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1.
Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporulated oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chromatography.  相似文献   

2.
Summary The activity of acid phosphatase produced in pure culture by the endomycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan (H. ericae LPA 2) was inhibited by high phosphorus levels, alkaline pH, fluoride, molybdate and mannosidase, and activated by concanavalin A. Over 80% of the enzyme activity was due to two wall-bound acid phosphatase isozymes with the characteristics of mannose-rich glycoproteins. Antiserum was raised against the major, low-molecular-weight wall isozyme and its activity tested by immunoblotting and ELISA. The antiserum cross reacted 100% with exocellular (excreted) and 28% with cytoplasmic cellular fractions of H. ericae (LPA 2) cultures, and showed high reactivity with other strains of H. ericae but not with fungal isolates from Erica hispidula L. or E. mauritanica L. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling confirmed its association with the fungal wall in pure culture and showed that the influence of a high phosphorus level, fluoride and molybdate is through inactivation of the enzyme. Intense acid phosphatase activity, sensitive to the latter inhibitors, was also present on external hyphae growing over a host or non-host root but it was weak or absent from intracellular hyphae where these developed within a host root. Indirect immunolabelling confirmed that this acid phosphatase was of fungal origin and that the specific inhibitory effect of host cells is due to inactivation of the enzyme rather than repression of its synthesis. Possible implications of fungal acid phosphatase in ericoid endomycorrhizal infection processes are discussed together with mechanisms that may be regulating the enzyme activity.  相似文献   

3.
Blood, withdrawn by heart puncture, from donor rabbits infected with Eimeria stiedai, was transfused by heart puncture, to uninfected recipient rabbits. Fifty-nine of 76 young coccidia-free recipients, given heparinized whole blood, erythrocytes or leucocytes from donors orally inoculated up to 27 days earlier, developed E. stiedai infections.  相似文献   

4.
Summary Soil organic matter was obtained from two agricultural soils using alkali extraction followed by acidification to produce humic and fulvic acids which were further fractionated by adsorption and gel chromatography.All the products inhibited the activity of phosphatase prepared from wheat roots, but to different extents. Humic acids produced a greater inhibition of enzyme activity than either the fulvic acids or water extracts of soil. Aspergillin, fromAspergillus niger, had a similar C, H and N content to humic acid and produced a similar inhibition of phosphatase activity.The inhibitions produced by corresponding fractions derived from the two soils were slightly different, but the trends between similar fractions from different soils were comparable. The lower mol. wt. components of humic acid inhibited phosphatase activity to a greater extent than higher mot. wt. fractions. Although fulvic acid comprised only low mol. wt. components it was less effective in inhibiting enzyme activity than those components of comparable mol. wt. present in the corresponding humic acid. Synthetic polymaleic acid, produced an inhibition of phosphatase activity similar to that caused by fulvic acid.  相似文献   

5.
Summary A strain of Escherichia coli exhibiting reduced activity of the periplasmic enzyme acid phosphoanhydride phosphohydrolase (pH 2.5 acid phosphatase) was isolated. The mutation designated appA1 was located at 22.5 min on the E. coli genetic map. Acid phosphatase purified from an appA transductant showed less than ten percent of the specific activity of an isogenic appA +strain. The mutant enzyme was highly thermolabile and its Km for paranitrophenyl phosphate was increased about 20-fold. The mutant protein cross-reacted with antibody to the wild-type enzyme and had the same molecular weight and concentration in extracts as the wild-type enzyme. These findings strongly suggest that appA is the structural gene of the acid phosphatase.Abbreviations PNPP paranitrophenyl phosphate - cAMP 3-5-cyclic adenosine monophosphate - Nitrosoguanidine N-methyl-N'-nitro-N-nitrosoguanidine - TCY tetracycline - KAN kanamycin - STR streptomycin  相似文献   

6.
Aluminium accumulates predominantly in the root apoplast where it binds to the pectin matrix of the cell wall with its negative charges. In this study, we investigated whether short-term Al treatment (2 h) affects the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells of Zea mays L. The pectin content of the cell cultures was modified by long-term adaptation to NaCl stress or long-term adaptation to the cellulose-synthesis inhibitor 2,6-dichlorbenzonitrile (DCB), and by short-term treatment for up to 15 min with pectolyase. At pH 4.5, neither acid phosphatase activity of commercial enzyme preparations nor of exudates from root-tips and suspension-cells of Zea mays L. were affected directly by Al. However, the exudation and the activity of apoplastic acid phosphatase was reduced to a greater extent by Al cells with high pectin content than in cells with normal pectin content. The strongest reduction of acid phosphatase exudation was observed in pectolyase-treated cells with the lowest pectin content. Al reduced not only the release of acid phosphatase from the suspension cells, but also the release of total proteins and pectins. However, no relationship existed between the magnitude of Al-induced reduction of protein and pectin release and the cell pectin contents. These results support the assumption that Al modifies cell-wall and plasma-membrane transport-properties for macromolecules and the activity of apoplastic enzymes thus modifying Al sensitivity.  相似文献   

7.
J. M. Palmer 《Planta》1970,93(1):53-59
Summary Prolonged washing of thin slices of Jerusalem artichoke (Helianthus tuberosus) did not result in any apparent increase in the activity of the phosphatase enzymes, although the washing process is known to stimulate the activity of many other enzymes. However, treatment of the tissue in either 3×10–5M 2,4-dichlorophenoxyacetic acid or 10–4M indole acetic acid resulted in a 3-fold increase in the phosphatase activity. Significant stimulations of activity were detectable one hour after placing the tissue in the auxin. Treatment of the tissue in either kinetin or gibberellic acid failed to stimulate the activity of the enzyme. The enhancement of phosphatase activity caused by auxins could not be prevented by adding cycloheximide to the treatment solution an it is concluded that the stimulation occurred as the result of the activation of enzyme already present in the tissue rather than by the de novo synthesis of new enzyme protein.  相似文献   

8.
Eight psychrotrophic strains belonging to four different genera were screened for the presence of cold-active alkaline phosphatase in sonicated cell homogenates. An approximately 1000-fold higher activity than E. coli was detected in two psychrotrophic strains of Sphingobacterium antarcticus and one mesophilic strain of Flavobacterium multivorum. The enzymes from the psychrotrophs showed maximum activity at 37°C and were also found to be active at 0°C. Alkaline phosphatase from one psychrotrophic Sphingobacterium lost 97% of its activity when it was heated for 10 min at 62°C. This enzyme was partially purified and characterised. The production of the enzyme was repressed when the organism was grown in the presence of phosphates and its activity was inhibited on preincubation with inorganic phosphates and ethylene diamine tetracetic acid. Potassium permanganate and potassium periodate did not inhibit the activity of the enzyme. The biotechnological importance of the enzyme is discussed.  相似文献   

9.
Phthalimide treatments at 125, 250 and 500 mg 1–1 to female plants of dioecious Morus nigra L. induced intersex and male flowers. Qualitative and quantitative analyses of acid phosphatase in male and female flower buds showed that the male flower had significantly higher levels of the enzyme activity than the female flower buds. The level of acid phosphatase activity significantly increased in intersex and male flowers induced on female plants after phthalimide treatments.  相似文献   

10.
11.
M. Godeh  J. Udvardy  G. L. Farkas 《Planta》1981,152(5):408-414
Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0–7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn2+ ions and partially restored by Co2+ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co2+ ions only. Zn2+ ions did not activate this enzymatically inactive protein. The Co2+-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.Abbreviations AA ascorbic acid - DEAE diethylamino ethyl - DHAA dehydroascorbic acid - EDTA ethylene-diaminetetra-acetate - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - HMP hexose monophosphate - P i inorganic phosphorus - pNPP p-nitrophenylphosphate - pNP p-nitrophenol - Tris Tris(hydroxymethyl)-aminomethane  相似文献   

12.
A composite plasmid was constructed using the broad‐host‐range vector pRK293 and the plasmid pPM9, the latter one harbouring a gene encoding the Nap A acid phosphatase from M. morganii. The recombinant construction was transformed and expressed in E. coli MC1061. Transformant clones were selected and characterized, showing that the relative orientation of both original plasmids with respect to each other affected the expression of the gene, with one of the plasmids (pT4) expressing significantly lower values of activity than the opposite orientation construction (pT5). Zymograms developed to detect acid phosphatase activity also corroborated the gene expression in the E. coli host. The genetic constructions (pT4 and pT5) were transferred to B. cepacia IS‐16 by conjugation. The same effect of the original plasmid orientation in the construction was corroborated in the B. cepacia IS‐16 strain. Compared with the strain lacking the recombinant plasmid, no signifi‐cant improvement of cell‐bound enzymatic activity was achieved by the exconjugant harbouring pT5. However, a significant increase in the extracellular enzyme activity was detected in the recombinant strains. Nometabolic load due to the presence of the recombinant plasmid was detected in both E. coli and Burkholderia hosts.  相似文献   

13.
R. Moore  D. B. Walker 《Protoplasma》1981,109(3-4):317-334
Summary In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.Supported in part by grants from the Academic Senate of UCLA, Sigma Xi, the American Philosophical Society, and the URC of Baylor University.  相似文献   

14.
Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.  相似文献   

15.
An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp. was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50. Both holoenzymes had a molecular mass of 103–108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric. Both enzymes had a pI ≈ 9.0 and were immunologically cross-reactive. There were minor differences in amino acid composition and in peptide maps following tryptic digest. The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0. Phosphatase I was more resistant to mechanical shear, γ-irradiation, high temperature, and toxins (F and formaldehyde). Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II. Phosphatase II had a lower K m and a lower V max for glycerol 2-phosphate hydrolysis. The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme. Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses. Revision received: 22 August 1997 / Accepted: 16 September 1997  相似文献   

16.
A large number of bacteria were searched for the activity of the synthesis of pyridoxine 5′-phosphate by the transphosphorylation between pyridoxine and p-nitrophenyl phosphate. Several properties of the transphosphorylation by the partially purified enzyme prepared from one of the isolated bacteria, Escherichia freundii K–1, were investigated accompanying with phosphatase activity. The behavior of the phosphotransferase and phosphatase activities in various reaction conditions were almost parallel. It was pointed out that the transphosphorylation might be catalyzed by the function of acid phosphatase. The phosphoryl donor specificity for the enzyme system was found to be broad.

The enzyme which catalyzed the transphosphorylation of pyridoxine accompanying with the hydrolyzation of phosphoryl donor substrates was purified and crystallized from the cell free extract of Escherichia freundii K–1. The purification procedures involved heat treatment, ammonium sulfate fractionation and DEAE-cellulose, hydroxylapatite, and CM-sephadex column chromatographies. The crystalline enzyme showed the sedimentation coefficient of 7.5 S and the diffusion coefficient of 6.15 × 10?7 cm2/sec. The molecular weight was calculated to be 120,000. Several properties of the purified enzyme were also investigated. It was recognized that the transphosphorylation of pyridoxine might be catalyzed by the action of acid phosphatase.  相似文献   

17.
A enzyme that catalyzed the specific formation of ascorbic acid-2-phosphate (AsA2P) from ascorbic acid (AsA) and adenosine-5′-triphosphate (ATP), was purified 3,200-fold to homogeneity from a cell extract of Pseudomonas azotocolligans. The purified enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and consisted of a single polypeptide with a molecular weight of about 30,000. Of phosphoryl donors tested, p-nitrophenylphosphate (p-NPP) and pyrophosphate (PPi) were as effective as ATP. Optimal pHs for the phosphorylating activity were around 4.0 and 5.5 when PPi and ATP were used as phosphoryl donors, respectively. The Km for AsA was 147 mm. The enzyme activity was inhibited by Cu2+, but not by sulfhydryl reagents.

The enzyme simultaneously had phosphatase activity at weakly acidic or neutral pH and the Km for p-NPP in the phosphatase activity was 0.38 mm. The enzyme was tentatively named “ascorbic acid phosphorylating enzyme.”  相似文献   

18.
Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ~40 times more active than the wild-type EAP. At the same time, the Km value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the kcat/Km ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.  相似文献   

19.
The degradation of yolk granules during the development of Artemia was studied. The results obtained suggest that lysosomes are involved in the process. In homogenates of embryos and larvae at different stages of development, the distribution of 2 lysosomal markers, acid phosphatase and cathepsin B, was studied by sucrose isopycnic gradient centrifugation. Three peaks of enzyme activity of densities > 1.3 and around 1.25 and 1.18 were observed. As revealed by electron microscope analysis, the 3 peaks were found to be associated with increasingly degraded yolk structures which stained for acid phosphatase. The process can be mimicked in vitro by incubating isolated yolk granules and lysosomes. The enzyme activity levels of the 3 peaks observed during development presented an oscillatory pattern, suggesting that degradation of yolk is cyclic. Five cycles of degradation were observed during the initial 60 hr of development.  相似文献   

20.
Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas β-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.  相似文献   

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