Biosynthesis of gastric mucus glycoprotein of the rat

We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.     

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone.

We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.     

Self-recognition of N-linked glycans with multivalent GlcNAc, determined as ceramide mimetic conjugate

Aminoceramide mimetic was synthesized and conjugated to N-linked oligosaccharides having multivalent GlcNAc by reductive amination. Ceramide mimetic conjugates with "complex-type" glycan having five or six GlcNAc termini (termed Os Fr. B-Cer) were purified, analyzed by thin-layer chromatography (TLC), and finally characterized by MS/MS analysis through liquid chromatography/mass spectrometry. Binding of Os Fr. B-Cer placed on solid phase polystyrene surface with [3H]cholesterol-labeled liposomes containing Os Fr. B-Cer, or containing various glycosphingolipids (GSLs) was determined. The binding of Os Fr. B-Cer liposomes to Os Fr. B-Cer coated plate was significantly higher than binding of GM3 liposomes. Other GSL liposomes showed no binding. Thus, self-recognition of Os Fr. B-Cer was clearly demonstrated using ceramide mimetic conjugates.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Use of active site-directed inhibitors to study in situ degradation of glycoproteins by the perfused rat liver

Use was made of the asialoglycoprotein receptor system in a perfused rat liver in order to study lysosomal degradation and subsequent metabolism of radioactive derivatives of asialo-ovine submaxillary mucin and asialo-alpha 1-acid glycoprotein. A trace of N-acetyl-D-[6-3H]galactosamine-labeled asialo-ovine submaxillary (4 micrograms) was completely taken up by the tissue in less than 20 min. After 3 h 24% of the radioactivity from the mucin reappeared on newly synthesized serum glycoproteins that were secreted into the perfusate. [6-3H] Galactose asialo-alpha 1-acid glycoprotein was also rapidly cleared by the liver; however, after 3 h greater than 60% of the radioactivity derived from this sugar labeled glycoprotein was secreted back into the perfusate as [3H]glucose. Rat livers perfused with 0.15 mM beta-D-galactopyranosylmethyl-p-nitrophenyltriazene lost 90% of their beta-D-galactosidase activity within 1 h while other representative glycosidases showed no change as followed by hydrolysis of p-nitrophenylglycosides. Livers pretreated with this triazene compound metabolized [3H]GalNAc asialo-ovine submaxillary mucin normally but were unable to process [3H]Gal asialo-alpha 1-acid glycoprotein as evidenced by a complete inhibition of [3H]glucose release following addition of the latter substrate. Metabolism of N-acetyl[14C]glucosamine asialo-alpha 1-acid glycoprotein was similarly inhibited by 70%. 125I-labeled asialo-alpha 1-acid glycoprotein catabolism was not affected by the chemically induced beta-D-galactosidase deficiency. Subcellular fractionation of inhibitor-treated livers accumulating radioactive carbohydrate showed the majority of the label was associated with a fraction enriched in lysosomes. Analysis of the trapped radioactivity by high resolution Bio-Gel P-4 chromatography revealed nearly intact oligosaccharides minus only the reducing N-acetylglucosamine of the chitobiose core. Direct comparison of these sugar chains with those isolated from human and canine GM1 gangliosidosis liver by silicic acid thin layer chromatography showed those isolated from rat liver to be identical to the major subset of oligosaccharides found in the human disease. In similar experiments in which the galactosyl triazene was replaced by swainsonine, an alpha-D-mannosidase inhibitor, catabolism of [14C]GlcNAc asialo-alpha 1-acid glycoprotein resulted in the accumulation of a single oligosaccharide of the structure. Man3[14C]GlcNAc1. These results demonstrate an endo-N-acetyl-beta-D-glucosaminidase is active in rat liver lysosomes.     

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.     

Oligosaccharide structures of human colonic mucin

Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.     

Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type 1.

Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide.     

Presence of covalently attached fatty acids in rat apolipoprotein B via thiolester linkages

Thiolester linked lipids in rat apolipoprotein B (ApoB) were examined by incubating reduced and carboxymethylated ApoB in 6 M urea buffer with [14C]methylamine at pH 8.5, 30 degrees C. It was observed that [14C]methylamine was covalently incorporated into ApoB, and there was a [14C]methylamine modified product which was lipid in nature. After extraction with organic solvents, the [14C]methylamine labeled product showed its Rf on TLC to be similar to that of the synthetic N-methyl fatty acyl amide. After purification on TLC and transesterification with 3 N methanolic HC1, methyl esters of C16:0, C18:0 and C18:1 fatty acids were identified by gas-liquid chromatography. These results suggest that rat ApoB, similar to human ApoB, contained covalently linked fatty acids through the high energy, labile thiolester bonds.     

Thin-layer chromatography-densitometry of minor acidic phospholipids: application to lipids from erythrocytes, liver, and kidney

A method for quantitative determination of acidic phospholipids by thin-layer chromatography (TLC) followed by densitometry is described. The total lipids were separated into neutral and acidic fractions by DEAE-Sephadex column chromatography. A clear-cut separation of acidic phospholipids was achieved by high-performance TLC with a solvent system of chloroform/acetone/acetic acid/formic acid/water (60/60/4/10/3). Each phospholipid band was quantitated by densitometry with the use of an internal standard. The lipid compositions of sheep and mouse erythrocytes and of rat liver and kidney were determined by the present method.     

Identification of the post-translational modifications of the core-specific lectin. The core-specific lectin contains hydroxyproline, hydroxylysine, and glucosylgalactosylhydroxylysine residues

The core-specific lectin (CSL) synthesized and secreted by rat hepatocytes and the rat hepatoma H-4-II-E shows affinity for mannose and N-acetylglucosamine residues in the "core" region of asparagine-linked oligosaccharides. The CSL undergoes two stages of post-translational modification which result in an increase in its Mr from 24,000 to 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have determined that the lectin undergoes hydroxylation of proline and lysine and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GlcGalHyLys). CSL metabolically labeled with [3H]lysine and [3H]proline contains hydroxylated forms of proline and lysine. The mature form of the lectin can also be metabolically labeled with [3H]galactose. alpha,alpha'-Dipyridyl, an inhibitor of collagen prolyl and lysyl hydroxylases, prevents the metabolic incorporation of [3H]galactose and the post-translational increases in the Mr of the CSL, indicating that both events are dependent upon hydroxylation of proline and lysine. Virtually all of the hydroxylysine present in the CSL is recovered as glucosylgalactosylhydroxylysine after alkaline hydrolysis. The post-translational modifications of the CSL place it in a select family of secreted proteins which contain collagen-like sequences, including the pulmonary surfactant proteins, complement component C1q, and the 18 S asymmetric form of acetylcholinesterase.     

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate.

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the mucin core protein by cell-free translation of messenger RNA from bovine submaxillary glands

Bovine submaxillary mucin was purified and subjected to chemical deglycosylation by treatment at 20 degrees C with either anhydrous hydrogen fluoride or trifluoromethane sulfonic acid. Virtually all of the sialic acid, galactose, fucose, and over 90% of the N-acetylhexosamines were removed by these treatments. The amino acid compositions of the deglycosylated and native mucins were similar indicating that chemical deglycosylation did not cause significant degradation of the protein. Antiserum specific for the deglycosylated bovine submaxillary mucin was produced by immunization of rabbits with the deglycosylated mucin. RNA was isolated from bovine submaxillary glands by extraction with guanidine hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ mRNA. The poly(A)+ mRNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine, [3H]leucine, [3H]threonine, [3H]proline, or [3H]serine as radiolabel and the translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum. A labeled product of molecular weight 60,000 was present in the immunoprecipitates obtained in the absence but not in the presence of the unlabeled competitor deglycosylated mucin. It is concluded that the primary translation product of the bovine submaxillary gland gene is a 60,000-dalton protein and that the monomer subunit of the mucin is about 170,000. Thus, in the native state the mucin consists of several self-associating subunits.     

The mucin biosynthesis stimulated by epidermal growth factor occurs in surface mucus cells, but not in gland mucus cells, of rat stomach

Although epidermal growth factor (EGF) accelerates gastric mucin biosynthesis, information on whether its activation is limited to the specific mucus-producing cells is lacking. In this paper, we investigated the effects of EGF on mucin biosynthesis and the expression of its receptor in distinct layers of rat gastric mucosa, including the possible participation of nitric oxide (NO). EGF enhanced the incorporation of [3H]glucosamine and [14C]threonine into the mucin in the full-thickness tissues of the gastric mucosa. This stimulation disappeared on the removal treatment of the surface mucosal layer chiefly consisting of surface mucus cells. The EGF-induced increase in [3H]-labeled mucin in the full-thickness mucosa was not suppressed by either NG-nitro-L-arginine (10(-5) M) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (10(-5) M). The EGF-receptor-mRNA expression was high in the surface mucosal layer but low in the deep and muscle layers of the stomach. These results suggest that EGF-induced stimulation of mucin biosynthesis is limited to the surface mucus cells of the rat gastric mucosa and is independent of the NO pathway.     

Further investigations into the structure of human gastric mucin: the structural configuration of the oligosaccharide chains

Human gastric mucin from aspirates has been degraded by proteolysis to afford a soluble glycopolypeptide which, when purified by gel filtration on Bio-Gel P 150 and chromatography on Ecteola cellulose, was essentially free from mannose. Structural information on the oligosaccharide chains attached to the polypeptide core was obtained by mild hydrolysis with acid, Smith degradation, and degradation with sodium phosphate-borohydride, both before and after the removal of fucose by controlled, acid hydrolysis. A number of oligosaccharides were isolated and purified by gel and paper chromatography. Minimal degradation due to base-catalysed peeling reactions was experienced with the sodium phosphate-borohydride technique. The oligosaccharides have been characterised and structures proposed for the various side-chains present in human gastric mucin.     

A synthesis of tritium-labeled juvenile hormone and radiometric analysis of the enzymatic hydrolysis level

A method of insect juvenile hormone III (JH-III) labeling by thermally activated tritium was developed. Purification of the labeled compound was performed on a TLC silica gel plate using a system of hexane-ethyl acetate 2: 1. The molar radioactivity of [³H]JH-III was 2.2 Ci mmol^–1. It was shown that tritium-labeled juvenile hormone can be used in the radiometric method for quantitative measurement of the activity of specific enzymes that hydrolyze juvenile hormone.