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S H Duke

Publications and source records attributed to S H Duke.

At least 19 recordsLinked to original sources
PubMed1990

Purification and Characterization of Pea Epicotyl beta-Amylase.

The most abundant beta-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this beta-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a beta-amylase by end product analysis and by its inability to hydrolyze beta-limit dextrin and to release dye from starch azure. Pea beta-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K(m) of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea beta-amylase is competitively inhibited by its end product, maltose, with a K(i) of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with alpha-cyclohexaamylose being a stronger inhibitor than beta-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea beta-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea beta-amylase is the multichain type. Possible roles of pea beta-amylase in cellular glucan metabolism are discussed.

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PubMed1990

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings.

The most abundant alpha-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This alpha-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an alpha-amylase by polarimetry, substrate specificity, and end product analyses. The purified alpha-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This alpha-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. beta-glucan) are not substrates of the enzyme. A very low amount of this alpha-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or alpha-amylase which is integrally associated with the chloroplast.

Journal Article↗
PubMed1990

Chloroplastic regulation of apoplastic alpha-amylase activity in pea seedlings.

Photobleaching of pea (Pisum sativum L.) seedling leaves by treatment with norflurazon (San 9789) and 7 days of continuous white light caused a 76- to 85-fold increase in the activity of the primary alpha-amylase, a largely apoplastic enzyme, over normally greening seedlings. Levels of chlorophyll were near zero and levels of plastid marker enzyme activities were very low in norflurazon-treated seedlings, indicating severe photooxidative damage to plastids. As levels of norflurazon or fluence rates were lowered, decreasing photobleaching of tissues, alpha-amylase activity decreased. Levels of leaf beta-amylase and starch debranching enzyme changed very little in norflurazon-treated seedlings. Infiltration extraction of leaves of norflurazon-treated and normally greening seedlings indicated that at least 57 and 62%, respectively, of alpha-amylase activity was in the apoplast. alpha-Amylase activity recovered from the apoplast of photobleached leaves of norflurazon-treated seedlings was 18-fold higher than that for green leaves. Inhibitors of photosynthesis (DCMU and atrazine) and an inhibitor of chlorophyll accumulation that does not cause photooxidation of plastid components (tentoxin) had little effect on levels of alpha-amylase activity, indicating norflurazon-caused loss of chlorophyll and lack of photosynthesis did not cause the large induction in alpha-amylase activity. An inhibitor of both abscisic acid and gibberellin synthesis (paclobutrazol [PP333]) and an analog of norflurazon which inhibits photosynthesis but not carotenoid synthesis (San 9785) caused only moderate (about five-fold) increases in alpha-amylase activity. Lincomycin and chloramphenicol increased alpha-amylase activity in light grown seedings to the same magnitude as norflurazon, indicating that the effect of norflurazon is probably through the destruction of plastid ribosomes. It is proposed that chloroplasts produce a negative signal for the regulation of the apoplastic alpha-amylase in pea.

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PubMed1990

Partial Characterization and Subcellular Localization of Three alpha-Glucosidase Isoforms in Pea (Pisum sativum L.) Seedlings.

Three isoforms of alpha-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two alpha-glucosidase isoforms (alphaG1 and alphaG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (alphaG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of alphaG1 and alphaG2 in apoplastic preparations accounted for 21% of the total acid alpha-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid alpha-glucosidase activity was due to alphaG1. The apparent K(m) values for maltose of alphaG1 and alphaG2 were 0.3 and 1.3 millimolar, respectively. The apparent K(m) for maltose of alphaG3 was 33 millimolar. The respective native molecular weights of alphaG1, alphaG2, and alphaG3 were 125,000, 150,000, and 110,000.

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PubMed1990

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function.

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or beta-limit dextrin containing gel and KI/I(2) staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar beta-amylase (EC 3.2.1.2) and the primarily apoplastic alpha-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily beta-amylase) activity and considerably higher ratios of beta- to alpha-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and beta-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of alpha-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher alpha-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in alpha-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the alpha-amylase that was greatly increased in activity was the primarily apoplastic alpha-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic alpha-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar beta-amylase activity.

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PubMed1989

Characterization of Pea Chloroplast D-Enzyme (4-alpha-d-Glucanotransferase).

Pea (Pisum sativum L.) chloroplast D-enzyme (4-alpha-d-glucanotransferase, EC 2.4. 1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M(r) of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's K(m) for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of alpha-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by D-enzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed.

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PubMed1988

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems.

Most of the activity of an alpha-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic alpha-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the alpha-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this alpha-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the beta-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.

Journal Article↗
PubMed1987

A special group of ultradian oscillations.

Based on results from our studies, including those on leaf movements, circummutations of shoots, levels of enzyme activity, and metabolites, and from an extensive review of the literature, we have identified a special group of ultradian oscillations. Collectively, these oscillations have periods that range from approximately 30 to 240 min. Their patterns seldom exhibit strict periodicity or constant waveform. They have been observed in various animals, human beings, plants, and microorganisms. These oscillations appear to be a ubiquitous phenomenon, present at various levels of organization, e.g., biochemical to behavioral, and provide a unifying paradigm for exploring biological dynamics.

Activity Cycles↗
PubMed1984

Electrophoretic transfer as a technique for the detection and identification of plant amylolytic enzymes in polyacrylamide gels.

An electrophoretic transfer technique was developed for the specific identification of isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase. Amylolytic enzymes are separated by native polyacrylamide slab gel electrophoresis and proteins in gels are electrophoretically transferred through starch-containing polyacrylamide gels. Each amylolytic enzyme degrades starch in the transfer gel to its characteristic limit dextrin as it moves through the gel. Various limit dextrins in the starch gel are identified by their characteristic color development in KI/I solution. Isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase can be easily identified in the same gel.

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PubMed1983

Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase.

The specific measurement of alpha-amylase activity in crude plant extracts is difficult because of the presence of beta-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70 degrees C for 20 min, HgCl(2) treatment, and the use of the alpha-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While beta-amylase can liberate no color alone, over 10 International units per milliliter beta-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at beta-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate beta-amylase interference: (a) the dilution procedure, the serial dilution of samples until beta-amylase levels are below levels that interfere; (b) the beta-amylase saturation procedure, addition of exogenous beta-amylase to increase endogenous beta-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in beta-amylase activity or inhibitory effects of the commercial beta-amylase. The beta-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as alpha-amylases in alfalfa and soybeans are heat labile. Whereas HgCl(2) proved to be a potent inhibitor of beta-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited alpha-amylase in barley malt. The reported alpha-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals.

Journal Article↗
PubMed1983

Differential leakage of intracellular substances from imbibing soybean seeds.

Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max [L.] Merr.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25 degrees C) of 3.0 and 15.8 kilocalories per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intracellular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.

Journal Article↗
PubMed1983

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings.

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO(3) (-), tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO(3) (-) and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO(3) (-) and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO(3) (-). The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO(3) (-), further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO(3) (-) requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO(3) (-) requirement for induction were quite different. K(m) values for NO(3) (-) were identical for both nitrate reductases.

Journal Article↗
PubMed1982

Ultrastructural localization of urate oxidase in nodules of Sesbania exaltata, Glycine max, and Medicago sativa.

The localization of urate oxidase (=uricase, E.C. 1.7.3.3) was determined cytochemically in nodules of Sesbania exaltata (Raf.) Cory, soybean (Glycine max [L.] Merr.) and alfalfa (Medicago sativa [L.] ), using the precipitation of peroxide (produced during the oxidation of urate) by cerium chloride. Cerium perhydroxide reaction product was noted only in the microbodies, a localization consistent with biochemical fractionation studies on urate oxidase. Urate oxidase was present not only in the uninfected cells of the cortical tissue, but also in both infected and interstitial cells in the central tissue, suggesting that at least this enzyme of ureide metabolism is not confined to interstitial cells. Urate oxidase cytochemistry of nodules from alfalfa (Medicago sativa L.), an amide producer, also resulted in microbody staining but the microbodies were infrequently noted in cell profiles.

Fabaceae↗
PubMed1982

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots.

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or beta-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato beta-amylase, while that of bacterial alpha-amylase was considerably higher. The identification of maltose and beta-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all beta-amylases.The pH optimum for both beta-amylase fractions was 6.0. Both light and heavy beta-amylases showed normal Michaelis-Menten kinetics, with soluble starch as substrate, and had respectively K(m) values of 5.9 and 6.8 milligrams starch per milliliter and V(max) of 640 and 130 international units per milligram protein. Arrhenius plots indicated that the energy of activation for the heavy beta-amylase remained relatively unchanged (12.7 to 13.0 kilocalories per mole) from 0 to 30 degrees C, whereas the energy of activation for the light amylase increased from 12.0 to about 28.0 kilocalories per mole at 8.7 degrees C as temperature was lowered. The light amylase was shown to be inhibited by maltose.

Journal Article↗
PubMed1981

Role of the testa in preventing cellular rupture during imbibition of legume seeds.

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.

Journal Article↗
PubMed1979

Low root temperature effects on soybean nitrogen metabolism and photosynthesis.

The influences of low root temperature on soybeans (Glycine max [L.] Merr. cv. Wells) were studied by germinating and maintaining plants at root temperatures of 13 and 20 C through maturity. At 42 days from the beginning of imbibition, 13 and 20 C plants were switched to 20 and 13 C, respectively. Plants were harvested after 63 days. Control plants (13 C) did not nodulate, whereas those switched to 20 C did and at harvest had C(2)H(2) reduction rates of 0.2 micromoles per minute per plant. Rates of C(2)H(2) reduction decreased rapidly in plants switched from 20 to 13 C; however, after 2 days, rates recovered to original levels (0.8 micromoles per minute per plant) and then began a slow decline until harvest. Arrhenius plots of C(2)H(2) reduction by whole plants indicated a large increase in the energy of activation below the inflection at 15 C. Highest C(2)H(2) reduction rates (1.6 micromoles per minute per plant) were at 58 days for the 20 C control. Root respiration rates followed much the same pattern as C(2)H(2) reduction in the 20 C control and transferred plants. At harvest, roots from 13 C-treated plants had the highest activities for malate dehydrogenase, glutamate oxaloacetate transaminase, and phosphoenolpyruvate carboxylase. Roots from transferred plants had intermediate activities and those from the 20 C treatment the lowest activities. Newly formed nodules from plants switched from 13 to 20 C had much higher glutamate dehydrogenase than glutamine synthetase activity.Photosynthetic rates on a leaf area basis were about three times as high in the 20 C control as compared to 13 C control plants. Photosynthetic rates of plants switched from 20 to 13 C decreased to less than half the original rate within 2 days. Photosynthetic rates of plants switched from 13 to 20 C recovered to rates near those of the 20 C control plants within 2 weeks. All leaf enzymes assayed at harvest, with the exception of nitrate reductase, were highest in activity in the 20 C control plants.

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