Changes in saliva of epileptic patients.

Unstimulated whole saliva samples of 27 indoor epileptic patients were studied on their protein composition using biochemical and immunochemical methods. A number of salivary proteins appeared at least partially to be hydrolyzed. In a number of saliva samples the concentration of carbohydrate-containing isoenzymes of amylase was reduced. In addition, the concentration of the 20 kD glycoprotein EP-GP was reduced by 60%. Sialic acid, the terminal sugar of the glycoproteins and mucins, was released for about 50% and in three salivas even nearly completely. Moreover, sialic acid- and fucose-containing epitopes could hardly be detected by monoclonal antibodies to human salivary mucins. As a consequence of this hydrolytic breakdown the saliva mediated aggregation of two S. sanguis strains had been reduced. In contrast, the aggregation of S. oralis had been maintained.

Adsorption to hydroxyapatite of partially deglycosylated human salivary mucins in competition with phosvitin and phytate.

The effect of phosvitin and phytate on the binding of native as well as partially deglycosylated human whole salivary mucins (HWSM) to hydroxyapatite was studied. Native HWSM preadsorbed onto hydroxyapatite was completely desorbed in the presence of greater than 500 micrograms/ml phosvitin. In contrast, in similar experiments, asialo-HWSM was desorbed approximately 10%. Desorption of preadsorbed asialo-afuco-HWSM in the presence of 1 mg/ml phosvitin was approximately 20%. Further deglycosylation of HWSM resulted in preparations which, after preadsorption to hydroxyapatite, were not desorbed upon subsequent incubation with phosvitin. With phytate, a less effective competitor of HWSM for the hydroxyapatite surface, essentially the same results were obtained, i.e. increase in deglycosylation of HWSM was concomitant with decrease in desorption by phytate. Using other incubation conditions (preadsorption of a phosphocompound, and simultaneous incubation of HWSM and phosphocompounds) essentially the same conclusion was obtained. The data indicate that the ability of salivary mucins to absorb to hydroxyapatite in competition with phosphocompounds appears to be enhanced by deglycosylation.

Coadsorption onto hydroxyapatite of human salivary mucins and phosvitin, a phosphoprotein from egg yolk.

The adsorption of human salivary mucins (HWSM, 0.4 mg/ml) onto hydroxyapatite (HAP) was studied in the presence of varying amounts of the phosphoprotein phosvitin by three different procedures. a. Preadsorption of HWSM onto HAP for 20 h, followed by 4 h coadsorption with phosvitin, resulted in a decrease of 50% in HWSM binding to HAP with 0.3 mg/ml phosvitin and a complete desorption with 1.0 mg/ml phosvitin. b. Preincubation of HAP with phosvitin for 20 h, followed by 4 h coadsorption with HWSM, resulted in decrease of 50% in HWSM binding to HAP with 0.15 mg/ml phosvitin and the adsorption of HWSM was prevented completely with 1.0 mg/ml phosvitin. c. Simultaneous incubation of HWSM and phosvitin gave the least adsorption of HWSM to HAP: a decrease of 50% with as little as 0.025 mg/ml phosvitin and a nearly complete desorption with 0.3 mg/ml phosvitin. Similarly, the adsorption of phosvitin was strongly inhibited by HWSM after either simultaneous adsorption or preadsorption with HWSM. However, after preincubation of HAP with phosvitin, desorption of phosvitin by HWSM was not achieved. Release of phosphate increased by preadsorption with HWSM followed by incubation with phosvitin, but was lowered by about 50% after preadsorption with phosvitin. After simultaneous incubation of HAP with both species, the adsorption did not result in release of phosphate ions. Calcium release was only substantial when phosvitin was in excess in solution. The smallest release of calcium ions was observed when HAP was preincubated with phosvitin, followed by coadsorption with HWSM.

Interaction of human salivary mucins with hydroxyapatite.

The interactions between hydroxyapatite (HAP) and several types of mucins (human whole salivary mucins, HWSM; ovine submandibular mucin, OSM; porcine gastric mucin, PGM) were compared using a quantitative assay. Of these mucins, HWSM displayed by far the highest binding to hydroxyapatite, followed by PGM and OSM, respectively. HWSM binding to hydroxyapatite was measured at pH 6.3 and 7.0. Data obtained appeared to fit empirically the Langmuirian adsorption isotherm. Apparent affinity constant (K-value) and maximum binding capacity (N-value), derived from these isotherms, indicated that upon lowering the pH the K-value increased from 0.7 to 0.8 ml/mg and the N-value from 2,050 to 2,640 micrograms/m2 hydroxyapatite. The pH-dependence of the HWSM-HAP interaction, measured over a large pH-range, at a fixed concentration of HWSM, indicated a two- to three-fold increase in binding upon lowering the pH from 7.5 to 5.5 At pH 5.5 the presence of HWSM induced an increased solubilization of hydroxyapatite, as deduced from the amounts of Ca2+ and phosphate-ions released. Addition of Ca2+ - and Mg2+ -ions (1mM) resulted in a decrease, both of HWSM binding and of the hydroxyapatite solubilization. These effects were more pronounced at the lower pH-values (pH less than 6.0).

Influence of phytate on the adsorption of human salivary mucins onto hydroxyapatite.

The effect of phytate on the adsorption of purified human salivary mucins (HWSM) onto hydroxyapatite (HAP) was studied using three incubation conditions. a. Preadsorption of HWSM onto HAP for 24 h, followed by 4 h coadsorption with phytate, resulted in at most a 25% decrease in HWSM binding. b. Preincubation of HAP with phytate for 24 h, followed by 4 h coadsorption with HWSM, resulted in a 40% decrease in binding of HWSM to HAP. c. Simultaneous incubation of HWSM and phytate with HAP resulted in a 50% decrease in HWSM binding. In contrast, the adsorption of phytate to HAP was not affected, irrespective of the incubation set up used. The adsorption of phytate to hydroxyapatite was accompanied by an increase in the phosphate concentration of the solution. The molar ratio of phosphate solubilized/phytate bound to HAP was approximately 2 to 3. On the other hand, under conditions when all phytate added became bound to HAP (i.e. HAP in excess over phytate), no increase in Ca-ions was observed. However, when free phytate was present (phytate in excess over HAP) approximately one mole of Ca-ions was released per mole free phytate. For comparison, the effect of phytate on the adsorption of porcine gastric mucin (PGM) and ovine submandibular mucin (OSM) was studied. Under conditions when HWSM binding to HAP was decreased by 10%, binding of PGM and OSM decreased by 65% and 100%, respectively.

Primary structure of O- and N-glycosylic carbohydrate chains derived from murine submandibular mucin (MSM).

The carbohydrate moiety of mouse submandibular mucin (MSM) contains mainly D-mannose and 2-acetamido-2-deoxy-D-glucose together with sialic acid, D-galactose, and 2-acetamido-2-deoxy-D-galactose. O-Glycosylically bound saccharides, obtained by treatment of MSM with alkaline borohydride, were shown by methylation analysis to have the structure: alpha-NeuAc-(2----3)-beta-Gal-(1----3)-GalNAc-ol. N-Glycosylically bound saccharides obtained from MSM by hydrazinolysis, and analysed by 500-MHz 1H-n.m.r. spectroscopy, were shown to have the following comprehensive structures. (Formula: see text).

Biochemical and immunochemical characteristics of the major protein of the murine parotid-granule membrane.

The membrane fraction (ParB) of the secretory granules of mouse parotid gland was isolated and characterized. The major phospholipids were phosphatidylcholine and sphingomyelin. The membranes contained one major protein, PMC, constituting at least 30 per cent of the total protein. PMC was purified: it is a small acidic protein with molecular weight of 12,000, containing one residue of phosphate per molecule. Using anti-PMC serum, PMC was detected only in the mouse parotid and saliva. Immunochemical characterization of organelle fractions indicated that PMC was mainly present in the secretory granule fraction; it was in part tightly bound to granule membranes. PMC was also present in both the 100,000 g parotid-tissue supernatant and the water-extract of the ParB granules. This dual localization was corroborated by immunofluorescent studies with anti-PMC serum which demonstrated that PMC was distributed uniformly over the acinar cells. The major protein component of these membranes is absent from other exocrine organs, e.g. pancreas, submandibular and sublingual glands.

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity.

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.

AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities.

The previously isolated female submandibular glycoprotein AM1 ( Nieuw Amerongen , A.V., Vreugdenhil , A.P. and Roukema , P.A. (1977) Biochim. Biophys. Acta 495, 324-335) has been shown to have hydrolytic activity using N-alpha-benzoyl-L-arginine ethylester (BAEE) and ChromozymR PK as a substrate. AM1 can be secreted in vivo by isoproterenol, and to a lesser extent by carbamylcholine and phenylephrine. Based on BAEE as a substrate, AM1 has an optimum pH of 7.8. In female submandibular glands, about one-third of total esterolytic activity resided in glycoprotein AM1, but in male submandibular glands, less than 3%. The Km value of glycoprotein AM1 is 50 microM and its Vmax is 117 mumol/min per mg glycoprotein AM1. The enzymatic activity is not inhibited by Ca2+, Mg2+ and Na+, slightly by Cu2+ and strongly by Hg2+ and phenylmethylsulfonyl fluoride. Glycoprotein AM1 is capable of hydrolyzing ChromozymR PK with a turnover value 20-fold lower than that for BAEE. With ChromozymR PK as a test substrate, glycoprotein AM1 was purified by a factor of 11. With this substrate, glycoprotein AM1 has an optimum pH between 6.6 and 7.6. Also with chromozymR PK as a substrate, the submandibular glands of female mouse showed a much higher activity of glycoprotein AM1 than the submandibular glands of the male mouse. About 75% of all enzymatic activity of female submandibular glands resided in glycoprotein AM1 and in male submandibular glands 22%. The Km value is 57 microM and its Vmax 6.7 mumol/min per mg glycoprotein AM1. From the biochemical characteristics and the localization of glycoprotein AM1, it has been concluded that glycoprotein AM1 is not identical to any of the other described murine submandibular esteroproteinases , such as kallikrein, gamma-subunit of the nerve growth factor, proteinase A and proteinase F.

Secretory granules of murine salivary glands. A morphological, biochemical and morphological, biochemical and immunochemical study of submandibular and parotid granules.

Two secretory granular fractions from murine submandibular glands (SM) and one fraction from murine parotid glands (Par) were isolated by centrifugation on two discontinuous sucrose gradients. From the parotid glands the granular fraction was layered on 1.9 M sucrose (ParB), and in addition a second fraction was layered on 2.1 M sucrose (SMc). The contamination of the granular fractions by other cellular organelles was determined. The membranes of both the Parb and SMb granular fraction contained a major protein fraction with a molecular weight of 12-14,000 designated PMC and MMC, respectively. Their total amino acid composition was similar, but not their carbohydrate composition and immunochemical properties. The major protein within the Parb granules was amylase, and in addition AM2-glycoprotein was also present. Both secretory components had exclusively an acinar localization in the parotid glands, indicating the acinar origin of the Parb granules. In the SMb granular fraction, consisting of large secretory granules, amylase activity was detected by enzymatic method, and in addition the nerve growth factor and epidermal growth factor were demonstrated by immunochemical methods. These activities could only be localized in the SM granular tubular (GCT) cells, pointing to a GCT cellular origin of the SMb granules. On the other hand, immunoreactive amylase, AM2-glycoprotein, and submandibular mucin (MSM) were present in the SMc granular fraction. In the SM tissue sections these components were localized in the acinar cells. So, it is likely that the SMc granules are seromucous acinar granules.

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.