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Sucrose synthetase from triploid quaking aspen callus tissue

by Graham, Larry Lester

Abstract (Summary)
Incubation of uridine triphosphate and 14 C-labeled glucose-l-phosphate with crude extracts of dark-grown triploid quaking aspen callus tissue led to the formation of two labeled products, uridine diphosphate glucose (UDPG) and sucrose. The following reaction scheme was suggested for their formation. G-1-P + UTP UDPG-yrophosphorylase UDPG + pyrophosphate UDPG + fructose sucrose synthetase_ sucrose + UDP Both enzymes, UDPG-pyrophosphorylase (E. C. 2.7.7.9) and sucrose synthetase (E. C. 2.4.1.13) are known to occur in plants; however, this thesis contains the first evidence for the presence of sucrose synthetase in any tree tissue, specifically in cultured tree callus tissue. Radioactive tracer studies showed that label could be incorporated into sucrose from uridine diphosphate-14 C-glucose and 1 4 C-fructose in the presence of the crude callus extract. There was some evidence that the formation of UDPG from sucrose and UDP was promoted by the crude callus extract. Both the UDPG-pyrophosphorylase and the sucrose synthetase activities in the crude extract obtained from the dark-grown triploid aspen callus sedimented at 100,000 x g, suggesting that these enzymes were membrane-bound. The sucrose synthetase from this tissue was purified by ultracentrifugation, solubilization with digitonin, salt fractionation, and gel filtration. Specific activities for the purified sucrose synthetase ranged from 0.143 to 0.243 micromole sucrose formed per milligram protein per minute. The purified sucrose synthetase was free of UDPG-pyrophosphorylase activity. A sedimentation coefficient of 11.13 x 10 13 sec- 1 was obtained for the purified sucrose synthetase. It had a molecular weight between 350,000 and 415,000 as determined by gel filtration on an agarose column. An optimum for the sucrose synthesis reaction was observed at pH 8. -2- The Michaelis constants for the purified sucrose synthetase in the sucrose synthesis direction were found. The value of K (UDPG) was 0.11 mM ([fructose] = -m 100 mM) and the value of K (fructose) was 5 mM ([UDPG] = 2 mM). High concentra- -m tions of UDPG inhibited the sucrose synthetase in its catalysis of the sucrose synthesis reaction. ADPG and GDPG could be utilized also as substrates by the enzyme; however, the effectiveness of these two substrates was much less than that of UDPG. The properties of the sucrose synthetase from aspen callus confirmed that it was a similar enzyme to that prepared from other plant sources. Sucrose synthetase activity was found also in crude extracts prepared from dark-grown loblolly pine, shortleaf pine, western hemlock, and eucalyptus callus tissues in addition to crude extracts prepared from light-grown Douglas-fir and triploid quaking aspen callus tissues. The importance of sucrose synthetase probably is not as a sucrose synthesizing enzyme. The current consensus is that it functions in vivo in the sucrosecleavage or UDPG-formation direction. This enzyme was very abundant in darkgrown quaking aspen callus tissue, accounting for nearly 5% of the protein in the tissue. Sucrose was the primary carbon source supplied to the callus tissue in the medium upon which it was grown. Because of its abundance in the tissue, sucrose synthetase must be very important to its growth and metabolism. Sucrose synthetase would appear to be the primary route for utilization of.sucrose by callus tissue via the formation of nucleotide diphosphate sugars. These compounds are, in turn, precursors in important biosynthetic processes within the tissue, such as cell-wall synthesis. -3-
Bibliographical Information:

Advisor:

School:Georgia Institute of Technology

School Location:USA - Georgia

Source Type:Master's Thesis

Keywords:institute of paper science and technology

ISBN:

Date of Publication:01/01/1975

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