Solubilization and purification of the glucosyltransferase involved in the biosynthesis of teichuronic acid by fragments of Micrococcus luteus cell membranes
Enzymes involved in the biosynthesis of teichuronic acid have been demonstrated in cytoplasmic membrane fragments recovered from lysozyme treated Micrococcus luteus cells. Solubilization of the glucosyltransferase activity was effected with aqueous solutions of Triton X-100, Nonidet P-40, Tween 20, or Thesit. Thesit proved most amenable for recovery of glucosyltransferase activity as well as spectrophotometric protein determinations. Recovery of the glucosyltranferase activity was aided during purification by inclusion of 15% glycerol, 0.75% Thesit, 20 mM magnesium ion and 2 mM 2-mercaptoethanol in all buffers. Glucosyltransferase activity was monitored by the transfer of (/sup 14/C)glucose from UDP-(/sup 14/C)glucose to an artificial acceptor. Although the natural acceptor is presumed to be an undecaprenyl diphosphate-activated oligosaccharide, alternate acceptors such as isolated cell wall fractions containing teichuronic acid served equally well. Highly purified teichuronic acid devoid of peptidoglycan was the most effective alternate acceptor. The glucosyltransferase was purified by ammonium sulfate precipitation followed by ion exchange chromatography on DEAE-cellulose yielding an overall 200-fold increase in specific activity.
- Research Organization:
- Univ. of Minnesota, St. Paul
- OSTI ID:
- 6374508
- Report Number(s):
- CONF-870644-; TRN: 87-033983
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ORGANIC ACIDS
BIOSYNTHESIS
TRANSFERASES
ENZYME ACTIVITY
FRACTIONATION
SOLUBILITY
AQUEOUS SOLUTIONS
CARBON 14 COMPOUNDS
CELL MEMBRANES
GLUCOSE
MICROCOCCUS LUTEUS
SOLVENT PROPERTIES
TRACER TECHNIQUES
ALDEHYDES
BACTERIA
CARBOHYDRATES
CELL CONSTITUENTS
DISPERSIONS
ENZYMES
HEXOSES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MEMBRANES
MICROCOCCUS
MICROORGANISMS
MIXTURES
MONOSACCHARIDES
ORGANIC COMPOUNDS
SACCHARIDES
SEPARATION PROCESSES
SOLUTIONS
SYNTHESIS
550201* - Biochemistry- Tracer Techniques