Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus.
A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.
Mooser
G
G
Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles 90089-0641.
Wong
C
C
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002918:Chymotrypsin; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D013291:Streptococcus / Q000201:enzymology*; D014357:Trypsin
D000596:Amino Acids; D005936:Glucans; D010446:Peptide Fragments; D005964:Glucosyltransferases; D002918:Chymotrypsin; D014357:Trypsin