Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were alpha-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr>25000). Over a 2h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional 'holes' of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and nonconfluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional 'holes' of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Summary We have previously shown that inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London) 306:67–69). This observation suggests that IP₃ might provide the missing link between activation of the muscarinic receptor and Ca²⁺ release from intracellular stores during stimulation. In order to localize the intracellular IP₃-sensitive calcium pool, IP₃-induced Ca²⁺ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl₂. IP₃-induced Ca²⁺ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl₂ precipitation there was a close correlation of IP₃-induced Ca²⁺ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=–0.64) and glutamate dehydrogenase (r=–0.75) and with the plasma membrane markers (Na⁺+K⁺)-ATPase (r=–0.81) and alkaline phosphatase (r=–0.77) in all fractions analyzed. IP₃-induced Ca²⁺ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca²⁺ from endoplasmic reticulum in pancreatic acinar cells.     

Affinity labeling of high-affinity α-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). ¹²⁵I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (K_d ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling ¹²⁵I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of M_r ≈ 150 000 revealed as a ¹²⁵I-α-thrombin cross-linked complex of M_r ≈ 180 000. Independent of chemical cross-linking, ¹²⁵I-α-thrombin also formed a covalent complex with a minor, 35 000 M_r, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of M_r 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 M_r binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta

Laminins assemble into trimers composed of α, β, and γ chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin α1 and α5 chains. Comparative pulse–chase experiments and deglycosylation studies in JAR cells established that the M_r 360,000 laminin α1 chain is glycosylated into a mature M_r 400,000 band while the M_r 370,000 laminin α5 chain is glycosylated into a M_r 390,000 form that upon secretion is further processed into a M_r 380,000 form. Hence, despite the shorter peptide length of α1 chain in comparison with the α5 chain, secreted α1 assumes a larger size in SDS–PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin α1 and laminin α5 chains in laminin-1 and laminin-10. In placenta laminin α1 chain (M_r 400,000) and laminin α5 chain (M_r 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin α1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin β2 chains. Surprisingly, a fraction of the laminin α1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin β1–β3 chains, possibly pointing to an unexpected complexity in the chain composition of α1-containing laminin isoforms.     

Prompt Protein Glycosylation during Acute Heat Stress

Constitutive patterns of protein synthesis and protein glycosylation are severely disrupted by acute heat stress. Stressed cells respond by preferential synthesis of specific proteins, e.g., the well-known family of heat shock proteins. We observed another response that rapidly occurs during heating periods as short as 10 min at 45°C. During that period, CHO cells began to glycosylate specific proteins, designated as "prompt" stress glycoproteins (P-SG), while constitutive protein glycosylation ceased. Labeling of P-SGs showed a dose response with time and with temperature and appeared regardless of the label used (D-[³H]mannose or D-[³H]-glucose). On SDS-PAGE, the major P-SG was characterized by M_r ≈ 67 kDa (P-SG67) and pI = 5.1. Other less prominent P-SGs appeared at M_r 160, 100, 64, 60, and 47 kDa; incorporated label showed little turnover during 24 h at 37°C. Prompt glycosylation was inhibited by tunicamycin, and label incorporated into P-SGs was sensitive to N -glycosidase F, but not to O-glycosidase. Analysis of enzymatically digested P-SG67 indicated that label had been incorporated into both high-mannose (Man₉G1cNAc) and complex-type oligosaccharides. Brefeldin A did not eliminate P-SG67 labeling, but caused the further appearance of novel, Brefeldin-associated P-SGs. Labeling of P-SG67 oligosaccharides occurred without significant concomitant protein synthesis, suggesting that addition of labeled oligosaccharides largely occurred on mature, rather than nascent proteins. The functional significance of prompt glycosylation remains to be defined, but we propose that this novel phenomenon is an integral part of the cellular heat stress response.     

Atmospheric deposition and solute export in giant sequoia — mixed conifer watersheds in the Sierra Nevada,California

Atmospheric depostion and stream discharge and solutes were measured for three years (September 1984 — August 1987) in two mixed conifer watersheds in Sequoia National Park, in the southern Sierra Nevada of California. The Log Creek watershed (50 ha, 2067–2397 m elev.) is drained by a perennial stream, while Tharp's Creek watershed (13 ha, 2067–2255 m elev.) contains an intermittent stream. Dominant trees in the area include Abies concolor (white fir), Sequoiadendron giganteum (giant sequoia), A. magnifica (red fir), and Pinus lambertiana (sugar pine). Bedrock is predominantly granite and granodiorite, and the soils are mostly Pachic Xerumbrepts. Over the three year period, sulfate (SO₄ ^2–), nitrate (NO₃ ^–), and chloride (Cl^–1) were the major anions in bulk precipitation with volume-weighted average concentrations of 12.6, 12.3 and 10.0 eq/1, respectively. Annual inputs of NO₃-N, NH₄-N and SO₄-S from wet deposition were about 60 to 75% of those reported from bulk deposition collectors. Discharge from the two watersheds occurs primarily during spring snowmelt. Solute exports from Log and Tharp's Creeks were dominated by HCO₃ ^–, Ca²⁺ and Na⁺, while H⁺, NO₃ ^–, NH₄ ⁺ and PO₄ ^3– outputs were relatively small. Solute concentrations were weakly correlated with instantaneous stream flow for all solutes (r² <0.2) except HCO₃ ^– (Log Cr. r² = 0.72; Tharp's Cr. r² = 0.38), Na⁺ (Log Cr. r² = 0.56; Tharp's Cr. r² = 0.47), and silicate (Log Cr. r² = 0.71; Tharp's Cr. r² = 0.49). Mean annual atmospheric contributions of NO₃-N (1.6 kg ha^–1), NH₄-N (1.7 kg ha^–1), and SO₄-S (1.8 kg ha^–1), which are associated with acidic deposition, greatly exceed hydrologic losses. Annual watershed yields (expressed as eq ha^–1) of HCO₃ ^– exceeded by factors of 2.5 to 37 the annual atmospheric deposition of H⁺.     

Application of 2-aminopyridine fluorescence labeling in the analysis of in vivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography

The present study describes a size-exclusion high-performance liquid chromatographic method for the separation and quantification of sulfated polysaccharides, such as dextran sulfate sodium (DSS). Pyridylamination of DSS was achieved without difficulty using 2-aminopyridine as a fluorometric label. In addition, 0.1–0.2 M phosphate buffer (pH 3.0) was found to be the mobile phase which produced the best separation. In vitro enzymatic degradation of the pyridylamino-DSS (PA-DSS₅₀₀₀, M_r 5000) using α-amylase and the in vivo metabolism in the rat feces after oral administration of PA-DSS₅₀₀₀ were then evaluated. Two small peaks of approximately M_r 380 and 600 appeared after co-incubation with α-amylase, indicating PA-DSS₅₀₀₀ may be considerably depolymerized. In vivo, however, PA-DSS₅₀₀₀ excreted in the feces was mainly of PA-DSS₅₀₀₀ polymer. No peaks of less than M_r 5000 were not clearly detectable in the feces because of background fluorescence attributable to gut lumen contents. This method of fluorometric analysis allows fairly selective detection of sulfated polysaccharides in biological materials.     

Effect of iron and other nutrient limitations on the pattern of outer membrane proteins in the cyanobacterium Synechococcus PCC7942

When cells of Synechococcus PCC7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of M _r 92000, 48000–50000 and 35000 appeared. Specific iron-limited outer membrane proteins (IRMPs) of M _r 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC6308. Under magnesium limitation polypeptides of M _r 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of M _r 32000 and one of over 100000 being induced, whereas calcium limitation had no apparent affect.Abbreviations EDDA ethylenediaminedihydroxyphenyl acetic acid - IRMP iron-regulated outer membrane protein - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride     

Comparison of molecular properties of rat plasma and erythrocyte selenium-dependent glutathion peroxidase

The molecular structure of plasma and erythrocyte selenium-dependent glutatione peroxidase (GSH-Px) was studied in rats drinking water containing [⁷⁵Se]selenious acid, 1.3 mg Se/L. Substantial differences were found using three-step fractionation, including gel filtration of crude plasma and erythrocyte lysate, gel filtration of⁷⁵Se-GSH-Px treated by mercaptoethanol, and SDS-electrophoresis. Native plasma⁷⁵Se-GSH-Px, which exhibited a molecular weight (M _r) of approx 700,000, could be destroyed by mercaptoethanol action, resulting in disintegration of enzyme into several different⁷⁵Se-protein fragments and release of part of low-mol-wt⁷⁵Se. Native erythrocyte⁷⁵Se-GSH-PxM _r, value was found to be 113,000; two⁷⁵Se-protein fragments arose after mercaptoethanol treatment without⁷⁵Se release from the enzyme. The⁷⁵Se-subunits of 22,500 and 21,900 were isolated from plasma and erythrocyte⁷⁵Se-GSH-Px, respectively. Another minor⁷⁵Se-GSH-Px was identified in erythrocyte lysate (M _r, 214,000, subunit 22,100), which was considered to be a dimer of the above-mentioned erythrocyte enzyme. It can be assumed, based on these data, that native plasma GSH-Px, in contrast to erythrocyte enzyme, represents a high-molecular wt complex composed of several tetramers linked with S—S bonds. A certain part of selenium present in this complex is probably not selenocysteine and may be released with the mercaptoethanol treatment.     

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent M_r is 250 000 by gel-filtration chromatography, and 225 000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent M_r is 140 000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10⁻⁶ M, as measured by fluorimetric titration.     

Wheat-germ agglutinin is synthesized as a glycosylated precursor

The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [³⁵S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with M_r 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of M_r 23 000 and the mature monomer of M_r 18000. When [³H]mannose was used in labeling studies, only the polypeptide of M_r 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of M_r 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Testosterone uptake by membrane vesicles of Pseudomonas testosteroni

Uptake of testosterone was demonstrated in membrane vesicles prepared from Pseudomonas testosteroni grown on testosterone. In contrast, membrane vesicles from uninduced cultures revealed no significant transport activity for steroids. The K_m of the reaction was 2 · 10⁻⁶M and the V 28.5 nmoles/min per mg protein. Steroid uptake was maximal within the pH range of 8 to 9 and at incubation temperatures between 30 and 37 °C. Transport of steroid was dependent upon NAD⁺ and was reduced by NADH, dinitrophenol, and inhibitors of electron transport, such as N_3⁻ · CN⁻ and amytal. The intravesicular steroid concentration was approx. 800 times the steroid concentration present in the medium at the start of the incubation.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Biochemical and physiological properties of a protein inducing protoplast release during conjugation in theClosterium peracerosum-strigosum-littorale complex

When mating-type minus (mt⁻) and plus (mt⁺) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt⁻ cells also proceeded without pairing in a medium in which mt⁻ and mt⁺ cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (M_r) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of M_rs 42000 and 19000, while the deglycosylated polypeptides had M_rs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·10⁹ and the concentration required for 50% of the maximum response (ED₅₀) as 4.1·10⁻⁹M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt⁻ cells of thisClosterium complex.     

The immunochemistry of neisserial LOS

The outer membrane glycolipids of Neisseria lack long polysaccharides and are properly termed lipooligosaccharides (LOS). A Neisseria strain makes from two to six LOS of M_r 3150–7100. Different species commonly make LOS of identical M_r and epitope content. Oligosaccharide (OS) differences account for physical heterogeneity. OS consist of a conserved triantenary basal oligosaccharide, two linear segments of (n) hexose residues that determine OS mass, and terminal sequences similar to those of glycosphyngolipids. Epitope expression is linked to physical heterogeneity and conditioned by the molecular environment of the outer membrane. Serotype epitopes are expressed on M_r-restricted LOS. LOS regulate complement activation onto the bacterial surface and, hence, immune lysis.     

Depolymerization of hyaluronan by sonication

High molecular weight hyaluronan (M _r 400 000) obtained from human umbilical cord was depolymerized by sonication for 10 h into small molecules and finally into molecules of constant size (M _r 11 000). The molecular size of the depolymerized hyaluronan was unaltered even under different conditions of sonication. After sonication, the main sugar residues at the reducing and non-reducing termini of depolymerized hyaluronan wereN-acetylglucosamine (86%) and glucuronic acid (98%), respectively. Hyaluronans derived from rooster comb (M _r 1×10⁶) andStreptococcus zooepidemicus (M _r 1.2×10⁶) were deploymerized into molecules of different but characteristic sizes by sonication. On the other hand, neither chondroitin sulfate nor glycogen was depolymerized by sonication. These results suggest that high molecular weight hyaluronan may have some weak linkages related toN-acetylglucosamine in the chain, which are extremely sensitive to sonication. At present, however, the nature of these linkages is still unclear.Abbreviations HA hyaluronan - PA 2-aminopyridine