Abstract
A procedure has been developed for the effective solubilization of UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase (cellulose synthase) by treatment of membranes from the bacterium Acetobacter xylinum with digitonin. Low concentrations of digitonin (0.1%, w/v) cause stimulation of the enzyme activity in membranes; treatment with higher concentrations of digitonin (1-10%) results in solubilization of up to 70% of the digitonin-stimulated activity. The digitonin-solubilized enzyme displays regulatory properties quite similar to those of the membrane-bound form of the enzyme, showing specific activation by GTP. GTP activation requires the presence of a protein factor which can be separated from the enzyme by washing the membranes prior to enzyme solubilization. Association of this protein factor with the membrane-bound enzyme is promoted by polyethylene glycol or by Ca2+; however, these compounds are ineffective in enhancing enzyme-factor association for the enzyme in the solubilized state. The observation that Ca2+ promotes enzyme-factor association in the membranes suggests that this cation, in addition to GTP, may play a role in the regulation of cellulose synthesis in vivo in A. xylinum.
Highlights
We report such a Progress in the study of the biosynthesis of cellulose (1,4P-D-glUCan) has been persistently hampered by the low rates of in vitro synthesis obtained usingcell-free preparations derived from plantor bacterial cells capable of abundant cellulose synthesis in uiuo [1,2,3,4]
We recently reported success in achieving high initial rates of synthesis of1,4-/3-D-glUCan from UDP-glucose using membrane preparations derived successful solubilization of the enzyme with digitonin, and compare the kinetic and regulatory properties of the digitoninsolubilized enzyme with those of the membrane-bound enzyme.We report that Ca2+can replace PEG 4000 in promoting association of the protein factor with the membrane-bound enzyme, and suggest that this cation could play such a role in promoting factor-enzyme association in uiuo
From the bacterium Acetobacter xylznum [5, 6]. The key to this success lay in the discovery of a complex regulatory system for the A. xylinum UDP-glucose:1,4-/3-D-glucan 4-p
Summary
UDP-[’4C]glucose (240 mCi/mmol) was obtained from Radiochemfound that theenzyme shows marked and specific activation ical Centre. Unlabeled UDP-glucose, PEG 4000, digitonin, GTP, and by GTP, a finding which shows somaenalogiesto therecently sodium borohydride were purchased from Sigma. Guanosine 5”(yreported GTP-mediatedregulation of yeast UDP-glucose:1,3- thio)-triphosphate was obtained from Boehringer. Purified exocellop-Dglucan synthase by Cabib’s group [7,8,9]. For the A. xy- biohydrolase from Trichoderma viride was a generous gift from Dr. Sharon Shoemaker, Cetus Corporation, Berkeley, CA. IsraelBinational Agricultural Research and Development Fund (BARD).The costa of publication of this article were defrayed in part by the payment of page charges. *marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom all correspondence should be addressed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
