Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Purification and characterization of hydroxycinnamoyl D-glucose. Quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas Lam

We have previously proposed a chlorogenic acid biosynthetic pathway which involves a transesterification reaction between hydroxycinnamoyl D-glucose and D-quinic acid. The proposed pathway was based on tracer experimental results (Kojima, M., and Uritani, I. (1972) Plant Cell Physiol. 13, 311-319). The enzyme that catalyzes the above reaction has been purified 160-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. The purified enzyme yielded one band of 26,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000 by gel filtration chromatography. Therefore, the enzyme seems to consist of a single polypeptide of 25,000-26,000 daltons. The isoelectric point of the enzyme was 8.6. The optimum pH of the enzyme reaction was 6.0. The enzyme did not require any metal for activity and showed a broad substrate specificity toward hydroxycinnamoyl D-glucose as donors. The Km and Vmax values were 3.7 mM and 8.5 units/mg of protein for t-cinnamoyl D-glucose, 3.9 mM and 15.1 units/mg of protein for p-coumaroyl D-glucose, and 14.3 mM and 38.1 units/mg of protein for caffeoyl D-glucose. The enzyme showed a strict substrate specificity toward D-quinic acid-related compounds as acceptors; the Km and Vmax values were 16.7 mM and 15.1 units/mg of protein for D-quinic acid, 250 mM and 19.0 units/mg of protein for shikimic acid, and there was no activity with either L-malic acid or meso-tartaric acid. The enzyme activity changed in a manner suggesting its involvement in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Sucrose Synthetase of Sweet Potato Roots

The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the enzyme was determined to be about 540,000 by the Sephadex gel filtration chromatography.     

Alpha-glucan phosphorylase from sweet potato: isolation and properties of the partially degraded enzyme.

Alpha-Glucan phosphorylase (EC 2.4.1.1.) was purified from sweet potato roots. Apparently homogeneous preparations obtained are partially degraded products from phosphorylase, as judged from the results of molecular weight determination, NH-2-termini analysis and pyridoxal-5'-P assay. Phosphorylase is shown to be degraded in the crude extract from sweet potato. The degradation is partly suppressed by EDTA and by salts and is accelerated by reducing agents. It is proposed that sweet potato phosphorylase in its intact form has a similar molecular structure and similar properties to the white potato enzyme. Both plant phosphorylases are preferentially cleaved by protease near the middle of their polypeptide chains without much loss of enzyme activity.     

Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus.

Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.     

Glucose 6-phosphate dehydrogenase from sweet potato

Glucose 6-phosphate dehydrogenase was purified about 290-foldfrom sweet potato root tissue. The molecular weight was estimatedto be 110,000 by Bio-Gel 300 column chromatography. A LINEWEAVER-BURKplot of the reciprocal rate against reciprocal glucose 6-phosphateconcentration was concave downwards. A HILL coefficient lessthan 1 was obtained at lower concentrations of glucose 6-phosphate(below 0.5 mM). These results suggest that binding of glucose6-phosphate to the enzyme occurs with negative cooperativity. (Received April 30, 1970; )     

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.     

Purification and properties of an unusual UDP-glucose dehydrogenase, NADPH-dependent, from Xanthomonas albilineans

Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K_m for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

甘薯提取物对谷氨酸所致PC12细胞损伤的保护作用初探

观察甘薯提取物对谷氨酸诱导的PC12细胞损伤的保护作用.将大鼠嗜铬细胞瘤细胞(PC12)分为空白对照组、模型组、甘薯提取物0.1、1.0、10.0 μg/mL低、中、高剂量组和1.0 μmol/L尼莫地平阳性药物对照组,用2.0 mmol/L谷氨酸造成PC12细胞损伤,MTT法测定损伤细胞的存活率,观察其细胞损伤的形态学变化,考察甘薯提取物对谷氨酸所致PC12细胞损伤的保护作用.结果表明甘薯提取物可明显提高谷氨酸诱导的PC12损伤细胞存活率,同时能够明显改善谷氨酸诱导的PC12损伤细胞的细胞形态变化,并呈现剂量相关性,具有一定的神经营养及保护作用.     

Sucrose Phosphate Synthetase from Various Plant Origins

Some properties of sucrose-P synthetases obtained from various plant tissues, including sweet potato roots, potato tubers and leaves of barley, rape and ladino clover were studied. The specific enzyme activity of the sucrose-P synthetase from sweet potato roots was much lower than that of the sucrose synthetase of the other tissues. The enzyme activity decreased gradually as the roots developed. The optimum pH did not differ between enzyme preparations from sweet potato roots and barley leaves. Manganese chloride exhibited a marked stimulative effect on the sucrose-P synthetase from sweet potato roots and potato tubers, whereas it was inhibited the barley leaf enzyme.

Kinetic studies of sucrose-P synthetase showed that the behavior of the enzyme to the substrates did not differ in the enzyme sources examined. The substrate saturation curve of the enzyme with respect to fructose-6-P was sigmodal in shape, giving a straight line with a slope of 1.35~1.5 (n value) in a plot of the data using the empirical Hill equation. On the other hand, enzymes from all the various tissues exhibited a hyperbolic substrate saturation curve for UDP-glucose, obeying the ordinary Michaelis-Menten type reaction. Manganese chloride had no effect on the Km for UDP-glucose, the S_0.5 for fructose-6-P and the n value of the enzyme from potato tuber tissues.     

Purification, cloning, and expression of a pathogen inducible UDP-glucose:Salicylic acid glucosyltransferase from tobacco

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.     

红薯水提物对外来入侵植物喜旱莲子草生长的化感影响

从植物化感作用的角度,探索利用具有较高经济价值的本地植物或伴生的本地物种对入侵植物进行抑制和清除,是一种控制外来入侵植物行之有效的方法。本研究以本地作物红薯和入侵植物喜旱莲子草为试验对象,探究红薯不同部位(根、茎、叶)3个浓度(0.025、0.05、0.1 g·mL^-1)的水提物对喜旱莲子草的化感作用。以形态学指标(无性系小株个数、节数、叶片数、叶面积、株高、总干重和根数)、化感响应指数、性状比值(肉质化程度、根冠比、比叶面积、叶生物量比、茎生物量比、根生物量比)、新生叶片中过氧化氢酶(CAT)、过氧化物酶(POD)、丙二醛(MDA)、超氧化物歧化酶(SOD)等作为衡量红薯对喜旱莲子草根状茎生长影响程度的参数。结果表明: 1)不同浓度不同部位红薯水提物对喜旱莲子草生长有不同影响。0.1 g·mL^-1根水提物显著抑制所有形态学指标,除总干重和根数外,其他形态学指标均随不同部位水提物浓度的升高而显著降低。2)所有处理的综合化感响应指数均为负值,说明红薯水提物对喜旱莲子草各指标具有负效应,抑制其正常生长。在所有处理中,0.1 g·mL^-1根水提物的化感抑制作用最强,化感响应指数为-0.73,其次为0.1 g·mL^-1茎水提物和0.05 g·mL^-1根水提物,化感响应指数均为-0.44。3)从性状比值可以看出,红薯水提物对肉质化程度、根冠比、比叶面积和叶生物量比有显著抑制作用,而对茎生物量比和根生物量比无显著性影响。4)红薯水提物显著增加新生叶片中丙二醛含量,显著降低超氧化物歧化酶含量,但对过氧化氢酶和过氧化物酶无显著影响。表明红薯水提物对喜旱莲子草根状茎生长有显著抑制作用。     

Isolation of the Sweet Potato Weevil Factor Causing Terpene Induction in Sweet Potato Roots

The larval homogenate of sweet potato weevil, Cylas formicarius, induced terpene formation in sweet potato root tissue. We indicated that the terpene-inducing factor of the larvae consisted of high molecular weight component(s) and low molecular weight component(s). The high molecular weight component(s) was heat-unstable and partially inactivated by pronase, indicating proteinacious properties. The low molecular weight components) was heat-stable.     

Starch phosphorylase inhibitor from sweet potato

A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K_i value of 1.3 × 10⁻⁶ molar when glucose-1-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role against the plant enzyme.     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

The conversion of 3-deoxyarabinoheptulosonate 7-phosphate to 3-dehydroquinate by sorghum seedling preparations

Partially purified preparations from etiolated sorghum seedlings catalyzed the conversion of DAHP to DHQ. The reaction catalysed by DHQ synthetase was stimulated by 0.1 μM to 0.1 mM NAD in the presence O-0.5 mM Co²⁺. NADH at 1 μM stimulated the reaction as much as 50% but became inhibitory at 100μM. Co²⁺ at 0.5mM stimulated enzyme activity 3-fold; Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ were not stimulatory. EDTA at 5 mM inhibited the reaction 95% but its effects were reversed by equal concentrations of Co²⁺. Phe, Tyr, Trp, t-cinnamate, several hydroxylated cinnamates, DHS, quinate, and shikimate at 0.3 mM failed to affect enzyme activity but slight inhibition occurred with DHQ and protocatechuic acid at 0.3 mM, inhibition being 14 % and 22 %, respectively. DHQ synthetase activity also was detected in spinach leaves and potato tuber tissue. Synthetase activity appeared to increase in response to injury of potato tuber and sweet potato root tissues.     

Synthesis and apparent turnover of Acid invertase in relation to invertase inhibitor in wounded sweet potato root tissue

Previously we showed that acid invertase activity increased and then decreased rapidly in wounded sweet potato (Ipomoea batatas Liam.) root tissue, and that the tissue contained a heat-stable, proteinaceous inhibitor with a molecular weight of about 19,500 daltons.     

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.