Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species |
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Authors: | Huber S C Pharr D M |
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Institution: | United States Department of Agriculture, Science and Education Administration, Agricultural Research and Department of Crop Science and Botany, North Carolina State University, Raleigh, North Carolina 27650. |
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Abstract: | High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I50 = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I50 = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I50 = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg2+ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour. |
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