Purification of lingual amylase from serous glands of rat tongue and characterization of rat lingual amylase and lingual lipase.
Lingual amylase and lingual lipase, two digestive enzymes that are secreted from lingual serous glands (von Ebner's), were simultaneously purified from rat lingual serous glands with hydrophobic chromatography used as the final step. This method, previously developed for the purification of lingual lipase, includes homogenization of rat lingual serous glands, 100,000 g centrifugation, ammonium sulfate precipitation of proteins, and extraction of lipids with acetone at -20 degrees C, followed by hydrophobic chromatography on ethyl agarose or Agethane. Amylase was eluted after the elution of proteins that did not interact with the hydrophobic gel at pH 6.3. Lingual lipase was eluted with a solution containing micelles of taurodeoxycholate, monoolein, and oleic acid. Analysis of each of the purified enzymes by SDS-polyacrylamide gel electrophoresis revealed one band at Mr = 59,000 for amylase and one band at Mr = 51,000 for lingual lipase. Isoelectric focusing of amylase indicated a strong band at pI = 5.0 and two very faint bands at pI = 4.9 and 4.8, possibly isozymes or deamidated protein. Amino acid and hexosamine analyses were performed on the enzymes after electroelution from SDS-polyacrylamide gels. Both lingual lipase and lingual amylase had a high content of dicarboxylic (free and amide) amino acids. For lingual lipase and lingual amylase, the % molar ratios of aspartic acid/asparagine were 15.35 and 15.10, and the % molar ratios of glutamic acid/glutamine were 7.07 and 7.20, respectively. Lingual amylase was very similar to rat parotid, pancreatic, and mouse salivary amylases, except that it contained more proline (11.03% molar ratio).(ABSTRACT TRUNCATED AT 250 WORDS)
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National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
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D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006595:Hexosamines / Q000032:analysis; D008049:Lipase / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D014059:Tongue / Q000201:enzymology*
D000596:Amino Acids; D006595:Hexosamines; D008049:Lipase; D000681:Amylases