Twenty different human and animal tissues were investigated for the presence of polyglycosylceramides. The glycolipids were isolated by peracetylation of dry tissue residues left after conventional lipid extraction, followed by extraction with chloroform and subsequent Sephadex LH-20, Sephadex LH-60 and silica gel chromatography. In most of the cases only trace amounts of complex glycolipids were found. Distinct bands of glycosphingolipids migrating on TLC plates in a region of brain gangliosides and below were observed in bovine erythrocytes, human leukocytes and human colon mucosa. Definite fractions of polyglycosylceramides were isolated from rabbit small intestine, dog small intestine, human placenta and human leukocytes. The polyglycosylceramides of dog and rabbit intestine were characterized by colorimetric analysis, methylation analysis, mass spectrometry and immunological assays. The dog material contained branched carbohydrate chains with repeated fucosylated N-acetyllactosamine units. Rabbit intestine polyglycosylceramides resembled rabbit erythrocyte polyglycosylceramides with Hex-Hex- terminal determinants but were more complex in respect of sugar composition and structure. The material isolated from dog intestine showed A, H, Le(x) and Le(y) blood group activities. Polyglycosylceramides of human erythrocytes, placenta and leukocytes showed strong binding affinity for Helicobacter pylori, while polyglycosylceramide fractions from rabbit and dog intestine were receptor-inactive for this bacterium or displayed only weak and poorly reproducible binding.
Extracts of Streptococcus mitis ATCC 903 were analysed for P-fructofuranosidase and a-glucosidase activities by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures. Three bands of activity were visualized in the gels after incubation with sucrose (PI 4.05, 4.25 and 4.85) and three other bands after incubation with p-nitrophenyl a-D-ghcopyranoside (PI 3-90,4.45 and 4.65). The enzymes responsible for the reaction with sucrose were identified as P-fructofuranosidases (EC 3.2.1 .26) for the following reasons : identical enzyme bands were visualized in the gels after incubation with raffinose; no enzyme bands appeared in the gel after incubation with the a-glucosides maltose, turanose, trehalose and melezitose ; and the soluble fraction hydrolysed sucrose to equimolar amounts of glucose and fructose. I N T R O D U C T I O NStreptococcus mitis comprises a significant proportion of the bacteria of dental plaque and saliva and on the buccal mucosa (Gibbons & van Houte, 1975). It is capable of sucrose metabolism, as are all other oral viridans streptococci (Facklam, 1977). However, in contrast to s. mutans, s. sanguis and s. salivarius, s. mitis is generally devoid of glucosyl-and fructosyltransferase activities (Facklam, 1977). Apparently S. mitis utilizes other enzymic reactions to bring sucrose into the glycolytic pathway.Two types of enzymes hydrolysing the glycosidic bond in sucrose have been demonstrated in micro-organisms (Myrback, 1960) : P-fructofuranosidases (P-D-fructofuranoside fructohydrolase, EC 3.2.1 .26 ; invertase) and a-glucosidases (a-D-glucoside glucohydrolase, EC 3 . 2 . 1 .20). Sucrose phosphorylase (sucrose : orthophosphate a-glucosyltransferase, EC 2 . 4 . 1 .7), which catalyses the phosphorylytic decomposition of sucrose, has been found in some bacteria (Silverstein et al., 1967).In the present investigation the soluble fraction of extracts of S. mitis was analysed by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures in order to separate and visualize enzymes active on P-fructofuranosidic and a-glucosidic linkages. The zymogram patterns showed three enzyme bands with substrate specificities characteristic of P-fructofuranosidase and three enzyme bands with a-glucosidase activity. Preparation of extracts. Bacteria were harvested 1 h after the exhaustion of the energy source or at the METHODS Growth medium and cultivation technique. Streptococcus mitis
An aminopeptidase isolated from the cytoplasmic fraction of a cell extract of Streptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-beta-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-beta-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0-7.2 and at 37-40 degrees C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co2+ ions. Hg2+ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co2+ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co2+ ions.
Streptococcus mitis ATCC 903 and Streptococcus sanguis 804 could utilize large peptides present in proteose peptone as their sole source of nitrogen but grew poorly in amino acid medium. By contrast, Streptococcus mutans BHT and S. mutans IB grew in amino acid medium but could not utilize large peptides. Streptococcus sp. strain Bravato had requirements for both peptides and amino acids. Peptide size did not seem important for the utilization of peptides by S. mitis except that dipeptides were not utilized. Large peptides were essential for depressed hyaluronidase synthesis in nongrowing S. mitis. Intracellular aminopeptidase activities were high in S. mitis ATCC 903 and Streptococcus sp. strain Bravato, intermediate in S. sanguis 804, and low in S. mutans BHT and S. mutans IB.
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