Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase.

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetyl-galactosamine 4-sulfate. Fractions, elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.

In vitro fatty acid acylation of mucus glycoprotein from sublingual salivary glands.

The enzyme activity which catalyzes the transfer of palmitic acid from palmitoyl-coenzyme A to sublingual gland mucus glycoprotein has been demonstrated in the detergent extracts of the microsomal fraction of rat sublingual and parotid salivary glands. The acyltransferase activity of this fraction was similar in both types of glands. Further subcellular fractionation performed on sublingual glands revealed that the enzyme is associated with the Golgi-rich membrane fraction. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained using 0.5% Triton X-100, 2 mM dithiothreitol, 25 mM NaF, and 10 mM MgCl2 at a pH of 7.4. Higher concentrations of NaF, MgCl2 and dithiothreitol, however, were inhibitory. The apparent Km of the sublingual glands microsomal enzyme for mucus glycoprotein was 0.55 mg/ml and for palmitoyl-CoA, 3.5 X 10(-5) M. A 15% decrease in the acyltransferase activity was obtained with the reduced and alkylated mucus glycoprotein and it showed no activity towards the proteolytically degraded glycoprotein. The 14C-labeled product of the enzyme reaction gave in CsCl density gradient a band at the density of 1.49 in which the 14C label coincided with the glycoprotein. The 14C label in this glycoprotein was susceptible to deacylation with hydroxylamine, and the released labeled material was identified as palmitate.

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid.

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.

Fatty acid acylation of mucin by gastric mucosa: effects of sofalcone and sucralfate.

The effects of antiulcer drugs, sofalcone and sucralfate, on the activity of gastric mucosal mucus glycoprotein fatty acyltransferase were investigated. The acyltransferase enzyme, contained in the detergent extracts of the microsomal fraction of rat gastric mucosa, was incubated with the deacylated gastric mucin and palmitoyl-CoA substrates in the presence and absence of drugs, and the formed fatty acid acylated glycoprotein product was quantitated. In the absence of drugs, the enzymatic activity increased proportionally with increased concentrations of both substrates and of enzyme, and gave an apparent Km value of 5.6 X 10(-7) M. Introduction of sofalcone to the reaction mixtures led to an enhancement in the rate of mucus glycoprotein acylation. The rate of enhancement was proportional to sofalcone concentration up to 1.0 X 10(-5) M, with an apparent Km value of 3.7 X 10(-7) M. In contrast to sofalcone, the acyltransferase activity was inhibited by sucralfate. The rate of inhibition of mucus glycoprotein acylation by sucralfate was of the competitive type and at 1.0 X 10(-4) M reached a value of 25%. The apparent KI value calculated from the double-reciprocal plots for sucralfate was 9.1 X 10(-7) M. As the acylation of mucin with fatty acids plays an important role in the maintenance of gastric mucosal integrity, the results suggest that stimulation of the fatty acyltransferase enzyme by sofalcone may be one of the beneficial effects of this drug towards ulcer healing.

Enzymatic sulfation of glycosphingolipids in rat parotid salivary glands.

The results of this study demonstrate that sulfatoglycosphingolipids of rat parotid salivary glands consist of galactosylceramide and lactosylceramide sulfates. The enzyme activity which catalyzes the formation of these compounds in vitro via transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate to galactosyl- and lactosylceramide is located in the microsomal fraction of parotid glands. This sulfotransferase exhibits its optimum activity at pH 6.8 and requires the presence of Triton X-100, Mg2+, and F1-.

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.

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.

Role of sulphation in post-translational processing of rat salivary mucins.

Segments of rat submandibular salivary gland were incubated in MEM supplemented with 10-800 microM sulphate in the presence of [3H]-glucosamine, [3H]-proline and [35S]-Na2SO4, with 0-8 mM chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulphate formation. Incorporation of glucosamine and sulphate depended upon the sulphate content of the medium and reached a maximum at 400 microM sulphate. The introduction of chlorate into the medium, while having no effect on the protein synthesis as shown by [3H]-proline incorporation, caused, at its optimal concentration of 4 mM, a 90% decrease in mucin sulphation and a 29% drop in mucin glycosylation. At low sulphate content in the medium and in the presence of chlorate the incorporation of sulphate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulphate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulphation in its carbohydrate chain. This effect of sulphate was, however, inhibited by chlorate. The results suggest that sulphation takes place at an early stage of mucin assembly and that sulphate availability is essential for the formation of the high molecular-weight mucin.

Enzymatic sulphation of mucus glycoprotein in rat sublingual salivary gland.

Sulphotransferase activity catalysing the transfer of sulphate ester group from 3'-phosphoadenosine-5'-phosphosulphate to salivary mucus glycoprotein was located in detergent extracts of the Golgi-rich membrane fraction of rat sublingual salivary glands. Optimum enzyme activity was obtained with 0.5 per cent Triton X-100, 20 mM NaF and 2 mM MgCl2, at pH 6.8, using desulphated sublingual salivary mucus glycoprotein. The enzyme was equally capable of sulphation of the proteolytically degraded and desulphated glycoprotein, whereas the acceptor capacity of intact salivary mucus glycoprotein was about four times lower. The Golgi enzyme preparation also catalysed the sulphation of galactosylceramide. However, the sulphation of mucus glycoprotein was not affected by the presence of this glycolipid, suggesting that the sulphotransferase involved in mucin sulphation is different from that responsible for the synthesis of galactosylceramide sulphate. The apparent Km of the sublingual-gland mucus glycoprotein sulphotransferase for salivary mucin was 7.7 microM. The 35S-labelled glycoprotein product of the enzyme reaction gave, in CsCl density gradient, a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein released the label into the reduced acidic oligosaccharide fraction. Upon thin-layer chromatography, two [35S]-oligosaccharides were detected. These were identified as penta- and heptasaccharides, each bearing a labelled sulphate ester group on the terminal N-acetylglucosamine residue. Based on the results of chemical and enzymatic analyses of the intact and desulphated compounds the following structures for these oligosaccharides are suggested: SO3----GlcNAc beta----Gal beta----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol and SO3----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol.

In-vitro biosynthesis of sulphatoglycosphingolipids by rat submandibular salivary glands.

A sulphotransferase activity, concentrated mainly in the microsomal fraction, which catalyses the transfer of sulphate group from 3'-phosphoadenosine-5'-phosphosulphate to galactosylceramide and lactosylceramide was demonstrated in rat submandibular and sublingual glands. However, the sulphotransferase activity of this fraction in submandibular glands was about ten times higher than in sublingual glands. Optimum enzyme activity was obtained using the detergent Triton X-100, F-, and Mg2+ at a pH of 6.8. The enzyme did not catalyse the transfer of sulphate to glucosylceramide, trihexosylceramide and triglucosyl glyceroglucolipid. The sulphotransferase exhibited similar affinity for both galactosyl- and lactosylceramide. The apparent Km of the enzyme for galactosylceramide was 3.8 X 10(-5) M, and for lactosylceramide, 4.3 X 10(-5) M. The results of compositional analysis and periodate-oxidation studies of the 35S-labelled products of the enzyme reactions established that in both [35S]-sulphatoglycosphingolipids the sulphate-ester group is located at C-3 of the galactose residue.

Enzymic acylation of mucus glycoprotein with palmitic acid in rat submandibular salivary gland.

The enzymic activity which catalyses transfer of palmitic acid from palmitoyl coenzyme A to mucus glycoprotein was found in Triton X-100 extracts of the microsomal fraction of rat submandibular and sublingual salivary glands. The acyltransferase activity of this fraction was 1.3-1.4 times greater in submandibular gland than in sublingual gland. Further subcellular fractionation of submandibular gland showed that the enzyme activity was associated with a Golgi-rich membrane fraction. Optimum enzyme activity for fatty acylation of mucus glycoprotein was at pH 7.4 using 0.5 per cent Triton X-100, 2 mM dithiothreitol 25 mM NaF and 10 mM MgCl2; higher concentrations were inhibitory. The apparent Km of the submandibular microsomal enzyme for mucus glycoprotein was 5.9 X 10(-7) M, and for palmitoyl-CoA, 3.3 X 10(-5) M. The 14C-labelled glycoprotein product of the reaction co-migrated on CsCl equilibrium, density-gradient centrifugation with submandibular mucus glycoprotein, and contained ester-bound palmitic acid. The fatty acyltransferase showed no activity with proteolytically-degraded glycoprotein; the acceptor capacity of reduced and S-carboxymethylated glycoprotein was only about 10 per cent lower than that of the intact mucus glycoprotein. This suggests that the acylation of salivary mucus glycoprotein with fatty acids occurs at its non-glycosylated, proteolysis-susceptible regions, and that the majority of these fatty acids are linked to the glycoprotein through hydroxyl esters.

Incorporation in vitro of [3H]glucosamine or [3H]glucose and [35S]SO42- into rat gastric mucosa. Presence of N-acetylhexosamine mono- and disulfates and galactose monosulfate in glycoprotein.

Rat gastric mucosa segments incorporate [3H]glucosamine and [35S]sulfate into glycosaminoglycans and glycoproteins. After 6-h incubation, the order of radioactivity incorporated from both precursors was glycoproteins greater than heparan sulfate greater than chondroitin sulfate. Pulse-chase studies indicate that much of the tissue-bound radioactivity was extruded into the medium by the 6th hour of incubation. The glycoprotein fraction was electrophoretically polydisperse and its subfractions exhibited a wide range of 3H/35S. Mild acid hydrolysis of the glycoprotein fraction afforded sulfated hexosamine fractions which migrated like N-acetylhexosamine mono- and disulfates by paper chromatography and by paper electrophoresis and exhibited 3H/35S which was in accord with the presence of mono- and disulfated compounds. The ratio of the monosulfated to the disulfated species was about 7 to 1. In separate studies, both of these compounds were found in the glycoprotein fraction elaborated into the medium, the glycoprotein from the combined tissue plus medium, and in the sulfated oligosaccharide isolated from the latter by reductive alkaline cleavage. Subsequent acid hydrolysis and paper chromatographic examination of the sugars liberated from each of these compounds revealed the presence of glucosamine as the principal amino sugar. The results of a periodate oxidation study of the N-acetylhexosamine monosulfate were in accord with N-acetylhexosamine 6-sulfate being the principal compound. About 20% of the 3H in the N-acetylhexosamine monosulfate and 10% of the 3H in the disulfated compound were found associated with galactosamine. Separate incorporation studies with [35S] sulfate and [3H]glucose not only confirmed the presence of N-acetylhexosamine mono- and disulfates in the glycoprotein fraction but also demonstrated the presence of galactose monosulfate therein.

The importance of PAPS in determining sulfation in gastrointestinal mucosa.

The in vitro sulfation of glycoproteins, mucopolysaccharides and lipids was studied with rat stomach, duodenum and colon mucosal scrapings. In the presence of Na2 35SO4, the incorporation of label was colon greater than stomach greater than or equal to duodenum. In the presence of [35S] 3'-phosphoadenosine 5'-phosphosulfate (PAP35S), the incorporation of label was colon = stomach greater than duodenum. The 35S was incorporated into glycoprotein, mucopolysaccharide and sulfatide. It is suggested that the availability of PAPS may be an important factor in determining the differences in sulfation previously observed histochemically in several species, namely that in vivo colonic mucosa far exceeds stomach mucosa in synthesis of sulfated polyanions.

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.

Determination of critical anthropometric parameters for design of respirators.

The objectives of this study were 1) to collect useful anthropometric data from 243 workers fit-tested in a continuing respirator fit-test program; and 2) to determine correlation between anthropometric data and Protection Factor obtained from quantitative fit-testing for half-mask respirators. Anthropometric data were collected from two direct facial measurements [nasal root breadth (NRB), and face-length (FL)] and five indirect facial measurements [nose length, nose protrusion, chin length, mouth width (MW), and face width (FW)] from front- and side-view slides of test subjects. For quantitative analysis, the anthropometric data collected in this study were normalized with relevant respirator dimensions (for four different brands and ten sizes). Results of linear regression analysis indicated that correlation coefficients between Protection Factor and anthropometric data (FL, MW, FW and NRB) were respectively - 0.04, 0.22, 0.30 and 0.04. These correlation coefficients are for white males without facial hair. The "critical" anthropometric parameters as apparent from the analysis were MW and FW. However, a person with certain combination(s) of multiple anthropometric parameters may provide a better correlation with Protection Factor.

Degradation of chondroitin 4-sulfate by rat stomach exoglycosidases, sulfohydrolase and hyaluronidase-like enzymes.

Rat stomach enzymes degrade chondroitin 4-sulfate (C4-S) by cooperative enzyme action yielding monosaccharides, SO4 (=) and oligosaccharides. Evidence for this was obtained by analyzing the effects of adding inhibitors and rate-limiting enzyme to incubation mixtures. A size preference appears to exist, since oligosaccharides of DP4-10 liberated by a gastric hyaluronidase-like enzyme is a poor substrate for desulfation by the gastric enzyme complex.

Identification of buccal mucosal mucin receptor.

A receptor for salivary mucin was isolated from epithelial cell membrane of buccal mucosa by a procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on Sepharose-bound wheat germ agglutinin. The receptor protein displayed an apparent molecular weight of 97kDa and exhibited binding specific to mucin in a concentration-dependent manner. The receptor, furthermore, showed a requirement for carbohydrate chains in mucin for binding, as their removal caused a marked (89%) reduction in binding capacity. Scatchard analysis revealed a linear plot with a single class of high affinity binding (Kd = 1.1 microM; Bmax = 7.68nmol/mg protein. The results provide for the first time evidence for the existence in buccal mucosa of a specific receptor for salivary mucin.

Lysolecithin affects the viscosity, permeability, and peptic susceptibility of gastric mucin.

The effect of lysolecithin and lecithin on gastric mucin viscosity, permeability to hydrogen ion, and degradation by pepsin was investigated. Preincubation with lysolecithin produced a marked decrease in the glycoprotein viscosity. This decrease was concentration-dependent and at 10 mM lysolecithin reached a value of 74%. A 25% increase in mucin viscosity was obtained with 10 mM lecithin. Permeability measurements showed that 10 mM lecithin increased the retardation ability of the glycoprotein to hydrogen ion by 11%, whereas a 12% decrease in the retardation capacity of the glycoprotein was obtained with 10 mM lysolecithin. The results of peptic activity assay indicated that whereas lecithin had no effect on the rate of mucin proteolysis, the lysolecithin exerted significant (75%) stimulatory effect. The results suggest that lysolecithin in the stomach weakens the integrity of the protective mucus layer by promoting peptic degradation, reducing the ability to resist acid penetration, and decreasing viscosity of its mucin component.

Effect of ethanol on the in vitro sulfation of salivary mucin.

In vitro sulfation of mucus glycoprotein by sulfotransferase from rat submandibular salivary gland and the effect of ethanol on this enzyme activity was investigated. Subcellular fractionation studies revealed that the enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein is associated with Golgi-rich membrane fraction. The sulfotransferase enzyme exhibited optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 4 mM MgCl2, and 25 mM NaF. The enzyme was equally capable of sulfation of the desulfated intact as well as proteolytically degraded desulfated glycoprotein preparations, whereas the acceptor capacity of the intact mucus glycoprotein was 70% lower. The submandibular gland sulfotransferase activity was inhibited by ethanol. The rate of inhibition of mucus glycoprotein sulfation was proportional to the concentration of ethanol up to 0.4 M, at which concentration a 39% reduction in the sulfotransferase activity occurred. The apparent Km value of the enzyme for salivary mucus glycoprotein was 11.1 microM, and the Kl in the presence of ethanol was 0.93 M. The synthesized 35S-labeled glycoprotein gave on CsCl equilibrium density gradient centrifugation 35S-labeled peak which coincided with that of the glycoprotein. Alkaline borohydride treatment of this glycoprotein led to the liberation of the label into the acidic oligosaccharide alditol fraction. As the inhibition by ethanol of sulfotransferase enzyme occurred below its isosmotic concentration to plasma, the observed effect could also be detrimental to salivary mucus glycoprotein sulfation in vivo.