Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases, and by the administration of Tunicamycin

Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunica-mycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).     

Light and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: Evidence for processing of the secretory material

Summary The subcommissural organ (SCO) of the rat was investigated by use of histochemical and immunocytochemical methods at the light and electron-microscopic levels. Consecutive thin methacrylate sections were stained with the pseudoisocyanin (Psi), immunoperoxidase (IMC; employing an antiserum against Reissner's fiber, AFRU), periodic acid-Schiff (PAS) and periodic acid-silver methenamine (SM) techniques, and reacted with six types of lectins. Psi, SM, concanavalin A (Con A) and IMC were also used for double and triple sequential staining of the same section. Increasing dilutions of AFRU (from 11000 to 1200 000) were used for immunostaining of serial paraffin sections. In addition, ultrastructural localization of (i) Con A-binding sites and (ii) immunoreactive secretory material was performed. Some of these procedures were also applied to the ophidian and canine SCO.Con A-positive, Psi-positive and immunoreactive materials coexisted within the same cisternae of the rough endoplasmic reticulum. The Golgi apparatus lacked Con A-positive and immunoreactive substances. Apical secretory granules and secreted material lying on the surface of the SCO showed (i) the highest affinity for AFRU, but were (ii) Con A-negative, and (iii) wheat-germ agglutinin-, PAS and SM-positive. Reissner's fiber displayed a low affinity for AFRU.It is suggested that the SCO secretes N-linked glycoproteins, the carbohydrate and protein moeities of which undergo (i) a maturation process before being released, and (ii) some kind of modification(s) after their release into the ventricle. The perivascular secretory cells of the dog SCO might secrete a material different from that secreted by the ependymal cells.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Direction de Investigaciones, Universidad Austral de Chile. The authors wish to thank Mrs. Elizabeth Santibáñez, Mr. Genaro Alvial and Mr. Luis Delannoy (Valdivia), and Mrs. Ragnhild Momberger (Giessen) for valuable technical cooperation     

Differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture

To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of ₁-protease inhibitor, ceruloplasmin, and ₂-macroglobulin, but had no effect on the export of fibronectin, -fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized ₁-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized ₁-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on ₁-protease inhibitor, ceruloplasmin and ₂-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.     

Binding interactions of glycoproteins with lectins

Summary Since plant lectins were used to help define differences between normal and transformed cell surfaces (reviewed in References^1–4), they have been employed in many other situations where their sugar-recognition specificities could be used to advantage. One of these applications has been the purification and characterization of enzymes and other proteins; this work is reviewed here in order to define some of the variables that affect binding of glycoproteins to lectins, as well as to demonstrate how this technique has been profitably exploited for isolation of purified glycoproteins, and for their better understanding.Abbreviations ConA concanavalin A - WGA wheat germ agglutinin - RCA₆₀ (RCA_II, mol. wt. 60,000) and RCA₁₂₀ (RCA, mol. wt. 120,000) Ricinus communis agglutinin - LCA Lens culinaris agglutinin - LTA Lotos tetragonolobus agglutinin - SBA soybean agglutinin - PNA peanut agglutinin - Me--Glc methyl--glucoside - Me--Man methyl--mannoside - Gal galactose - GlcNac N-acetylglucosamine     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

Summary The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested, Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Linkage studies on α-glycerophosphate and isocitrate dehydrogenases in Aedes albopictus (diptera: Culicidae)

Linkage studies were carried out on -glycerophosphate dehydrogenase (-GPDH) and isocitrate dehydrogenase (IDH) in the mosquito Aedes (Stegomyia) albopictus. Only one locus coding for -GPDH was revealed on agar gels by applying adult homogenates. Two loci for IDH were observed using either fourth-instar larvae, pupae, or adults. This study was restricted to the more anodal Idh-2 of the two loci, and -Gpdh. Both -Gpdh and Idh-2 encode dimeric enzymes. Thirteen backcrosses indicated that the -Gpdh and Idh-2 loci are arranged in linkage group 2 in the following order: p (pigmented pupa)—(ca. 2 map units)—Wb (white-body)—(7.5–17.8)—Idh-2—(13.1)—-Gpdh. Females exhibited more recombination than males.This work was supported by a Grant-in-Aid for Co-operative Research from the Ministry of Education, Japan.     

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries)

Summary Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called interstitial cells or supporting cells. In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.     

Homology of a 130-kb region enclosing the α-globin gene cluster,the α-locus controlling region,and two non-globin genes in human and mouse

The human -globin gene cluster (30 kb) is embedded in a GC-rich isochore very close to the telomere of Chromosome (Chr) 16p. The -Locus Controlling Region (-LCR) is located upstream of the adult -globin genes and has been shown to be essential for their expression. In this study we have been looking for expressed genes in the region upstream of the -globin cluster to understand the role of the LCR-like element in the expression and replication timing of flanking gene clusters. We show that the upstream -globin region is conserved over a 75-kb range and includes at least two oppositely transcribed non-globin genes, here referred to as Mid1 and Dist1. Complementary DNA sequences of 250 bp and 2.5 kb from Mid1 (coordinate-68) and Dist1 (coordinate-90 to-99), respectively, were isolated from human and mouse. The deduced partial amino acid sequences of these cDNAs are 81% and 95% identical for the Mid1 and Dist1 gene respectively. We have cloned a mouse cosmid contig which includes Dist1, Mid1, and the entire murine -globin cluster. The murine homolog of the -LCR was mapped upstream of the mouse globin genes at approximately the same position as in the human locus. Our results indicate that, in mouse and human, the -globin loci and their flanking sequences are homologous over a range of at least 130 kb. The structural homology of this region in both mammals suggests also a functional one and indicates the mouse as a potential model for studying the role of the -LCR controlling element in the regulation of expression and replication timing of the flanking gene clusters.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers M99623, M99624, M99625, and M99626.     

Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections

Summary The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)-and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as post-Golgi elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Major organ-specific glycoproteins in isolated brain and kidney membranes identified as Na,K-ATPase subunits by combined glycan-, lectin-, and immunoblotting

In the present work combined glycan-, lectin-, and immunoblotting of isolated brain and kidney membranes shows that the and subunits of Na,K-ATPase are the most abundant glycoproteins. Further,Datura stramonium andGalanthus nivalis agglutinins recognize the Na,K-ATPase subunits in a mutually exclusive manner in membranes from human, rabbit and rat brain or human, rabbit, rat, pig and dog kidney indicating the presence of species-independent organ-typical glycoforms. The glycosylation status is not related to the ouabain-sensitivity. Taken together, the data reveals organ-specific glycoforms of Na,K-ATPase which might have roles for organ identification and recognition.Abbreviations NKA Na,K-ATPase (EC 3.6.1.37) - PAGE polyacrylamide gel electrophoresis in dodecylsulfate - Con-A Concanavalin A - DSA Datura stramonium agglutinin - GNA Galanthus nivalis agglutinin - MAA Maackia amurensis agglutinin - PNA Peanut agglutinin - SNA Sambucus nigra agglutinin - WGA Wheat germ agglutinin Abbreviations used in figures K kidney - B brain - Cr Crude - De Detergent-treated - Fe fetuin - Ct creatinase - I-blot immuno-blot - L-blot lectin-blot     

The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.     

Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins

Glycoproteins (gps) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. They are present on the cell surface and function as receptors in various life processes. Many exist in soluble or gel form and serve as biological lubricants or as barriers against microbial invasion. During the past two decades, eleven mammalian structural units have been used to express the binding domain of applied lectins. They are:F, GalNAc1 3GalNAc;A, GalNAc1 3Gal;T, Gal1 3GalNAc;I, Gal1 3GlcNAc;II, Gal1 4GlcNAc;B, Gal1 3Gal;E, Gal1 4Gal;L, Gal1 4Glc;P, GalNAc1 3Gal;S, GalNAc1 4Gal andTn, GalNAc1 Ser(Thr). ExceptL andP, all of the units can be found in glycoproteins.Tn, which is an important marker for breast/colon cancer and vaccine development, exists only inO-glycans. NaturalTn gp, the simplest mammalianO-glycan, is exclusively expressed in the armadillo salivary gland. Antifreeze gp is composed of repeating units ofT.Pneumococcus type XIV capsular polysaccharide has uniformII disaccharide as carbohydrate side chains. Asialo human ₁-acid gp and asialo fetuin provide multi-antennaryII structures. Human ovarian cyst gps, which belong to the complex type of glycoform, comprise most of the structural units. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding properties of Gal/GalNAc reactive lectins have been classified according to their highest affinity for structural units and their binding profiles are expressed in decreasing order of reactivity. Hence, the binding relationship between glycoproteins and Gal/GalNAc specific lectins can be explored.     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.     

Reversible cross-linking of crystalline bacterial surface layer glycoproteins through their glycan chains

After periodate oxidation and incubation with dithiodipropionic acid dihydrazide cross-linking of the crystalline surface layer (S-layer) glycoproteins of Clostridium thermohydrosulfuricum L111-69 and Bacillus alvei CCM 2051 was achieved specifically through the glycan chains. The cross-linked S-layers were used for the immobilization of chemically synthesized, spacer-linked, tumour-associated T-disaccharide [Gal(13)GalNAc]. Electron microscopical evaluation of the resulting conjugates showed densely packed, multilayered S-layer structures loaded with the immobilized ligand. After reductive cleavage of the disulphide bond of dithiodipropionic acid by dithiothreitol, monomeric haptenated S-layer conjugates could be obtained. Both the cross-linked and the monomeric type of conjugate might be useful for assessment of specific immune responses, which, in general, can be elicited by those artificial antigens. Correspondence to: P. Messner     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Comparison of several parameters related to the secretory activity of the subcommissural organ in European green frogs

Summary In European green frogs the secretory activity of the subcommissural organ (SCO) was investigated and quantified measuring three parameters considered to be closely related to the cellular processes of synthesis and release of secretory material by the cells of the SCO: (1) the amount of stained secretory material in the SCO; (2) the amount of secretory material in the SCO labelled by a radioactive precursor; and (3) the growth rate of the liquor (cerebrospinalis) fibre (LF). A significant negative linear correlation appears to exist between the growth rate of the LF, on the one hand, and the amount of stained secretory material as well as the amount of radioactively labelled secretory material, on the other hand. A significant positive linear correlation exists between the amounts of stained material and radioactively labelled secretory material. The occurrence in the SCO of European green frogs of a larger amount of stained and/or of radioactively labelled secretory material is probably an expression of a lower (LF-producing) secretory activity. In the light of these observations the suitability of the three parameters as a measure of the secretory activity of the SCO is discussed.