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Metabolism and Enzymology

UDP-Glucose: (1,3)--Glucan Synthase from Daucus carota L. ¹

Characterization, Photoaffinity Labeling, and Solubilization

Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, New Jersey 08903, Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536

The membrane-bound UDP-glucose--(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5'--[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.

¹ This research was supported in part by grants DMB 85-02723 from the National Science Foundation (BPW), 87-CRCR-1-2414 from the U.S. Department of Agriculture (BPW), GM 35766 from the National Institutes of Health (BEH), the Charles and Johanna Busch Foundation (BPW), The George H. Cook Honors Program, and the New Jersey Agricultural Experiment Station with State and Hatch Act Funds. New Jersey Agricultural Experiment Station, Publication No. D-10546-1-88.

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