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A B Bennett

Publications and source records attributed to A B Bennett.

71 records · Page 4Linked to original sources
PubMed1987

Regulation of climacteric respiration in ripening avocado fruit.

Ripening of avocado fruit is associated with a dramatic increase in respiration. In vivo(31)P nuclear magnetic resonance spectroscopy revealed large increases in ATP levels accompanying the increase in respiration. Both glycolytic enzymes, phosphofructokinase, and pyrophosphate: fructose-6-phosphate phosphotransferase were present in avocado fruit with the latter activity being highly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate levels increased approximately 90% at the onset of ripening, suggesting that the respiratory increase in ripening avocado fruit may be regulated by the activation of pyrophosphate:fructose-6-phosphate phosphotransferase by an increase in fructose 2,6-bisphosphate.

Journal Article↗
PubMed1987

Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase.

An anion-sensitive H(+)-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H(+)-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO(3) (-), N,N'-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H(+)-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K(m) for the H(+)-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H(+)-ATPase are very similar to those described for a number of plant cell tonoplast H(+)-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.

Journal Article↗
PubMed1987

Transport Properties of the Tomato Fruit Tonoplast : II. Citrate Transport.

Citrate transport across the membrane of tomato fruit tonoplast vesicles was investigated. In the tonoplast vesicles, [(14)C]methylamine uptake was stimulated 10-fold by MgATP and strongly inhibited by NO(3) (-). Under identical experimental conditions, [(14)C]citrate uptake was inhibited by 5 millimolar free Mg(2+), and this inhibition was reversed in the presence of ATP, presumably by ATP chelation of free Mg(2+). No evidence was obtained in support of energy-linked ATP stimulation of citrate uptake. Citrate uptake showed saturation kinetics, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid and by other organic acids. The pH-dependence of uptake suggested that citrate(3-) was the transported species. Our results indicate that citrate transport across the tomato fruit tonoplast occurs by facilitated diffusion of citrate(3-). The carrier shares some features in common with anion channels in that it is relatively nonspecific for organic acids and is inhibitable by 4,4'-diisothyocyano-2,2'-stilbenedisulfonic acid.

Journal Article↗
PubMed1987

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits.

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant.

Journal Article↗
PubMed1986

Biotinylated proteins as molecular weight standards on Western blots.

Protein molecular weight standards were biotinylated by reaction with biotinyl-N-hydroxysuccinimide ester. The biotinylated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The resolved protein bands were detected by formation of a streptavidin-biotin/horseradish peroxidase complex and reaction with 4-chloro-1-naphthol and hydrogen peroxide. The biotinylated proteins are easy to prepare and are useful as molecular weight standards with most procedures employing immunodetection of proteins following transfer to nitrocellulose paper.

Biotin↗
PubMed1986

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

Journal Article↗
PubMed1986

Synthesis and processing of cellulase from ripening avocado fruit.

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.

Journal Article↗
PubMed1986

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L.

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.

Adenosine Triphosphatases↗
PubMed1985

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products.

Two distinct membrane fractions containing H(+)-ATPase activity were prepared from red beet. One fraction contained a H(+)-ATPase activity that was inhibited by NO(3) (-) while the other contained a H(+)-ATPase inhibited by vanadate. We have previously proposed that these H(+)-ATPases are associated with tonoplast (NO(3) (-)-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP(2-), and Mg(2+) concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg(2+)) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP(2-) in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP(2-) complex.The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H(+)-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg(2+) could modulate ATPase activity at both the tonoplast and plasma membrane.

Journal Article↗
PubMed1984

Sink to source translocation in soybean.

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [(14)C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [(14)C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.

Journal Article↗
PubMed1984

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase.

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO(3) (-)-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO(3) (-)-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H(+) transport, with the transport process showing the same differential sensitivity to NO(3) (-) and vanadate as the ATPase activity.Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO(3), stimulated by anions (Cl(-) > Br(-) > acetate > HCO(3) (-) > SO(4) (2-)), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H(+) transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H(+)-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H(+)-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

Journal Article↗
PubMed1984

H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase.

The H(+)/ATP stoichiometry was determined for an anion-sensitive H(+)-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A(340)) or by monitoring the release of (32)P from [gamma-(32)P]ATP. Inasmuch as this anion-sensitive H(+)-ATPase is strongly inhibited by NO(3) (-), initial rates of H(+) influx and ATP hydrolysis were measured in the absence and presence of NO(3) (-) to account for ATPase activity not involved in H(+) transport. The NO(3) (-)-sensitive activities were calculated and used to estimate the ratio of H(+) transported to ATP hydrolyzed. These measurements resulted in an estimate of the H(+)/ATP stoichiometry of 1.96 +/- 0.14 suggesting that the actual stoichiometry is 2 H(+) transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H(+) across the tonoplast measured in vivo, our result suggests that the H(+)-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.

Journal Article↗
PubMed1984

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos.

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and gamma-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.

Journal Article↗
PubMed1983

Derepression of amino Acid-h cotransport in developing soybean embryos.

The uptake of the unnatural amino acid alpha-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH(4)NO(3), or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H(+) cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos.

Journal Article↗
PubMed1983

Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots.

When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root microsomal membranes. The distribution of the NO(3) (-)-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H(+)-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 mum vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO(3) (-)-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H(+) from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K(+) > NH(4) (+) > Rb(+) > Cs(+) > Li(+) > Na(+) > choline(+)) whereas the NO(3) (-)-sensitive ATPase was stimulated by anions (Cl(-) > Br(-) > C(2)H(3)O(2) (-) > SO(4) (2-) > I(-) > HCO(3) (-) > SCN(-)). Both ATPases required divalent cations. However, the order of preference for the NO(3) (-)-sensitive ATPase (Mn(2+) > Mg(2+) > Co(2+) > Ca(2+) > Zn(2+)) differed from that of the vanadate-sensitive ATPase (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO(3) (-)-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO(3) (-)-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO(3) (-)-sensitive ATPase was stimulated by gramicidin and was associated with NO(3) (-)-inhibitable H(+) transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO(3) (-)-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO(3) (-) sensitivity is a better criterion by which to identify this ATPase than either Cl(-) stimulation or H(+) transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.

Journal Article↗
PubMed1982

Localization of a proton-translocating ATPase on sucrose gradients.

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing beta-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.

Journal Article↗