P.A. Roukema

The murine sublingual and submandibular mucins. Their isolation and characterization.

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S20,W values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected. Both mucins consisted for about 1/3 of protein and 2/3 carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively. The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [35SO4] sulfate.

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE‐CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M‐sucrose or in 0.15 M‐NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP‐350 on account of its elution with 350 mM‐NaCl on a DEAE‐cellulose column. From DEAE‐cellulose chromatography it is evident that compounds comparable to GP‐350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP‐350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose‐containing materials during the purification procedure of GP‐350. The complete absence of other glucose‐containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP‐350 accounts for at least 3 per cent of the total saline‐extractable protein and about 20 per cent of the total saline‐extractable protein‐bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP‐350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).

Comparison of the secretory process in the parotid and sublingual glands of the mouse: I. Regulation of the secretory processes

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.

Aggregation of 27 oral bacteria by human whole saliva

Twenty-seven oral strains of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (19) were tested for aggregation by human whole saliva, as well as the effect of culture medium, Ca-ions, and bacteria concentration thereupon. Of the media tested, GF-broth gave rise to less interference by autoaggregation or higher aggregation titers than BHI and TSB, and was used throughout this study. In most cases, Ca-ions (1 mM) only enhanced the rate of induced aggregation, whereas raising the bacteria concentration increased the rate of both induce- and autoaggregation. The final titers, ranging from 1–64, were hardly affected by these parameters, except those of S. rattus HG 59 and S. mutans HG 199, which were respectively increased and decreased by Ca-ions. Saliva-induced aggregation was observed for 21 strains of A. viscosus, A. naeslundii, A. israelii, B. gingivalis, B. intermedius, S. cricetus, S. mutans, S. rattus, S. sanguis, and S. sobrinus, mostly within 15 min to 3 h. Seventeen of these strains also showed autoaggregation, usually well after the onset of induced aggregation. Any potential induced aggregation of B. gingivalis HG 91 was always masked by autoaggregation, as well as that of the S. mutans strains under a particular set of conditions. The aggregation rate and titer varied considerably in a mutually unrelated and strain-dependent way. These microtiterplate data were matched by the 5 spectrophotometric patterns observed for saliva-bacterial interaction, which moreover, gave the better differentiation between induced and autoaggregation. In conclusion, most strains tested can show rapid saliva-induced aggregation in a strain-dependent way, yet strongly affected by the experimental conditions and interference from autoaggregation.

Localization of amylase and mucins in the major salivary glands of the mouse.

SummaryAntibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.

Immunofluorescence study on the cellular localization of GP-350, a sialoglycoprotein from brain

The indirect immunofluorescence technique has been employed to determine the cellular localization of GP-350, a sialoglycoprotein isolated from calf brain. The results indicate that GP-350 is localized mainly, or perhaps exclusively, in neuronal structures. This has particularly been studied in the cerebellum, the cerebral cortex, the medulla oblongata, the corpus callosum, the caudate nucleus, and the cauda equina from calf. A part of the fluorescence could be observed in the neuronal cell bodies of these areas,e.g. the Purkinje cells, the pyramidal cells and the stellate cells. Although, the axonal structures were more brightly fluorescent than the perikaryon of the neuronal cells. No fluorescence was visible in glial cells or in gliomas. In human sciatic nerve fluorescence was present in long parallel nerve fibers. In the adenohypophysis and in the neurohypophysis of calf the fluorescence was detected in many, densely-packed, ring-shaped and oval cellular structures; in the pineal gland similar fluorescent structures could be observed, but far less frequently.

GP‐350, A SIALOGLYCOPROTEIN FROM CALF BRAIN: ITS SUBCELLULAR LOCALIZATION AND OCCURRENCE IN VARIOUS BRAIN AREAS

Abstract— A membrane‐bound form of GP‐350, a sialoglycoprotein from calf brain has been shown to occur in the crude mitochondrial fraction (P2). After subfractionation on a discontinuous sucrose gradient, consisting of 0.8 and 1.2 m‐sucrose, GP‐350 was immunologically only detectable in the synaptosomal fraction. After further separation of the lysed synaptosomal fraction on a discontinuous sucrose gradient (0.4, 0.8 and 1.2m) GP‐350 could be detected only in the 0.8–1.2 m‐sucrose interface.

PHYSICO‐CHEMICAL CHARACTERISTICS AND REGIONAL DISTRIBUTION STUDIES OF GP‐350, A SOLUBLE SIALOGLYCOPROTEIN FROM BRAIN

The apparent molecular weight of GP‐350, a sialoglycoprotein from calf and rat brain, has been determined by SDS‐polyacrylamide gel electrophoresis. The electrophoretic mobility corresponds to the mobility of a polypeptide with a molecular weight of 11,600 ± 200. On this basis it can be calculated that only one sialic acid residue is present/GP‐350 molecule.

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis, Streptococcus oralis, and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.

THE REGIONAL DISTRIBUTION OF CYTIDINE 5′‐MONOPHOSPHO‐N‐ACETYL‐NEURAMINIC ACID SYNTHETASE IN CALF BRAIN

—The enzyme cytidine 5′‐monophospho‐N‐acetylneuraminic acid synthetase was studied in different parts of the calf brain. Characterization of partial purified enzyme preparations from cortical grey matter and corpus callosum by means of pH optima and Km values, showed the enzyme of grey and white brain areas to be identical. Unexpectedly the regional differences of the enzyme activities per g wet tissue and per mg protein were very slight. From the presence of the enzyme in pure white brain areas, which are known to be poor in neuronal perikarya, and the fact that the enzyme is localized in the cell nucleus, we concluded that cytidine 5′‐monophospho‐N‐acetylneuraminic acid is produced in glia cell nuclei and that it is very likely that biosynthesis of sialo‐glycoproteins and/or ganglio‐sides occurs within glia cells.