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The initiation of the processing of apomucin was investigated using mucus glycoprotein synthesizing polysomes from rat gastric epithelial cells. The polysomes were isolated from cells labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine, purified on Helix pomatia-Sepharose affinity column, dissociated to release peptidyl-tRNA, and chromatographed on DEAE-HPLC column to separate peptidyl-tRNA complexes from the free and ribosomal RNA and proteins. The analysis of the HPLC purified peptidyl-tRNA revealed that complexes were labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine. Digestion of the peptidyl-tRNA with RNase released 3H and 14C labeled peptides, while alkaline degradation destroyed the complex and rendered the [3H]palmitic acid extractable with hexane. The treatment of the 3H and 14C labeled peptidyl-tRNA complexes with alpha-N-acetylgalactosaminidase led to the release of radiolabeled N-acetylgalactosamine, whereas alkaline borohydride reduction produced N-acetylgalactosaminitol. The fatty acid residues have been detected in peptidyl-tRNA containing 2,000Da peptides, whereas N-acetylgalactosamine was discernible on 5,000Da peptides.

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Mucus coat was isolated from oral epithelial surfaces of caries-resistant and caries-susceptible subjects, analysed for content and composition of lipids and mucus glycoproteins, and evaluated for physico-chemical characteristics. The mucus coat from caries-resistant subjects had a protein content similar to that of the caries-susceptible group but a higher content of carbohydrate and a lower content of lipids and covalently bound fatty acid. The carbohydrate component was mainly mucus glycoprotein, which accounted for 28.4% of the dry weight of caries-resistant mucus and 25.3% of caries-susceptible mucus. By chromatographic analysis on Bio-Gel A-50, both types of preparations had high (Mr approximately 2000 kdalton) and low (Mr approximately 300 kdalton) molecular-weight mucus glycoproteins. In the caries-susceptible mucus coat these two glycoproteins were in similar proportions, whereas the low molecular-weight glycoprotein predominated in caries-resistant mucus. In both preparations, the high molecular-weight glycoprotein was characterized by a high content of carbohydrates, associated lipids and covalently bound fatty acids, whereas the low molecular-weight glycoprotein was richer in protein and contained lesser amounts of associated and covalently bound lipids. Although the low molecular-weight glycoprotein showed only minor compositional differences with caries status, the high molecular-weight glycoprotein of the caries-resistant group had a 2.5 times lower content of covalently bound fatty acid, a 1.3 times lower content of associated lipids and contained 1.2 times more sulphate and sialic acid then that of the caries-susceptible group.(ABSTRACT TRUNCATED AT 250 WORDS)

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1. The effect of 70 mg/l and 35 mg/l MS 222 an anaesthetic on the enzymes: superoxide dismutase (SOD), catalase (C) and peroxidase (P) were estimated in erythrocytes of Cyprinus carpo, a freshwater fish and Dicentrarchus labrax, a marine fish. 2. The end of the summer, at 16 degrees C MS 222 in concentration 70 mg/l caused an enhancement of the SOD and peroxidase activities and a decrease of the catalase activity. 3. In the autumn at 22 degrees C SOD and peroxidase activities in erythrocytes of Dicentrarchus labrax are normally higher than at 16 degrees C. On the contrary MS 222 causes no significant modification of enzymatic activities measured, but an increase in the dispersion of the results. 4. At 13 degrees C in the spring, MS 222 has no immediate influence on the activity of these enzymes, whilst at the same temperature at the beginning of winter, SOD is the only one activated. 5. It seems that in experiments concerning peroxide metabolism enzymes the use of anaesthetic MS 222 is not advisable.

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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.

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The effect of 16,16-dimethyl prostaglandin E2 (DMPGE2) on the sulfation of mucus glycoprotein in gastric mucosa was investigated. The enzymatic activity which catalyzes the transfer of the sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to gastric mucus glycoprotein was located in the detergent extracts of Golgi-rich membrane fraction of antral and body mucosa of rat stomach. The sulfotransferase activity of this fraction from body mucosa, however, was 35% higher than that from the antrum. The enzyme exhibited optimum activity at pH 6.8 using 0.5% Triton X-100 and 30 mM NaF. The apparent Km of the enzyme for sulfation of mucus glycoprotein was 10.5 microM, and the sulfate ester was found incorporated into the carbohydrate chains of the glycoprotein. Introduction of DMPGE2 to the reaction mixtures led to an enhancement in the rate of mucus glycoprotein sulfation. The rate of enhancement was proportional to the concentration of DMPGE2 up to 1.0 x 10(-4) M and was of the competitive type, with an apparent Km value of 6.7 microM. Since sulfated mucins play an important role in gastric mucosal defense and the increase in their sulfation occurred at levels of prostaglandin present in gastric mucosa, the observed effect may be of significance to gastric mucosal defense in vivo.

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1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [3H]palmitic acid and [35S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells. 2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes. 3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [3H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da. 4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30-43 amino acids and those of 18-25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10-15 amino acids. 5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occurring in an immediate vicinity of the signal peptide fragment.

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A preparation of peptidyl-tRNA from intact microsomes of mucin-synthesizing polysomes of sublingual salivary gland cells contained fatty-acylated galactosamine-free and galactosamine-enriched peptidyl-tRNA fractions, whereas trypsin-chymotrypsin treated microsomes yielded predominantly the acylated galactosamine-enriched peptidyl-tRNA complexes. Radioscanning and chemical analyses revealed that palmitate was substituted on all nascent peptides, except those shorter than 20 amino-acid residues. In contrast, the [35S]-methionine label was detected only on galactosamine-free peptides containing up to 70 amino acids. On SDS-polyacrylamide gel, the peptides released from galactosamine-enriched tRNA complexes separated into a multitude of bands ranging in size from 6000 to 60,000 dalton, whereas the total preparation afforded peptides ranging from 2000 to 60,000 dalton. Pulse-chase experiments, using radiolabelled methionine, palmitic acid and N-acetylgalactosamine, combined with chemical characterization of the radiolabelled fatty acids and carbohydrates from purified peptidyl-tRNA, confirmed that the N-terminal fatty acylation and the initial O-glycosylation with N-acetylgalactosamine are the co-translational processes taking place as soon as peptide is sufficiently large to be acylated, trimmed, and translocated to the luminal site of endoplasmic membrane.

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1. Sedimentation of chromatin DNA and isolated deproteinized DNA was compared in neutral and alkaline sucrose density gradients after incubation of chromatin or DNA with various concentrations of heparin. 2. Irrespective of the molecular weight of DNA, an increase in the sedimentation constant of DNA was found with increasing concentration of the polyanion employed.

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