Purification and Characterization of Sucrose Synthetase from the Shoot of Bamboo Leleba oldhami

Abstract

UDPG is doubtless the precursor of all sugar nucleotides mentioned above. From the physiological properties of bamboo shoot and the results of a preliminary survey, we suspected that UDPG could be supplied by the reaction catalyzed by sucrose synthetase in the direction of sucrose cleavage. Sucrose synthetase from bamboo shoot may be a regulatory enzyme which is more important for synthesis of UDPG rather than for synthesis of sucrose. Here, we report the properties of sucrose synthetase purified from the shoot of bamboo L. oldhami, especially those relevant to the postulated physiological functions of the enzyme explained above. EXPERIMENTAL PROCEDURESA series of studies aimed at the elucidation of the biochemical mechanism of cell wall maturation in higher plants have been conducted in our laboratory. The shoot of bamboo Leleba oldhami has been chosen as the main research material. Previous investigations revealed the types of glycosidic linkages present in bamboo cell wall polysaccharides (14), the distribution of sugar nucleotides in the tissue (12), and the changes of polysaccharide constituents accompanied with the growth and maturation of the plant (13). Unlike many other plants, bamboo shoot is incapable of utilizing GDP-D-glucose, which could not be detected in the plant, as the precursor for synthesizing cell wall polysaccharides. UDPG, the most abundant soluble nucleotide in bamboo shoot (12), is a good precursor for synthesizing a 83-1,3-glucan (15). This synthetic ability is strongly inhibited by UDP-D-xylose (and moderately by xylose and xylobiose), which has been demonstrated to be a good precursor of bamboo shoot pentosans (16). The pentosan synthesis from the precursor UDP->-xylose, which is rapidly isomerized to a mixture of UDP->-xylose and UDP-Larabinose by the pentosan synthetase preparation, is inhibited by UDP-D-galactose, the precursor of bamboo shoot galactan. MATERIALSThe shoots of bamboo L. oldhami grown in the vicinity of Taipei were sampled. Only the edible part was used as the enzyme source. All of the commercially available chemicals except UDP were used without purification. The UDP obtained from the Sigma Co. was contaminated with UDPG and UMP, the former of which interfered with the assay of sucrose synthetase by the UDPG dehydrogenase coupled method. These contaminants were removed by paper chromatography according to the method of Paladini and Leloir (10). The filter paper used for this purification procedure was prewashed in sequence with 1 M oxalic acid and distilled H20 and dried. Calcium phosphate gel was prepared according to Keilin and Hartree (4). METHODSThe protein content was estimated with Lowry's phenol method (6) using crystalline BSA as the standard.In density gradient centrifugation analyses, a linear gradient of sucrose from 5 to 20% and of glycerol from 8 to 33% (specific gravities from 1.0173 to 1.0806) were employed. The density gradient columns were centrifuged in a swinging bucket rotor at 112,000g and 4 C for 10 hr. Yeast alcohol dehydrogenase (S20, a = 6...

Elevation in the Sucrose Content of the Shoot Apical Meristem of Sinapis alba at Floral Evocation

“…If the flux through the pathway from sucrose through glycolysis is elevated at evocation, the probability is that sucrose hydrolysis is rate limiting. The SO.5 for sucrose synthetase (-50 mM) (24) and the Kmfor invertase (-3-10 mM) (22) are in the range of our estimate for sucrose concentration in Sinapis meristem (10 and 17 mM for control and evoked meristems, respectively). Thus, the sucrose concentration increase upon evocation that we report could serve to speed sucrose hydrolysis.…”

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

“…Sucrose synthase is a homotetramer in all plant species analysed so far, including mung bean (Delmer 1972), rice (Nomura and Akazawa 1973), bamboo (Su et al 1977), sugarcane (Buczynski et al 1993), barley (Guerin and Carbonero 1997) pea (Dejardin et al 1997) and maize (Su and Preiss;1978;Echt and Chourey 1985;Chourey et al 1986Chourey et al 1988. It is possible that the mutant SS2 protein is also a homotetramer composed of only the larger subunit which was detectable by polyclonal but not the monoclonal antibodies.…”

Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings

“…The sucrose cleavage and synthetic activities of RSS were assayed as described by Su et al (13) and Klann et al (14), respectively. The nucleotide specificity of the enzyme was determined as reported (11).…”