PE Activity Research Articles

Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.

Structure and function relationship of Pseudomonas exotoxin A: An immunochemical study

We have raised antisera against Pseudomonas exotoxin A (PE) and domains Ia and III to study the structure-function relationships of PE. Anti-PE antibody (AbPE) was shown to abolish the ADP-ribosylation activity of PE. However, neither antidomain Ia antibody nor antidomain III antibody inhibited the ADP-ribosylation activity of PE. This suggests that the inhibition of ADP-ribosylation by AbPE results from the binding of AbPE to the region between domains Ia and III. Since the binding of AbPE to PE did not inhibit NAD hydrolysis in the absence of elongation factor 2, the inhibitory effect of AbPE on ADP-ribosylation may be due to steric hindrance rather than a direct action on the catalytic function. Thus, the interface between domain Ia and III may be the site of entry of elongation factor 2 during ADP-ribosylation. The antibodies were also used to study both the inhibitory effects of PE on protein synthesis and its cytotoxic activity. Either AbPE or antidomain Ia antibody, but not antidomain III antibody, was able to reverse the inhibition of protein synthesis by PE and to block its cytotoxicity. In addition, rabbits immunized with domain Ia acquired tolerance against 100 micrograms of PE injected subcutaneously. These results suggest that domain Ia is the cell-binding domain of PE and may be used for vaccination against PE-mediated diseases.

32] Use of exotoxin A to inhibit protein synthesis

Publisher Summary This chapter describes the usage of exotoxin A to inhibit protein synthesis. The importance of Pseudomonas exotoxin A (PE) as one of the prime virulence factors of Pseudomonas aeruginosa is well established. The toxin is characterized on both the structural and biochemical level. Pseudomonas exotoxin A has a molecular weight of 66,583. PE exerts its toxic effect by specifically stopping protein synthesis both in vitro and in vivo . At the biochemical level, it acts in a manner identical to fragment A of diphtheria toxin—for example, by the adenosine diphosphate (ADP) ribosylation of cytoplasmic elongation factor 2 (EF-2). PE is produced by P. aeruginosa as a proenzyme and must be activated to express enzyme activity. A wide range of cells are susceptible to the cytotoxic action of PE, with mouse fibroblasts (L929, LM, 3T3) being the most sensitive cell lines. One simple assay to measure PE activity is to assess inhibition of protein synthesis.

Postharvest creasing of Robinson tangerines as affected by harvest date, pectinesterase activity and calcium content

SummaryRobinson tangerines were harvested on 8 and 22 October. The more mature fruits of the 22 October harvest were susceptible to postharvest creasing when stored for 2 weeks at 21°C, whereas the less mature fruit (8 October) did not manifest this disorder. Freshly harvested fruit of the 8 October harvest had an average pectinesterase activity (PE µmg−1 protein) of 1.11, and after 2 weeks storage this activity increased to 2.10. Fruit of the 22 October harvest showed an initial PE of 2.19 which increased to 2.48 after storage. Ca contents in the peel of both the 8 October (1 765 µg g−1 fr. wt) and 22 October (1 762 µg g−1 fr. wt) harvests were similar, and decreased to about the same level after storage, namely 1 549 and 1484 µg, respectively. Postharvest creasing appears related to both fruit maturity and initial PE activity.

Effect of Total Solids Level on Heat Inactivation of Pectinesterase in Orange Juice

ABSTRACTPectinesterase (PE) heat inactivation at various Brix did not obey first order reaction kinetics. Inactivation of PE at 10 ‐ 35° Brix did follow straight line semilogarithm plots of enzyme activity versus time. However, at 40° Brix and above the semi‐log graph between PE activity versus time was unclear. At high Brix levels a protective effect seemed to occur because the rate of enzyme inactivation declined. Increasing enzyme concentration in the 40° Brix sample showed that the curve was S‐shaped or sigmoidal. Increasing pH from 4 to 7 decreased the “Thermal Death Time” from 18 set to 4 sec.

The Inhibition of Tomato Fruit Ripening by Silver

Mature green tomato fruit, infiltrated with STS (up to 10 μmol) while still attached to the plant, ripened unevenly to give extensive green areas on an otherwise red background. Pericarp wall tissue from the two contrasting areas was analysed for various organic constituents. Both the green and, to a certain extent, the red tissue from treated fruit showed differences from normal in AIS, acidity, and PE activity. PG activity, which usually increases rapidly as tomatoes ripen, was low in the green but not significantly different from normal in the red tissue from STS-treated fruit. TEM examination revealed that electron-dense particles were present in the cell walls of phloem elements in vascular bundles of the green tissue, but these deposits were not found in the red tissue from the same fruit. X-ray microanalysis of the particles suggested that they contained concentrations of silver and sulphur. The results are interpreted as suggesting that silver is affecting those sites in the cell that would subsequently be involved in promoting the synthesis of PG.

Exercise: a vital component for patients on the Biostator.

Exercise was incorporated into the treatment of diabetic patients on the Biostator. As the purpose of the Bio stator is to determine the patient's in sulin requirements over a 24 hour pe riod, activity and exercise are impor tant to normalize that 24-hour period as much as possible. Individual exercise programs were developed for each of the patients on the Biostator. The most common ex ercise vehicle chosen was a bicycle ergometer. Physical Therapy devel oped these programs based on the pa tient's "Home Activity Assessment. " Nursing and Physical Therapy then monitored the patient's compliance with the exercise program during the "run" on the Biostator. The Biostator print-out (see Table I, p. 13) was used in a retrospective study of the effect of exercise on the patient's blood glucose levels. It was found that the median decrease in blood sugar immediately after cessa tion of exercise was - 6mgldl and for 15 minutes after cessation of exercise was — 12mg/dl. Empirical data is im portant to the patient so that they may balance exercise, diet, and insulin to keep blood glucose levels in control.

Why Your Company Should Support PES Activities

Why Your Company Should Support PES Activities

Physiological Factors in the Maturation and Ripening of Mango(Mangtfera IndicaL.) Fruit in Relation to the Jelly-Seed Physiological Disorder

SummaryPectinesterase (PE) activity remained constant in freshly picked cv Sensation mango fruit during all stages of maturity except in tree-ripened fruit and therefore cannot be used as a measure of physiological maturity. PE activity was only associated with ripeness, showing a sharp decrease after 14 days storage of immature fruit and 7 days storage of mature fruit, as well as in tree-ripened fruit. Physiological maturity of Sensation fruit can be associated with this drop in PE activity 7 days after harvest, combined with a sharp increase in the mass/volume ratio, at which time c. 15% of the fruits were sinking or slowly rising rather than floating in water at 25°C. It was concluded that the Sensation mango cultivar must be harvested before any external yellowing appears but with an internal yellow colour of c. 10%. From this time until the first appearance of jelly-seed is about 18 days.

The Maceration of Vegetable Tissue by a Strain of Bacillus subtilis

Pectate lyase (PAL EC 4.2.2.2), pectinesterase (PE EC 3.1.1.11), L‐arabinanase, D‐xylanase, D‐galactanase and neutral protease activities were identified in culture filtrates prepared from a strain B3 of Bacillus subtilis isolated from carrot. The PAL was purified by ion‐exchange chromatography and iso‐electric focusing and its properties examined. PAL had a pI of 9·85 and a molecular weight of 33000. Optimum activity occurred at pH 8–9 and 60–65°C. Calcium and to a lesser extent strontium were stimulatory while ethylenediamine tetraacetic acid led to inactivation. Thin layer chromatography separations of the end products of reactions and viscosity measurements suggested that the enzyme acted in a random manner. When examined over a range of pH values both culture filtrate and the purified PAL produced two distinct peaks of maceration (pH 6–6·5 and 8–9) against carrot or potato tissues. Evidence was obtained that although the presence of lyase was the sole external factor responsible for the maceration of carrot at pH 6·0, it acted in conjunction with a heat‐labile, high molecular weight factor extractable from carrot tissue. Carrot extracts were unable to macerate carrot but liberated reducing groups from polygalacturonic acid and it is suggested that the factor may be, in part at least, carrot polygalacturonase. Maceration at pH 8·5 was largely accounted for by PAL and PE activities.

Efeito de reguladores do crescimento nas características tecnológicas das frutificações do tomateiro (Lycopersicon esculentum Mill cv. Gigante Piedade)

No presente trabalho foi estudado o efeito de reguladores de crescimento (CCC, Alar, Giberelina e Ethephon) nas características do tomate. Os reguladores foram aplicados uma só vez, em pulverização foliar aos 38 dias após a semeação. Foram efetuadas duas colheitas, aos 60 dias e aos 100 dias após a aplicação dos reguladores. A qualidade foi avaliada através de análises físicas (Brix e cor), químicas (pH, ácido ascórbico e atividade de pectinesterase) e sensorials (cor, "flavor", aspectos interno e externo). Dentro das condições deste experimento os autores concluíram que a aplicação daquelas substâncias não teve influência no pH, na cor, nos conteúdos de sólidos solúveis e de ácido ascórbico dos frutos. Houve pequena influência na atividade da pectinesterase porém, devida à interação entre tratamentos e época de colheita. O "flavor" não foi afetado pelos tratamentos, mas os tomates da segunda colheita foram significativamente melhores. O aspecto interno e o externo foram bastante afetados, tendo o Ethephon sido o pior e o CCC, o melhor. Em termos de qualidade geral, os tratamentos classificaram-se, de acordo com a preferência dos julgadores, em ordem decrescente de qualidade como segue: CCC, Ethephon, Testemunha, Giberelina e Alar.

Pectinesterase Activity in Controlled Atmosphere Stored Avocados1

Abstract Pectinesterase (PE) activity was determined in avocado fruit (Persea americana Mill) at various times during controlled atmosphere storage (CA) and subsequent softening. A reduction in the initial softening time as related to storage temperature or ethylene accumulation in the CA unit was associated with a decrease in the PE activity of firm fruit at time of removal from storage. It is suggested that PE activity in fruit could be used to monitor a change in softening time during CA storage.

Pectinesterase, Polygalacturonase, Cx-Cellulase Activities and Softening of the rin Tomato Mutant1

Abstract Pectinesterase (PE), polygalacturonase (PG), and cellulase (Cx form) activities and softening of 4 physiological maturities were compared in normal and rin tomatoes (Lycopersicon esculentum Mil cv. Rutgers). PG, PE, and Cx-cellulase activities increased during ripening of normal fruits. In rin fruits, PG activity was not detected, PE activity remained relatively constant, and Cxcellulase activity increased during ripening. The lack of softening in rin fruits appears to be associated with the lack of PG activity.

青果物の鮮度保持に関する研究 (第9報)

Changes of the activity of chlorophyllase and PE during the ripening of tomato fruit were measured, and effect of packaging them with a polyethylene bag on the change of the activity was studied.1) The optimum concentration of acetone, the optimum pH and the optimum temperature in reaction mixture for measurement of the activity of chlorophyllase in tomato fruit were about 45%, 7.0 and 25°C respectively.2) The activity of chlorophyllase in tomato fruit on the plant increased with the ripening of them and reached the maximum at about the full-ripe stage, and then decreased.3) In tomato fruit detached from the plant, the activity of chlorophyllase also increases with the ripening. In the packaged fruit stored at a room temperature, the activity of chlorophyllase was maintained on the original level, and the content of chlorophyll was kept under control and did not decrease. On the other hand, in the non-packaged fruit stored at 10°C, the enzyme activity fairly increased with the ripening of fruit and chlorophyll was decreased.4) The activity of PE increased with the ripening of fruit.5) The activity of PE in tomato fruit detached from the plant also increased with the coloring and softening of them, but its increase was controlled when fruit was packaged with a polyethylene bag.

Measurement and Partial Characterization of Pectinesterase in Apple Fruits1

Abstract Pectinesterase (PE) assay of apple tissue was accomplished by using a dialysis cell containing a Visking (cellulose) membrane. Production and separation of methanol from the pectin substrate occurred during the dialysis. Agitation was needed for best separation of methanol from the reaction mixture. Microquantities of methanol were detected subsequently by direct injection into a gas-liquid Chromatograph equipped with a flame-ionization detector. Apple PE, prepared as an acetone powder, exhibited the characteristics of optimal pH between 6.5 and 7.5, an optimal reaction temperature at 55°C, and an activation energy of 5800 calories. The enzyme was completely inactivated at 80° for 10 min or 90° for 5 min. About 40% of the natural PE activity was inhibited in the presence of 15% sucrose.

Irradiation Effects on the Ripening of Kent Mangoes

SUMMARY— Kent mango fruit irradiated with 0, 100, 200, and 300 Krad were ripened at 20°C for 0, 4, and 8 days.Irradiated mangoes were less firm than control fruit immediately after irradiation, but fruit softening due to ripeness was more pronounced than softening induced by irradiation. Irradiated fruit contained higher water‐soluble and lower Versene‐vinsoluble pectic fractions as compared to unirradiated fruit. NO differences were observed in the content of the Versene‐soluble fraction among all irradiation dosages. Higher PE activities were exhibited by the irradiated fruit throughout the ripening period. Irradiated fruit contained higher AIS and lower soluble solids than control fruit. No differences in sucrose content due to irradiation were observed, but there were less reducing and total sugars at the 300 Krad dose as compared to other dosages.Changes in pectic fractions and PE activity were more pronounced immediately following irradiation in comparison to those occurring during ripening. The increase in soluble solids as well as sucrose, and the decrease in AIS, titratable acidity, reducing sugars and firmness during ripening proceeded at faster rates in control fruit than in the irradiated fruit. These variable rates of change were reflected by the significant irradiation dose and storage duration interactions.Results suggest that irradiation induces a delay in ripening of Kent mangoes.

Papaya Pectinesterase inhibition by Sucrose

SUMMARYPectinesterase (PE) was inhibited by sucrose at the same concentrations that delayed gelation of papaya purée. The inhibitory effect was linear with sucrose concentration throughout the range investigated (up to 50% sucrose). Conditions for optimum PE activity, pH 7.5 and 0.21M NaCl, were not affected by inhibitory concentrations of sucrose. Evidence was obtained against competitive inhibition, transferase activity, and an effect on the binding of PE as possible mechanisms for inhibition. Other sugars tested, including glucose, maltose, and corn syrups of varying degrees of hydrolysis, as well as glycerol were also inhibitory.

Seasonal Changes Occurring in the Pectinesterase Activity and Pectic Constituents of the Component Parts of Citrus Fruits II. Pineapple Orangesa

SUMMARYPectinesterase activity, 3 pectic fractions, and other characteristics were determined periodically on 5 component parts of Pineapple oranges during a 9.month maturation cycle for 2 seasons. Generally, PE activity was greatest in the peel, membrane, and juice sacs in March, April, and May, when the Brix/acid ratio was highest. However, the activity varied in similar components for like months during the 2 seasons. The order of component parts for PE in most cases, from highest to lowest activity, was juice sacs, membrane, peel, seeds, and juice. Total PE in the average whole orange attained maximum activity in March and April. Over 52% of the activity present was found in the juice sacs, which represented about 22.5% of the whole fruit. Water‐soluble pectin increased slightly in the peel and membrane, remained somewhat irregular in the juice sacs, and decreased to a constant level in the seeds throughout the growing season. Ammonium‐oxalate‐soluble pectin in the peel decreased slightly, and in the other components was either irregular or increased slightly, during maturation. Quantity of protopectin was greatest in the membrane and generally decreased toward the end of the sampling period in the various components. Total pectin and weight of the average whole orange was greater in the 1961–62 season than in the preceding season.

Seasonal Changes Occurring in the Pectinesterase Activity and Pectic Constituents of the Component Parts of Citrus Fruits.

SUMMARYPectinesterase activity, 3 pectic fractions, and other characteristics were determined periodically on 5 component parts of Valencia oranges during a 7‐month maturation cycle for 2 seasons. Usually, PE activity for peel, membrane, and juice sacs was least in December, when the Brix/acid ratio was low, and highest in June, when this ratio was greatest. The order of component parts for PE in most cases, from highest to lowest activity, was juice sacs, membrane, peel, seeds, and juice. Water‐soluble pectin generally remained constant in peel and juice sacs, increased slightly and then remained constant in the membrane, and was irregular throughout the cycle in the seeds. The trend of ammonium‐oxalate‐soluble pectin in the components was to increase during maturation. Protopectin in the component parts usually increased to a peak and then gradually decreased for the remainder of the season, except that protopectin in the juice sacs decreased throughout the sampling period. In this component, protopectin evidently was at its maximum by the first picking in December. Total pectin remained constant in the juice and seeds, and slowly declined in the other 3 components with maturation. Membrane contained the highest source of protopectin and total pectin throughout the season.

The Effects of Several Fungal Pectic Enzyme Preparations on Grape Musts and Wines

Both juice yield and browning rate increased with increasing amounts of enzyme, and also with increasing reaction time. The effect on sedimentation rate, compactness of sediment, and clarity was variable. Juice yield was increased by all the enzymes tested. The differences in sedimentation rate and compactness of sediment were small. All the enzymes showed a general tendency to increase clarity and browning rate. There was no relation between PE or PG activity and juice yield, clarity, or browning rate. Of chief interest is the comparison of the effect of the various enzymes on five different musts. The data clearly show that the effect of pectic enzymes not only varies with the individual musts, but that the enzymes differ among themselves in their effects on a single must. This not only explains the variable results obtained by wineries using pectic enzymes, but points out the necessity of developing methods for predicting their effect on a given must before the ezymes may be used with confidence in the results.