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Articles

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species ¹

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, Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27650

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.

A heat-stable proteinaceous factor was identified in desalted crude leaf extracts that increased ATP sensitivity of the partially purified enzyme. Apparently, during purification a dissociable factor, that is required for maximum sensitivity to low concentrations (<1 millimolar) of Mg:ATP, is lost from the enzyme.