Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

The isolation and partial characterization of a glycoprotein isolated from human gastric aspirates and from extracts of gastric mucosae

The isolation and partial characterization of a glycoprotein isolated from individual gastric aspirates and extracts of gastric mucosae solubilized with N-acetylcysteine is described.The isolated glycoproteins and the glycoproteins from proteolysed gastric aspirates showed virtually the same carbohydrate and amino acid composition. The results indicate that they consist of a protein core to which are attached carbohydrate side-chains composed of four sugars: N-acetylgalactosamine N-acetylglucosamine, galactose, fucose showing a ratio of 1 : 3 : 4 : 2. Superimposed on this basic structure were additional sugar residues, the blood-group determinants. The results also suggest that the carbohydrate side-chains are linked by an alkali-labile O-glycosidic linkage to the threonine and serine residues of the protein core, N-acetylgalactosamine forming the link.     

Studies on the mucin derived from human colloid breast carcinoma

1. A non-diffusible mucoid, showing a single peak in the ultracentrifuge, was isolated from human colloid breast carcinoma by treatment with trypsin and pepsin. The material contained threonine, leucine (isoleucine), valine, proline, glycine and glutamic acid in the approximate molar proportions 5:1:1:2:1:1. Smaller amounts of aspartic acid and serine were also found. For each 5 threonine residues, 6 N-acetylgalactosamine and 3–4 galactose residues were present. 2. The mucoid possessed reducing properties by the Park & Johnson (1949) procedure; these were attributable to the action of mild alkali, as employed in this procedure. Mild alkaline treatment by the Aminoff, Morgan & Watkins (1952) procedure gave rise to a diffusible N-acetylgalactosamine chromophore that gave an enhanced colour with Ehrlich's reagent. That galactosyl-(1→3)-N-acetylgalactosamine residues were liberated was supported by periodate studies. 3. Alkaline liberation of hexosamine residues was accompanied by a specific destruction of threonine. After 40 min. at 100° in 0·18 n-lithium hydroxide, both moieties had almost completely disappeared from the ninhydrin-positive components formed on subsequent acid hydrolysis. Glycine and α-oxobutyric acid were present in the acid hydrolysate, showing that both possible pathways of a β-elimination reaction were involved. Formation of diffusible peptide on very mild alkaline treatment was attributable to the rupture of the original peptide core, necessitated by the second of these two pathways. 4. Hydroxamate formation on treatment with hydroxylamine showed the presence of carbohydrate linkage to glutamic acid or aspartic acid residues or both. This could account for the single N-acetylgalactosamine residue not linked to threonine. 5. The native mucin contained sialic acid, which was cleaved by the acid environment used in the treatment with pepsin. A statistical model of the mucin would require each prosthetic group to be linked, via N-acetylgalactosamine, to threonine, which would occupy every alternate position among the amino acids in the peptide core.     

Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes

Phenol extraction of horse, sheep, cow, pig and human erythrocyte membranes and human milk fat globule membranes gave glycoprotein fractions, all of which were shown by gas chromatography to contain the reduced disaccharide β-d-galactosyl $\text{(1?3)-N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosaminital}$ after treatment with alkaline borohydride. Cow and pig erythrocyte membrane glycoproteins were found however to contain much lower amounts than the erythrocyte membrane glycoproteins of the other species tested. After gel filtration, a tetrasaccharide was isolated from horse and sheep glycoproteins containing the disaccharide plus two molecules of sialic acid. Periodate oxidation together with paper chromatography of alkaline degraded fragments showed these two molecules of sialic acid to be linked to positions C3 and C6 of the galactosyl and N-acetylgalactosamine residues respectively. Evidence was obtained for a similar structure from pig and cow erythrocyte glycoproteins and human milk fat globule membrane glycoproteins although the complete structure was not elucidated.In all native glycoprotein fractions, the unsubstituted disaccharide β-d-galactosyl $\text{(1?3)-N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosamine}$ was found to be present to different extents.Haemagglutination inhibition tests against human anti-T serum, Arachis hypogoea and Vicia graminea by desialylated glycoproteins showed the presence of the T-antigen, confirming the chemical findings. Inhibition was found to be proportional to the chemically detected amounts of disaccharide in each fraction. Evidence for a second carbohydrate chain in horse, sheep and human erythrocyte glycoproteins with a sialic acid substituted N-acetylgalactosamine residue as the terminal sequence was obtained using the agglutinin from Helix pomatia.     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.     

Purification and immunofluorescent localization of rat submandibular mucin

Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S⁰_20,w 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1×10⁶. Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.     

Purification and properties of the major sialoglycoprotein of the milk fat globule membrane

The major periodate-Schiff positive component (glycoprotein-2) of bovine milk fat globule membranes (MFGM) has been purified by extraction of washed cream with chloroform/methanol followed by chromatography on Sephadex G-200 in sodium dodecyl sulfate. The glycoprotein is > 95% pure by polyacrylamide electrophoresis in dodecyl sulfate and shows the same prominent component at gel percentages of from 5 to 12.5. The molecular weight obtained by extrapolation of the apparent molecular weights on these gels to higher gel percentages was 70,000. An apparent molecular weight of 105,000 was obtained by gel filtration in 1% dodecyl sulfate on Sepharose 4B. The glycoprotein contains 50% carbohydrate by weight, with sialic acid (30.5%), N-acetylglucosamine (22.3%), galactose (15.9%), N-acetylgalactosamine (14.0%), mannose (11.1%), and fucose (5.8%) being the major monosaccharides. Leucine, glutamic acid, and glycine are the major amino acids. Affinity chromatography of deoxycholate-solubilized MFGM indicates that glycoprotein-2 is not the major concanavalin A receptor of these membranes.     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56° C and pH 2.0. Its plasma membrane possessed 21–22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched α 1 → 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunction with water turgor, may contribute to the rigidity of the membrane and the cation binding.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Immunochemical and chemical investigations of the structure of glycoprotein fragments obtained from epiglycanin,a glycoprotein at the surface of the TA3-Ha cancer cell

The structures of the carbohydrate chains present in fragments of a large-molecular-weight glycoprotein, epiglycanin, cleaved from the surface of viable TA3-Ha murine mammary carcinoma ascites cells and purified by gel filtration, were studied by immunochemical and chemical methods. Inhibitory activities for neuraminidase-treated and untreated glycoprotein material in the hemagglutination of NN-specific human erythrocytes by eight purified lectins were determined. Excellent inhibition was obtained in the Bauhinia purpurea, Arachis hypogaea, Iberis amara, and Wistaria floribunda systems, and weak inhibition against the Ricinus communis and Glycine max lectins. No activity against hemagglutination by the Phaseolus vulgaris and Phaseolus limensis lectins was observed. These results, when compared with those obtained by periodate oxidation, alkaline borohydride reduction, and partial methylation, suggest the possible presence of six different carbohydrate chains of 1 to 5 components in length, having as terminal groups N-acetylneuraminic acid, galactose, and 2-acetamido-2-deoxygalactose. All chains are attached to a single polypeptide chain by O-glycosyl bonds involving a 2-acetamido-2-deoxygalactose residue and a serine or threonine residue. It is suggested that the native molecule of epiglycanin of molecular weight 500,000 contains more than 500 carbohydrate chains attached to a single polypeptide chain of ≈ 1,300 amino acid units.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.     

Immunological studies in ulcerative colitis: extract, fractionation, and immunological characterization of germ-free rat colon antigen

Intestinal mucins from germ-free rats contained antigens reactive with sera from patients with ulcerative colitis, in addition to human blood group A- and H-like antigens. A crude antigen extract was obtained by phenol-water extraction at 65 °C. Two intestinal glycoproteins were purified from the extract by fractionated ethanol precipitation, ion-exchange chromatography, and gel filtration. The two glycoproteins (2aI and 4aIIb) were homogeneous in regard to electrical charge and molecular size. Both were glycoproteins of the blood group substance type. Component 2aI was very rich in N-acetylgalactosamine and threonine and low in N-acetylglucosamine and sialic acid(s). It had strong blood group A-like activity, weak blood group H activity, and no colon antigen activity as defined by patients' sera. Component 4aIIb was rich in sialic acid(s). About 40% of the sera from patients with ulcerative colitis reacted with this component. No blood group A- or H-like activity could be demonstrated. Colon antigen activity was sensitive to periodate oxidation, but resistant to boiling at neutral pH. It was very sensitive to acid hydrolysis. In fact, colon antigen activity was significantly reduced when subjected to weak acid hydrolysis under conditions which only appeared to release sialic acids.     

Further studies on the peptido-galactomannan from the yeast form of Cladosporium werneckii. Identification of O-acetyl substituents and isolation of the peptide components following beta-elimination.

The pathogenic yeast Cladosporium werneckii produces a surface peptido-phosphogalactomannan (PPGM) with a peptide backbone rich in serine and threonine to which three types of carbohydrate chains are linked. These chains are: Type a, glactomannan units linked through phosphodiester bonds to produce long chains of molecular weight about 50,000; type b more numerous short mannosyl oligosaccharide units, and type c, more infrequent, long galactomannan chains. The first two are linked to the peptide through alkali-labile bonds to serine and threonine of the peptide whereas type c chains are linked through alkali-stable bonds (Lloyd, K. O. (1972) Biochemistry, 11, 3884–3890). The PPGM sample can be separated into three or four major components by diethylaminoethyl (DEAE-) Sephadex chromatography. By means of sequential degradations with alkali and acid, the structural basis for this heterogeneity has been demonstrated. It is due to the presence of different proportions of the three types of chains in the various fractions. The presence of O-acetyl groups, mainly on the a chains, was demonstrated in PPGM by chemical analysis and by proton and ¹³C nuclear magnetic resonance spectroscopy. Purified a chains were isolated from PPGM following Pronase digestion. By taking advantage of the alkali lability of the carbohydrate-protein linkages it has been possible to cleave the peptide moiety away from the carbohydrate. By sequential chromatography on Bio-Gel P-100, Dowex 1 and Bio-Gel P-100, five modified peptide fractions were isolated. The molecular weights of these fractions varied from 9,500 to 18,500 as judged by polyacrylamide-gel electrophoresis. Unlike the original peptide, the modified peptides contained low amounts of serine and threonine. The predominant amino acids were alanine, glycine, aspartic acid and glutamic acid which together make up between 51 and 55% of the peptides. The high content of the last two amino acids accounts for the acidic nature of the peptides. It appears that each fraction consists of a slightly heterogeneous population of peptides very similar in amino acid composition. It is not clear whether the compositional and size heterogeneity exists in the original peptide or whether it arose during the isolation procedure.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

Carbohydrate compositions of the rabbit plasminogen isozymes.

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.