Akira Tsunemitsu

Heterogeneity of Peroxidase Related to Antibacterial Activity in Human Parotid Saliva

The peroxidase that is related to the thiocyanate-peroxidase-hydrogen peroxide antibacterial system in human parotid saliva was separated into three subfractions by diethylaminoethanol (DEAE)-cellulose column chromatography. Heterogeneity was confirmed by isoelectric focusing. Three subfractions contributed to the inhibition of the growth of Lactobacillus casei ATCC 7469.

Lysis of Streptococci by Lysozyme from Human Parotid Saliva and Sodium Lauryl Sulfate

Egg-white lysozyme (EC 3.2.1.17) has little effect on bacteria (including Streptococcus mitis and Streptococcus salivarius) indigeneous to the oral cavity of man (R. J. GIBBONS, J. D. STOPPELAAR, and L. HARDEN, J Dent Res 45:877, 1966). Recently, it has been shown that relatively lysozyme-resistant Streptococcus faecalis can be lysed by brief incubation with lysozyme, followed by the addition of sodium lauryl sulfate (SLS) or sodium chloride (R. H. METCALF and R. H. DEIBEL, J Bact 99:674, 1969). In our previous work (Y. IWAMOTO ET AL, J Dent Res 49:1104, 1970), lysozyme from human parotid saliva was purified successfully. It had a specific activity three and a half times greater than that of hen egg-white lysozyme and a composition that was different, especially in regard to tyrosine, serine, and glutamic acid contents. There have been no reports on the lysis of streptococci by parotid lysozyme. The purpose of this study was to observe the lysis of streptococci through the synergistic effect of SLS and lysozyme from human parotid saliva. Streptococci used in this study were S salivarius ATCC 9222, S mitis ATCC 9811, Streptococcus sanguis ATCC 10556, S sanguis ATCC 10557, and Streptococcus mutans strains GS-5, HS-6,* AHT, and FA-1.t Cells were inoculated from the thioglycolate agar stabs into a modified medium (K. S. BERMAN, R. J. GIBBONS, and J. NALBANDIAN, Arch Oral Biol 12:1133, 1967). After incubation for several hours at 37 C, cells in the exponential phase cultures were centrifuged and washed twice in cold distilled water. They were suspended in aliquots of 1/15 M Tris-HC1 buffer, pH 9.0. Lysis experiments were carried out according to the methods of Metcalf and Deibel (J Bact 99:674, 1969). Cells were resuspended in 4 ml lysing medium; the mixture contained TrisHC1 buffer (pH 9.0) in a final concentration of 1/15 M and 60 [tg of the purified parotid lysozyme. The reaction mixture was incubated at 37 C in a water bath with shaking. After ten minutes incubation, 0.1 ml of 0.32 M SLS was

Purification and Properties of Acid Phosphomonoesterase from Human Parotid Saliva

Acid phosphomonoesterase was separated from human parotid saliva and purified. The purified enzyme was proved homogeneous on sedimentation and disk electrophoresis. The optimal pH of the enzyme was 3.3. The enzyme had no substrate specificity for phosphomonoesters and its activity was inhibited by SnF2 or NaF.

The Presence of Uridine-5'-diphospho-galactose : N-Acetylglucosamine Galactosyltransferase in Human Saliva

It is known that the oligosaccharide side chains of salivary glycoprotein are composed of sialic acid, fucose, galactose, N-acetylglucosamine, and mannose (LEACH, Dental Plaque, 1970). Recently a few glycosyltransferases including this enzyme have been shown to have an important role in the biosynthesis of oligosaccharides of glycoproteins in some mammalian tissues and fluids (KIM ET AL, Biochim Biophys Acta 244: 505, 1971), but no investigations have been made in saliva. In this paper we present evidence that a UDP-galactose:N-acetylglucosamine galactosyltransferase is present in human saliva. Parotid and submaxillary-sublingual saliva samples were freshly obtained from eight men ranging in age from 23 to 30. Parotid fluid was collected by the method of Keene (J Dent Res 42: 1041, 1963) and submaxillary-sublingual fluid by the method of Brock and Brotman (NY Dent J 28: 116, 1962) Enzyme assays were performed by a modification of the method of Babad and Hassid (I Biol Chem 241: 2672, 1966). The reaction mixture contained 2 ttmoles of Tris-HCl (pH, 7.5), 1 jtmole MgCl2, 0.5 moles MnCl2, 5 jumoles mercaptoethanol, 0.45 Amoles N-acetylglucosamine and 32.2 jumoles (274 mCi/ mmolea; radiochemical purity verified by paper chromatography as > 99%) UDP-galactose-C14 (26,000 dpm) to which was added 0.05 ml of saliva sample in 0.5% Triton X-100b with a final volume of 0.2 ml. Incubations were conducted at 37 C for 20 minutes. The reaction was stopped by placing the reaction mixture on an anion exchange column consisting of about 1 ml of packed Bio-Rad

Heterogeneity of exo-type of β-N-acetylglucosaminidase in human parotid saliva

The exo-type of β-N-acetylglucosaminidase (NAGase) in human parotid saliva was separated into two fractions, NAGase A and B, by diethylaminoethanol (DEAE)-cellulose column chromatography. The heterogeneity was confirmed by isoelectric focusing and disc electrophoresis. Isoelectric points of NAGase A and B were at pH 7.2 and 5.0, respectively. NAGase A was more heat-stable than NAGase B. A slight difference in pH optimum was found between NAGase A and B. Both components had almost the same Km values for p-nitrophenyl N-acetylβ-glucosaminide, p-nitrophenyl N-acetyl-β-galactosaminide, phenyl N-acetyl-β-glucosaminide and phenyl N-acetyl-β-galactosaminide.

Purification and Amino Acid Analysis of Human Parotid Saliva Lysozyme

Lysozyme from human parotid saliva was purified to the point where it was judged, by the criteria of ultracentrifugation and disk electrophoresis, to be homogeneous. The parotid lysozyme had a specific activity 3.5 times greater than that of hen egg-white lysozyme and a composition that was different, especially in regard to tyrosine, serine, and glutamic acid content. The molecular weight was determined to be around 14,300. When tryptophan residues were modified with N-bromosuccinamide, no lytic activity was found, which indicates that the tryptophan residues may play an important role in the maintenance of the parotid lysozyme activity.

Transglycosylation by the exo-type of β-N-acetylglucosaminidase from human parotid saliva

Abstract The transglycosylation by the exo-type of purified β-N-acetylglucosaminidase from human parotid saliva and Aspergillus oryzae was investigated by means of time course of transfer product, thin-layer chromatography and gas chromatography. It has been shown that the transglycosylation is virtually absent in the exo-type of β-N-acetylglucosaminidase from human parotid saliva, while the enzyme from A. oryzae has strong transglycosylation activity.

Isolation and Characterization of β-N-Acetylglucosaminidase from Human Parotid Saliva:

Beta-N-acetylglucosaminidase in human parotid saliva was separated into two subfractions by diethylaminoethanol cellulose column chromatography. One subfraction of the enzyme was isolated and purified. Disk electrophoresis showed that the purified enzyme was homogeneous. The molecular weight of this enzyme was estimated to be about 153,000 by gel filtration and dodecyl sulfate polyacrylamide gel electrophoresis. N-acetyl-β-galactosaminides also were hydrolyzed by this enzyme at the same site.

Hydrolysis of Glycol Chitin by an Endo- and Exo-Type of β-N-Acetylglucosaminidase from Human Parotid Saliva

Since it was shown that chitin, a polymer of N-acetyl-p-D-glucosaminide bounded by 1,4 linkages, could be lysed by egg white lysozyme (BERCER and WEISER, Biochim Biophys Acta 26: 517, 1957), the so-called lysozyme (EC 3.2.1.17) is considered to be an endo-type of ps-N-acetylglucosaminidase (NAGase). More recently, an exo-type of NAGase (EC 3.2.1.30) from human parotid saliva was purified successfully (WATANABE ET AL, J Dent Res 52: 782-790, 1973) . In a preliminary study, we confirmed that the exotype of NAGase hydrolyzes the chitin oligosaccharides (dimers, trimers, and tetramers of Nacetyl-3-glucosaminide).a The purpose of this study was to observe the hydrolysis of chitin through the synergistic effect of the endoand exo-type of NAGase from human parotid saliva. Hydrolysis experiments were carried out by a modification of the methods of Hamaguchi et al (J Biochem (Tokyo) 48: 351, 1960). The reaction mixture contained 10 mg glycol chitin,5 16 ,ug purified endo-type of NAGase from human parotid saliva (IWAMOTO ET AL, J Dent Res 49: 1104, 1970), 8 ,ug purified exo-type of human parotid NAGase in 0.05 M citrate-0.1 M phosphate buffer (pH, 4.5) in a final volume of 1 ml and a drop of toluene to inhibit bacterial growth. After incubation for 4, 8, or 16 hours at 37 C, the liberated N-acetylglucosamine was measured by the methods of Reissig et al (J Biol Chem 217: 959, 1955) . Optical density readings were made with a spectrophotometere at 585 nm, against a blank that contained all components except enzyme. As indicated in the illustration, glycol chitin was hydrolyzed by the endo-type of NAGase gradually, whereas the exo-type did not hydrolyze it at all. In a system that contained the endoand exo-type of NAGase, initially the hydrolysis of the glycol chitin increased. After 16 hours incubation, liberated N-acetylglucosamine concen-