antagonists in some neurons. These results demonstrate the involvement of both N-methyl-D-aspartate- and non-N-methyl-D-aspartate receptors in synaptic responses of cat motor cortex neurons in vivo, and that the synaptic pharmacology of the thalamic input may differ from that of the local recurrent pathways.

Structure of linkage region between chondroitin polysulfates and peptides.

Three different types of chondroitin polysulfate-peptide, chondroitin sulfate D-peptide, chondroitin sulfate E-peptide, and chondroitin sulfate K-peptide, all contained xylose, galactose, and serine in a molar ratio of about 1 : 2 : 1. After treatment with alkali in the presence of NaBH4 and PdCl2, they produced alanine and xylitol in amounts equivalent to the decrease in the amount of serine. Consequently, it was proved that these chondroitin polysulfates are all linked to peptides by O-glycosidic bonds between xylose and serine, as in chondroitin sulfates A and C. It is suggested that the carbohydrate-peptide linkage regions have the same structure in all the chondroitin sulfates, regardless of differences in the structure of the polysaccharide chains, such as the position of sulfate groups and the degree of sulfation.

Fluorescence spectral studies on the specific interaction between sulfated glycosaminoglycans and potato lectin.

On excitation at 280 nm, Solanum tuberosum agglutinin (STA) produced a fluorescence emission spectrum with a maximum at 347 nm, which is typical of tryptophanyl residues. The fluorescence emission maximum was shifted toward shorter wavelength and quenched by the addition of specific sugars. The changes were analyzed precisely and quantitatively by measuring the fluorescence difference spectra. This method required far smaller amounts of samples than UV-difference spectroscopy to obtain the binding parameters. The binding constants of chitin sulfate, keratan sulfate, and highly sulfated keratan sulfate were calculated to be 1.8 X 10(5) M-1, 3.4 X 10(5) M-1, and 5.2 X 10(5) M-1, respectively. The results indicate that consecutive and non-consecutive, alternating (1 leads to 4)-N-acetyl-beta-D-glucosaminyl residues in the glycosaminoglycans can interact freely with STA in spite of the existence of the sulfate groups at C-6 of the N-acetyl-D-glucosaminyl residues and extra sulfate groups at C-6 of the galactosyl residues linked with N-acetyl-6-O-sulfo-beta-D-glucosamine.

Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent.

The optimal conditions for the activation of Sepharose by epichlorohydrin and subsequent immobilization of ligands were investigated. Under the optimal conditions for activation, namely, 30% Sepharose-5% epichlorohydrin-0.4 M NaOH, 40 degrees C, 2 h, the maximum amount of epoxy group was introduced into Sepharose with low cross-linking. The absorbents obtained by using N-acetyl-D-glucosamine, tri-N-acetylchitotriose, and glycoprotein as a ligand exhibited no nonspecific adsorption and good permeability for the high molecular substance to be purified, and were stable in an alkaline solution. Solanum tuberosum agglutinin was specifically adsorbed on a tri-N-acetylchitotriose-Sepharose column and was quantitatively recovered by elution with 0.2 M ammonia solution. Furthermore, the column could be repeatedly used under these conditions without reduction of its capacity.

Tryptophan residues and the sugar binding site of potato lectin.

Potato lectin (Solanum tuberosum agglutinin, STA) was found to contain fluorescent tryptophan residues highly exposed to solvent. The binding of chitin oligosaccharides to STA induced fluorescence quenching, a shift of the fluorescence maximum to shorter wavelength, a decrease in the quenching constant of iodide ion and a decrease of the number of tryptophan residues modifiable by N-bromosuccinimide. The results suggested that one tryptophan residues is located at or near a sugar binding site of STA, and that its environment is altered from hydrophilic to relatively more hydrophobic upon interaction with specific sugars. The binding constants of STA with chitin oligosaccharides were determined by measuring the peak-trough heights in the fluorescence difference spectra induced by various concentrations of sugars. The inhibition constants of chitin oligosaccharides for the hemagglutinating activity of STA were obtained by the method of Pitts and Yang [(1981) Biochem. J. 195, 435-439] and the results were in good agreement with those obtained by the fluorescence spectral method. Standard and unitary free energy changes (delta G0 and delta Gu) and standard enthalpy changes (delta H0) were also obtained. These values decreased with sugar chain length up to at least the tetramer. Thus, it was assumed that there are at least 4 subsites, A, B, C, and D, in the sugar binding site of STA. The contributions to the binding energy (delta Gu) were -17.0, -12.6, -7.3, and -4.4 kJ/mol at subsites A, B, C, and D, respectively, and the bindings of chitin monomer (GlcNAc), dimer, trimer, and tetramer were assumed to occur at subsite A, AB, ABC, and ABCD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization of protein ligands on new formyl-spacer-carriers for the preparation of stable and high capacity affinity adsorbents.

New procedures to immobilize high concentrations of protein ligands by reductive amination on two types of formyl-carriers (I & II) having different spacer lengths were investigated in order to prepare stable and high-capacity adsorbents essential for efficient affinity chromatography. Formyl-carrier (I) was prepared by reductive amination with glutaraldehyde of the amino-carrier obtained on amination of an epoxy-activated carrier. Formyl-carrier (II) was prepared by sodium metaperiodate (NaIO4) treatment of a glyceryl-carrier obtained on hydrolysis of an epoxy-activated carrier. Especially high concentrations of protein ligands were immobilized on formyl-Sepharose 4B (I) under very mild conditions (pH 7.0, 4 degrees C). A series of lectins, one of the most useful classes of group-specific ligands, was successfully immobilized by the procedures. Concanavalin A-Sepharose 4B (I) thus obtained exhibited an adsorption capacity five times greater than that of concanavalin A-Sepharose 4B made by Pharmacia Fine Chemicals, and could be repeatedly used over twenty times without a significant reduction in its adsorption capacity.

Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.

Lectin binding sites related with rat ascites hepatoma cell adhesion.

In order to elucidate the correlation between cell surface lectin binding sites and the degree of cell adhesiveness, quantitative lectin binding assays were performed using three types of rat ascites hepatoma cell lines (free cell, mixed cell, and island-forming cell types). The lectin binding site patterns showed no remarkable differences among the intact tumor cell lines, but treatment of the cells with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or neuraminidase induced remarkable differences in the modulation of the number of lectin binding sites. TPCK-trypsin treatment caused a marked decrease in the number of peanut agglutinin binding sites on the island-forming and mixed cell types, concomitant with disaggregation of the cells, showing that trypsin sensitive binding sites are involved in the cell-cell adhesion. Neuraminidase treatment caused a decrease in wheat germ agglutinin binding sites and an increase in castor bean agglutinin binding sites, and these effects were greater for the free cell type. These results indicated that alpha-sialyl-beta-D-galactosyl residues are more abundant on the cell surface of the free cell type than the other cell types. Therefore, it was suggested that electrostatic repulsion due to negative charges of the cell surface sialic acid contributes to the low cell adhesiveness of the free cell type.

Immunochemical and spectral studies on Vicia faba agglutinin.

Affinity chromatographic purification of Vicia faba agglutinin (VFA) was performed with a Sephadex G-150 column according to the method of Allen and Johnson [Biochim. Biophys. Acta (1976)]. VFA has 10 tryptophanyl residues per molecule on the assumption that its molecular weight is 50,000 daltons. Equilibrium dialysis with methyl a-D-[glucose-14C(U)]glucopyranoside showed that VFA has two sugar binding sites per molecule with a binding constant of 220 M-1. Upon interaction with specific sugars, VFA induced UV-difference spectra which are typical of the perturbation of tryptophanyl residues. Therefore, the binding constants of VFA with specific sugars could be calculated from the intensity changes in the difference spectra induced by various concentrations of the sugars. The results obtained were in good agreement with the results of hemagglutination inhibition assays. 3-O-Methyl-D-glucose had the highest binding constant (1.9 x 10(3) M-1) among the sugars examined. The binding constants of VFA with glucose, mannose, methyl a-D-glucopyranoside, methyl a-D-mannopyranoside, and maltose were 290, 900, 220, 500, and 220 M-1, respectively, which are lower than those of concanavalin A. VFA did not bind with mucopolysaccharides containing 2-acetamide-2-deoxy-a-, or -beta-D-glucopyranosyl residues, such as heparin, heparan sulfate, and hyaluronic acid. The far UV-CD spectrum of VFA was similar to that of concanavalin A.

Isolation and characterization of a lectin from the fruit of Clerodendron trichotomum.

An agglutinin of Clerodendron trichotomum fruit (CTA), found to be specific for N-acetyl-D-galactosamine and D-galactose, was isolated and characterized. The fruit extract was decolorized first by passage through a Toyopearl column and a phenyl-Sepharose column. Then the lectin activity was adsorbed on p-aminophenyl N-acetyl-alpha-D-galactosaminide- or p-aminophenyl beta-D-galactoside-Sepharose, and eluted as a sharp peak with 0.2 M lactose. The purified CTA was found to be homogeneous by SDS-polyacrylamide gel electrophoresis, gel chromatography and ultracentrifugal analysis, and was determined to be a glycoprotein homodimer with a molecular weight of 56,000 daltons. Hemagglutination-inhibition assay indicated that CTA is most specific for N-acetyl-D-galactosaminide with a hydrophobic aglycon.

Dermatan sulfate-reactive lectin from chicken liver.

A lectin highly reactive with dermatan sulfate (DS-lectin) was purified from adult chicken liver by gel filtration on Toyopearl HW-55 and subsequent affinity chromatography on new adsorbents which were prepared by immobilizing heparin or dermatan sulfate via the reducing ends on hydrazino-Toyopearl. The DS-lectin behaved as a single protein on polyacrylamide gel electrophoresis. On excitation at 280 nm, the DS-lectin emitted fluorescence centered at 336 nm, which was attributable to tryptophan residues and could be quenched by the addition of specific saccharides. The affinity constants of the DS-lectin with specific saccharides were calculated from the changes in intensities of fluorescence-difference spectra induced by the saccharides. Dermatan sulfate and protuberic acid, which is composed of L-iduronic acid and D-glucuronic acid (1:2), had the highest affinity constants among the polysaccharides tested. Partially N-desulfated heparin had a higher affinity constant than that of native heparin while dextran sulfate showed no affinity. D-Glucuronic acid and N-acetylneuraminic acid induced weak but significant quenching, but not N-acetylgalactosamine or cellobiose. These results were essentially in good agreement with those of hemagglutination inhibition tests and indicated that DS-lectin has a strong affinity for L-iduronic acid residues and probably carboxyl groups in the saccharides, while sulfate groups on the saccharides interfere with the specific interaction.

Amination and subsequent derivatization of epoxy-activated agarose for the preparation of new affinity adsorbents.

We have found a simple procedure to convert epoxy-activated agarose into amino derivatives using ammonia solution. The amino derivatives of agarose were succinylated with succinic anhydride and then activated with N-hydroxysuccinimide according to the method of Cuatrecasas and Parikh. Formula: (See Text). Coupling of the ligand to the activated agarose (shown above) was performed under mild conditions. The adsorbent thus obtained has no charged group in the linkage region between the ligand and agarose, thus reducing the nonspecific adsorption, and the bonds formed (ether and amide bonds) are stable even in an alkaline medium. Lens culinaris hemagglutinin-Sepharose 4B prepared by this method was successfully used for the affinity chromatography of solubilized human red blood cell membrane in detergent solution and was stable when elution was performed with borate buffer, pH 9.8.

Hydrophobic properties of porcine fibronectin and its functional domains.

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.

Detection of lectin-sugar interaction by ultraviolet difference spectroscopy.

It was found that the addition of specific sugars to solutions of several lectins induced ultraviolet difference spectra. The difference spectra of lectins from Lens culinaris, Sophora japonica, Solanum tuberosum and wheat germ have two peaks at 292 nm and 284-287 nm which are characteristic of the tryptophanyl residue. The difference spectrum of Arachis hypogaea agglutinin has two peaks at 285 nm and 279 nm which seem to be characteristic of the tyrosyl residue. In addition to the identification of these amino acid residues as being in or near the sugar binding sites of the lectins, the binding constants of these lectins with the specific sugars can be easily determined from the intensities of the difference spectra at various concentrations of sugars. This method may be useful for the simple and direct determination of the binding constants of lectins with various naturally occurring sugars which have no chromogenic groups.

Immunological characterization of human vitronectin and its binding to glycosaminoglycans.

The cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65 kDa polypeptides, was found to have different ratios of amounts of 75 and 65 kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochem. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitronectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40 mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13 kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75 kDa polypeptide in plasma.