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

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

Interaction of model IgG complexes with lectin from Ricinus communis in solutions studied by light scattering method]

In order to demonstrate the participation of galactose-containing carbohydrate epitopes on the surface of model IgG complexes (MIC) during their interaction with high molecular weight ligands, MIC were obtained. The interaction of MIC with Ricinus communis agglutinin (RCA) was studied. The time-dependent changes in the intensity of light scattering in solutions containing MIC of different molecular masses were measured after addition of RCA. It was shown that the efficiency of MIC interaction with RCA depends on the molecular mass of the former. The binding of RCA to MIC is highly specific, it being completely abolished after addition of lactose (1-15 mM). It was found that the final lactose concentration necessary for the complete inhibition of MIC interaction with RCA to take place, depends on the molecular mass of MIC. The data obtained point to the accessibility of IgG oligosaccharide antennae within the composition of MIC for the binding to high molecular weight ligands as well as to the increased density of galactose-containing epitopes on the surface of MIC resulting from the increase in their molecular mass.     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots

Unreduced human immunoglobulin G (IgG) which was not aggregated showed anomalous apparent molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It migrated mainly as three distinct bands with apparent molecular masses from 190 to 240 kDa on gels containing 8% polyacrylamide, when denatured at 37 degrees C. Generation of this banding pattern has two reasons: (a) the pattern is a superposition of bands originating from the four IgG subclasses that differ in molecular masses and structures; and (b) the complexity of the band pattern is further increased, because IgG myeloma proteins of the IgG1 and IgG2 subclass migrated as doublets, while IgG3 and IgG4 formed primarily one band with slightly different apparent molecular masses. These properties were independent of the type of light chain in all myeloma proteins studied. Generation of doublets suggests heterogeneities of monoclonal proteins. The two separable protein populations from IgG1 differ in their susceptibility to reduction. Reduction at 37 degrees C cleaved the larger into heavy and light chain, while it generated heavy chain dimer and light chain from the smaller species. Hence, it is possible that monoclonal IgG1 are comprised of at least two subpopulations of molecules with different S-S bonds. Doublet formation of IgG2 remains unexplained, since both species were equally sensitive to reduction. Knowledge on the anomalous properties of IgG on SDS-PAGE is a prerequisite to run immunoblots from unreduced cellular antigens without confounding cell-associated IgG with cellular antigens.     

Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.     

Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells

Summary After purifying plasma membranes from isolated maize sperm cells by aqueous polymer two-phase partition, peripheral and integral proteins were solubilized from the plasma membrane with Triton X-114 and separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining revealed 10 bands (19–68 kDa) of peripheral membrane proteins and about 40 bands (12–120 kDa) of integral proteins. Peroxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51,48, 44, 36, and 32 kDa, and 6 integral polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands differed from those of somatic samples. Staining specificity was demonstrated by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peripheral 68 kDa polypeptide was further separated into 4 spots by isoelectric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membrane of maize sperm cells via the fluorescein isothiocyanate (FITC) technique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous labelling of the surface of isolated viable maize sperm cells with Con A-FITC.Abbreviations FITC fluorescein isothiocyanate - Con A Canavalia ensiformis agglutinin - HRP horseradish peroxidase - RCA Ricinus communis agglutinin - WGA Triticum vulgaris agglutinin     

Scale-associated glycoproteins of Scherffelia dubia (Chlorophyta) form high-molecular-weight complexes between the scale layers and the flagellar membrane

Flagellar scales were isolated from the flagellate green alga Scherffelia dubia. The flagellar scales consist mainly of acidic polysaccharides (70%) and glycoproteins (10%), and monosaccharide analyses show that the scales contain high amounts of unusual 2-keto-sugar acids. Approximately, 72 mol% of total carbohydrate is 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methylmanno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of at least 18 different scale-associated proteins (SAPs), ranging in apparent molecular mass from 77 kDa to over 300 kDa. Lectin blot analyses performed in combination with glycosidase treatment, showed that SAPs contained N-glycans of the highmannose type and the hybrid type, as well as a complex type that was not immunologically related to higher-plant complex glycans. Most of the SAPs were present in two or possibly three high-molecular-weight complexes. In these complexes, individual polypeptides are cross-linked by disulfide bridges. A polyclonal antibody was raised against a SAP of 126 kDa (SAP 126), a glycoprotein present in a high-molecular-weight complex. The SAP126 antibody was used to localize the protein between scale layer and flagellar membrane. We suggest that these high-molecular-weight complexes link scales to the flagellar membrane.Abbreviations AAA Aleuria aurantia agglutinin - DSA Datura stramonium agglutinin - DTT dithiothreitol - GNA Galanthus nivalis agglutinin - RCA Ricinus communis agglutinin - SAP Scale-associated protein - TBS Tris-buffered saline Dedicated to Professor Eberhard Schnepf on the occasion of his 65th birthday.This work was supported by the Deutsche Forschungsgemeinschaft and an Alexander von Humboldt Foundation research award to L. Perasso. We thank G. Noat for providing the anti--glucosidase and anti-pectin methyl esterase antibodies.     

Isolation and characterization of gastric microsomal glycoproteins. evidence for a glycosylated β-subunit of the H+/K−-ATPase

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

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Specific Lectin Binding to Beta1 Integrin and Fibronectin on the Apical Membrane of Madin-Darby Canine Kidney Cells

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.     

Characterization of the protein components of Nephila clavipes dragline silk

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.     

Wheat-germ-agglutinin and Ricinus communis-agglutinin-binding sites of BHK cells compared with each other and with 140 kDa fibronectin receptors.

We compared the wheat-germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) binding sites of baby-hamster kidney (BHK) cells. There were 1.01 X 10(8) WGA-binding sites per cell (Kd = 0.027 nM) and 6 X 10(6) RCA-binding sites per cell (Kd = 0.014 nM). Binding of WGA or RCA to BHK cells resulted in more than 75% of the cell-surface binding sites becoming associated with the cytoskeleton (i.e. resistant to extraction with detergent), although no more than 10% of these sites were associated with the cytoskeleton before addition of the lectins. After binding of WGA to the cells, the cell surface was cross-linked so extensively that it remained intact even after detergent extraction of the treated cells, and could be observed by electron microscopy. A similar cross-linking effect did not occur after binding of RCA to cells, which may be because there were so many more binding sites for WGA than for RCA. The composition of WGA- and RCA-binding molecules was analysed by lectin affinity chromatography of metabolically radiolabelled BHK cells. We found that in the WGA-binding-molecule preparations there were eight major polypeptides, ranging in molecular mass from 93 to 340 kDa, and that the RCA-binding molecules were a subpopulation of the WGA-binding molecules. A polyclonal antibody against the 140 kDa fibronectin (FN) receptors of Chinese-hamster ovary (CHO) cells immunoblotted a 145 kDa polypeptide component in both WGA- and RCA-binding-molecule preparations. The results indicated that the 145 kDa component was present in at least two FN-receptor complexes that differed in glycosylation, only one of which was able to bind to RCA affinity columns. The oligomeric nature of the FN-receptor complex, which contained three polypeptides with molecular masses of 120-145 kDa, was demonstrated by using anti-(CHO-cell FN receptor) antibodies to immunoprecipitate extracts prepared from radioiodinated BHK cells.     

Novel water stress protein from a desiccation-tolerant cyanobacterium. Purification and partial characterization

A desiccation-tolerant cyanobacterium Nostoc commune accumulates a novel group of acidic proteins when colonies are subjected to repeated cycles of drying and rehydration. The proteins occur in high concentrations; they have isoelectric points between 4.3 and 4.8 and apparent molecular masses between 30 and 39 kDa. The purification of three of these proteins with molecular masses of 33, 37, and 39 kDa is described. The amino-terminal sequence of the 39-kDa protein is Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu. Peptide mapping of the 39- and the 33-kDa proteins, using different protease, gave similar patterns of digestion fragments. The amino acid compositions of the proteins isolated were similar, and each cross-reacted with a polyclonal antibody raised against the largest (39-kDa) protein. The results indicate that the microheterogeneity observed was generated by in vivo proteolysis of the 39-kDa protein. It is suggested that this protein is a water stress protein with a protective function on a structural level.     

Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.     

The identification of membrane glycoconjugates in Leishmania species

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Preparation of Chitooligosaccharide-alditols by Hydrogenation with a Ruthenium Catalyst

The existence of glycosylated DNA-binding proteins was demonstrated in a whole cell extract from a filamentous fungus, Aspergillus oryzae. The proteins were specifically eluted from a DNA-cellulose column by the eluate containing shared double-stranded DNA and were detected by wheat germ agglutinin (WGA)-probing. The apparent molecular masses of these proteins on SDS-PAGE were 140 kDa, 115 kDa, 105kDa, 68 kDa, and 60 kDa. The labeling of the proteins by uridine 5′-diphosphate(UDP)-[¹⁴C]galactose using galactosyltransferase showed the same electrophoretic pattern with the WGA-probing. The [¹⁴C]- galactose-labeled saccharides were released from the proteins by mild-base treatment but not by N-glycopeptidase F digestion, indicating the O-glycosidic linkage of the saccharide chain attachment to proteins. The [¹⁴C]galactose-labeled saccharides co-migrated with galactose-(β1 → 4)-N-acetylglucosaminitoI on a silica gel plate. Thus, it was seen that several proteins which had the DNA-binding activity were modified by N-acetylglucosamine monosaccharide through an O-glycosidic linkage in A. oryzae.     

Mapping proteins from various structural regions of human kidney by two-dimensional electrophoresis]

The protein composition of various structural divisions of human kidney was studied using two-dimensional electrophoresis. Two-dimensional electrophoregrams of the cortical substance of human kidney revealed 165 polypeptide fractions within the pH range of 4.5-7.5, having molecular masses of 10 to 330 kDa. Electrophoresis of glomerular proteins gave 155 fractions with M(r) = 15-300 kDa, whereas fractionation of glomerular basement membrane proteins gave 40 fractions with M(r) = 30-330 kDa within the same range of pH. The M(r) values for all fractions and the relative electrophoretic mobility in the forward direction were determined. A comparative analysis of the electrophoregrams was conducted. The data obtained were used to construct two-dimensional maps of the cortical substance and glomerular proteins of human kidney.     

Purification of basic FGF receptors from rat brain

Receptor molecules for basic fibroblast growth factor (bFGF) were isolated from rat brain by a novel and rapid procedure and characterized. Purification was performed by wheatgerm agglutinin (WGA) gel affinity chromatography in combination with bFGF gel affinity chromatography, utilizing a novel elution method involving heparin. The eluted proteins were active in binding bFGF and were separated as two bands with respective molecular masses of 140 kDa and 110 kDa on SDS-PAGE. More than half of this bFGF-binding activity was lost after 16 h at 4 degrees C. Thus, bFGF receptors were purified as labile glycoconjugates.