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Metabolism and Enzymology

CHAPS Solubilization and Functional Reconstitution of -Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue ¹

Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, New Jersey 08903

A method for the solubilization and reconstitution of red beet (1,3)--D-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.

³ Present address: Agricultural College of Athens, Department of Food Science and Technology, Votanikos, Athens 11855 Greece.

¹ This research was supported in part by Grant DMB 85-02523 from the National Science Foundation, a Biomedical Research Support Grant, the Rutgers Research Council and the New Jersey Agricultural Experiment Station with State and Hatch Act Funds. New Jersey Agricultural Experiment Station, Publication No. D-10538-1-87.

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