Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion

Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mM-glucose, 500 mM-sucrose or 500 mM-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0.5%) and glucosamine (1.5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.     

Carbohydrate content of insoluble elastins prepared from adult bovine and calf ligamentum nuchae and tropoelastin isolated from copper-deficient procine aorta

1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13-0.35%, w/w) and amino sugars (0.01-0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.     

Physico-chemical and immunochemical characteristics of molecular forms of carcinoembryonic antigen (CEA) not bound with concanavalin A]

A molecular form of CEA non-binding Con A (CEAnC) was isolated from colon adenocarcinoma metastases in liver as a fraction of CEA having no affinity to Con-A-Sepharose. CEAnC was shown to be immunochemically identical to CEA, but to differ substantially with regard to the amino acid and sugar composition, and structure of the sugar moiety, possibly containing non only N-, but also O-glycosyl carbohydrate chains. The antigens studied were also found to possess different spatial structures. The differences between CEA and CEAnC suggest CEAnC to be a new molecular form of CEA.     

Identification of a common antigen of herpes simplex virus bovine herpes mammillitis virus, and B virus.

In immunoelectrophoretic analyses one common antigen was demonstrated in antigen preparations from herpes simplex virus types 1- and 2- (HSV-1 and HSV-2), bovine herpes mammillitis (BHM) virus-, and B virus-infected cells solubilized by Triton X-100. The antigen was also demonstrated in solubilized purified HSV-1 and BHM virus. The common antigen was identified as antigen 11 of HSV-1 or HSV-2. Differences were found in the polypeptide composition of the related antigens when isolated from the four different herpesviruses, but a glycopolypeptide with a molecular weight of 125,000 was present in each of the four different antigen preparations, indicating that this polypeptide carried the common antigenic determinants.     

Host-Pathogen Interactions: XIII. Extracellular Invertases Secreted by Three Races of a Plant Pathogen Are Glycoproteins Which Possess Different Carbohydrate Structures

The invertase present in the culture fluid of races 1, 2, and 3 of Phytophthora megasperma Drechs. var. sojae A. A. Hildebrand (Pms) were purified until they gave but a single band, whether stained for protein or carbohydrate, after isoelectric focusing in flat bed gels. The sugar compositions of multiple preparations of the purified invertases from each race of this fungal pathogen were determined by quantitative gas chromatography of their alditol acetates. The invertases are composed of about 25% carbohydrate. Mannose and glucosamine make up more than 97% of the carbohydrate portions of the invertases of all three Pms races analyzed, but the ratio of mannose to glucosamine is clearly not the same in each race. The glycosyl linkage compositions of the glucosamine-containing mannans of multiple preparations of the Pms invertases were determined by GC-MS analysis of the partially methylated alditol acetate derivatives. The results of these analyses demonstrate clear quantitative differences between the glycosyl components of the different Pms races. The existence of race-specific carbohydrate structures in the differentially virulent Pms races suggests that these carbohydrates may be involved in determining the specificity of hostpathogen interactions.     

Carbohydrate determinants associated with carcinoembryonic antigen (CEA)

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.     

Isolation and characterization of <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) singer extracellular lectins

Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 +/- 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 +/- 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.     

The apparent molecular weight of androgen-binding protein (ABP) in the blood of immature rats differs from that of ABP in the epididymis

When androgen-binding protein (ABP) in unfractionated immature (20-day old) male rat serum was covalently labeled with the site-specific photoaffinity ligand [3H]17 beta-hydroxy-4,6-androstadien-3-one and analyzed on 5.6% polyacrylamide tube gels containing SDS (SDS-PAGE), a protein of Mr 33,700 +/- 1200 was shown to be specifically labeled. Rat epididymal ABP from unfractionated cytosol analyzed under identical conditions exhibited two androgen-specific peaks of radioactivity, Mr 49,900 +/- 600 and Mr 44,100 +/- 800, which correspond to the previously described subunits of ABP. The apparent molecular weight differences between serum and epididymal ABP were further assessed on preparations of serum ABP that had been partially purified by chromatography on Affi-Gel blue (to remove albumin) and on Sephadex G-150 (to remove other proteins). When these preparations of ABP were photolabeled and analyzed by SDS-PAGE as above, two subunits of Mr 61,700 +/- 1300 and Mr 47,100 +/- 700 were resolved. Serum and epididymal ABP were further purified by androgen affinity chromatography. When these preparations were subjected to SDS-PAGE on slab gels containing 10% polyacrylamide and identified by fluorography of photolabeled ABP or by immunochemical localization following electrophoretic transfer to nitrocellulose, differences in the apparent molecular weight of ABP from the two sources persisted. Immunochemical localization studies on ABPs that had been desialylated with neuraminidase indicated that there was an increased mobility of the subunits, as one would anticipate from removal of carbohydrate. Differences in apparent molecular weight of ABPs from the two sources are likely due to differences in glycosylation.     

Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.     

Isolation of a novel metal-binding protein from rat testes. Characterization and distinction from metallothionein

This study was undertaken to further establish the nature of the low molecular weight metal-binding proteins in rat testes. In all cases, control testes were compared to livers of zinc-treated rats, which are known to contain high concentrations of metallothionein. Gel filtration of testicular and hepatic cytosol revealed a major zinc- and/or cadmium-binding protein in the low molecular weight range in both tissues. This protein could be partially purified from either source by a combination of heat treatment and sequential acetone precipitation. When such partially purified preparations were further fractionated by high performance liquid chromatography using a linear gradient of 25%-40% acetonitrile in 0.1% trifluoroacetic acid, two major forms with similar retention times were seen in each tissue. The utility of this high performance liquid chromatography system for separating isoforms of metallothionein was verified by separation of commercially available purified rabbit hepatic metallothionein into a total of five separate forms. Amino acid analysis of the two proteins derived from rat liver was consistent with the known amino acid composition of metallothionein. However, the two testicular forms separated by high performance liquid chromatography were notably different in amino acid composition from metallothionein, with a distinctly lower content of cysteine. These results indicate that the major low molecular weight cadmium/zinc-binding proteins in rat testes are not metallothioneins.     

Isolation and properties of the alpha-latrotoxin receptor.

The receptor protein of alpha-latrotoxin (alpha LTx, a neurotoxin with 'pure' presynaptic action isolated from black widow spider venom), was solubilized by Triton X-100 from bovine brain membranes and purified by affinity chromatography on alpha LTx-Sepharose. The purified receptor preparation contained four major polypeptides of molecular masses 200 (alpha), 160 (alpha'), 79 (beta) and 43 (gamma) kd according to SDS electrophoresis with molecular ratio alpha 1 alpha' 1 beta 2 gamma 2. The alpha- and alpha'-subunits are glycoproteins binding to wheat germ lectin and can be separated under non-denaturing conditions by anion exchange chromatography. Purified to homogeneity, both of them, though differing in the carbohydrate composition, retain the alpha LTx-binding activity and give closely related peptide maps. Anti-alpha antibodies recognize the alpha'-subunit as well. These results suggest that alpha LTx receptor is present in purified preparations in two very close forms containing the alpha- or alpha'-subunit. Beta and gamma proteins do not specifically bind alpha LTx and their physiological role is unclear. They form a complex with solubilized alpha- and alpha'-subunits independently of alpha LTx presence. The receptor proteins were purified to homogeneity by high performance gel filtration in the presence of SDS, their amino acid composition was determined.     

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.     

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

Localization of the antigenic determinants in carcinoembryonic antigen: the role of the carbohydrate component

The majority of topographic antigenic determinants of the carcinoembryonic antigen (CEA) representing a glycoprotein were shown to contain sugar residues of the CEA carbohydrate chains. Periodate oxidation of CEA followed by reduction afforded a corresponding CEA derivate (CEA-POR), which retained 3% antigenic activity of CEA. In secondary and tertiary structures CEA-POR was proved to be close to CEA pre heated to 80 degrees C. Formation of borate complexes with sugar residues of CEA decreased the CEA antigenic activity to 5% while a but did not affect the spatial structure of its protein core.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.     

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.     

Immunochemical studies of mammalian brain acetylcholinesterase.

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro-complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.     

Purification of catalytically active hepatic NADPH cytochrome P450 oxidoreductase from the rhesus monkey, Macaca mulatta.

Hepatic NADPH cytochrome P450 oxidoreductase capable of supporting polysubstrate monooxygenase (PSMO) reactions was purified from microsomes obtained from phenobarbitone (PB) pretreated rhesus monkey. Two preparations of the enzyme purified by affinity and molecular exclusion chromatographic techniques demonstrated specific content of 19.5 and 37.9 nmol cytochrome c reduced/min/mg protein and subunit molecular weight of 66 and 80 kDa, respectively. Both forms supported oxidation of NADPH and reduction of cytochrome c and DCIP but only 80 kDa preparation supported PSMO reactions. The reconstituted system consisted of hepatic P450, NADPH cytochrome P450 oxidoreductase, cytochrome b5 all purified from PB pretreated rhesus monkey and dilauroyl phosphatidylcholine or microsomal lipid. Eighty kDa preparation supported the metabolism of aminopyrine and tolbutamide by hepatic P4502C and erythromycin, ethylmorphine and nifedipine by hepatic P450 3A, respectively. The turnover of these substrates increased in the presence of partially purified cytochrome b5 from the rhesus monkey. To best of our knowledge this is the first report on the purification of monkey hepatic NADPH cytochrome P450 oxidoreductase capable of supporting in vitro PSMO by different isozymes of P450.