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1.
We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.  相似文献   

2.
ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-32P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn2+ or Mg2 and was inhibited or had greatly reduced activity with 5 mM Ca2+, Co2-, Cu2+ or Zn2+. Incorporation was much lower when [γ-32P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-32P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [32P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.  相似文献   

3.
Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P2). Its activity is inhibited by Ca2+ and Zn2+ and is activated by Mg2+, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.  相似文献   

4.
Abstract: The association of Mg2+ ions with mitochondria isolated from guinea pig cerebral cortex is investigated and resolved into two components, that bound to the surface of both the outer and the inner membranes and that transported into the mitochondrial matrix. When rotenone-treated mitochondria are preincubated in a Mg2+ -containing medium, Mg2+ binding can be measured and actual Mg2+ transport determined after the addition of succinate. Mg2+ uptake as well as retention within mitochondria is an energy-dependent process linked to substrate oxidation. EGTA completely prevents Mg2+ uptake, while the Ca2+ uniporter inhibitor Ruthenium Red, along with prevention of Mg2+ uptake, induces a slow efflux of accumulated Mg2+ ions. These findings suggest that both inward and outward Mg2+ movements follow Ca2+ fluxes across the mitochondrial membrane. Modulation of Mg2+ movements by mitochondria is therefore suggested to occur within nerve terminals.  相似文献   

5.
The Mg2+-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg2+ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg2+ has three different roles: (i) Mg2+ stimulates transport activity of the PPase. (ii) Mg2PPi inhibits PPase-mediated H+ transport, (iii) MgPPi* (= MgPPi2-+ MgHPPi-) is the substrate with an apparent K1/2= 5–10 μM, with no discrimination between MgPPi2- and MgHPPi-.  相似文献   

6.
Dehydrins are hydrophilic proteins that accumulate during embryogenesis and osmotic stress responses in plants. Here, we report an interaction between citrus dehydrin Citrus unshiu cold-regulated 15 kDa protein (CuCOR15) and DNA. Binding of CuCOR15 to DNA was detected by an electrophoretic mobility shift assay, a filter-binding assay and Southwestern blotting. The binding was stimulated by physiological concentrations of Zn2+, but little stimulation occurred when other divalent cations, such as Mg2+, Ca2+, Mn2+, Ni2+ and Cu2+, were substituted for Zn2+. Ethylenediaminetetraacetic acid cancelled the Zn2+-stimulated binding. A binding curve and competitor experiments suggested that the DNA binding of CuCOR15 exhibited low affinity and non-specificity. Moreover, tRNA competed with the DNA binding. Histidine-rich domains and a polylysine segment-containing domain participated in the DNA binding. These results suggest that CuCOR15 can interact with DNA, and also RNA, in the presence of Zn2+. Dehydrin may protect nucleic acids in plant cells during seed maturation and stress responses.  相似文献   

7.
Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na2SO4. Na2SO4 was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na+ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na2SO4 reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na+, K+, Mg2+ and Ca2+ content were correlated to initial Na+ concentration in the medium, not salt type. Accumulation of Na+ was accompanied by loss of K+ and Mg2+. Six to seven times less sulfate was measured in callus grown on Na2SO4 than chloride in callus grown on similar concentrations of NaCl.  相似文献   

8.
Deoxyribonuclease activities in Myxococcus coralloides D   总被引:2,自引:1,他引:1  
Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+); however, other metal ions (Fe2+, Ni2+, Zn2+) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).  相似文献   

9.
Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4)2SO4 precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)-1 min-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+). In the presence of MnCl2+ other divalent cations (Mg2+, Ca2+, Ba2+, Hg2+ and Cd2+) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μ M , the Km for PRPP was 0.29±0.06 m M and the Ki for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 m M  相似文献   

10.
The binding of plasmin to Streptococcus uberis strain 0140 J was optimal in the pH range 5·0–5·5. Plasmin binding decreased exponentially with increasing NaCl concentration (0–0·8 mol l−1), reaching a minimum at NaCl concentrations exceeding 0·55 mol l−1. Neither K+, Mg2+ nor the metal chelator EDTA had any effect on the interaction. Plasmin binding was prevented, in a concentration-dependent manner, by the amino acids lysine, arginine and ε-aminocaproic acid. Bound plasmin was also eluted from the bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium in a time- and temperature-dependent fashion, the rate of plasmin loss increased with increasing temperature over the range 4–55 °C, and the elution of plasmin from live and heat-killed bacteria was similar. Cell-bound plasmin was only partially inhibited by the physiological inhibitor α2-antiplasmin whereas the serine protease inhibitor aprotinin, and the active site titrant p -nitrophenyl- p -guanidiniobenzoate, inhibited the activity of the cell-bound plasmin by more than 95%.  相似文献   

11.
An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca2+ concentration required for PLI and PLII activity was determined to be 0·5 mmol l−1. The Ca2+ requirement could not be replaced by other metal cations such as Mg2+, Cu2+, Zn2+, Fe3+ and Co2+. Optimal pH for activity was determined to be between 8·5 and 9·0. The K m values for sodium polygalacturonate were 1·28 and 1·11 mg ml−1 for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl2 or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.  相似文献   

12.
A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H+ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H+-ATPase (EC 3.6.1.35) and ATP-dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H+ transport. The increase of ATP-dependent H+ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl-sensitive calli, plasma membrane H+-ATPase from calli tolerant to 50 m M NaCl showed a lower Km for Mg2+-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H+-ATPase for the substrate ATP and stimulates the H+-pumping activity of this enzyme without modifying its phosphohydrolytic activity.  相似文献   

13.
The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg2+. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg2+ were chromatographically resolved from spinach leaves. The Mg2+-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P2, was highly specific, and was strongly inhibited by fructose-2,6-P2. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg2+. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P2, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P2: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P2, and with fructose-1,6-P2, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg2+-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg2+-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.  相似文献   

14.
Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd2+ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd2+ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na+ and Cl was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na+/Cl ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca2+ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K+ and Mg2+ was only little affected by NaCl. Excretion of Li+ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li+ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .  相似文献   

15.
Abstract: Purified chromaffin granules from bovine adrenal medulla bound a small group of medullary cell cytosol proteins at micromolar levels of Ca2+ and physiological levels of K+, Mg2+, and Mg-ATP. The bound proteins had molecular weights of 33,000-37,000 and 70,000-71,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and did not correspond with any previously reported cytosolic components of chromaffin cells. The new proteins were eluted from intact granules or resealed granule membranes at 0.1 μ M Ca2+; binding was half-maximal at 2.6 μ M . Adsorption and elution in this manner resulted in a high degree of purification of the new proteins that were minor components of the original cytosol. Partially purified fractions enriched in the 33,000-37,000 and 70,000-71,000 proteins bound 45Ca2+ at submicromolar levels in the presence of millimolar Mg2+. Calmodulin was also bound by the granule membranes and was present in trace amounts in cytosol eluates from granules, but it did not bind to the new proteins in the presence of calcium ions. The possible significance of the new proteins to calcium-mediated secretion from chromaffin cells is discussed.  相似文献   

16.
Abstract Crystalline 3-methylaspartase (EC 4.3.1.2) from Escherichia coli strain YG1002 that had been isolated from soil was characterized. The enzyme activity was induced when the organism was grown statically on medium containing ( S )-glutamic acid. Its molecular mass is about 84 kDa, and it may be composed of two identical subunits of 42 kDa. The enzyme requires both divalent and monovalent cations such as Mg2+ and K+, respectively. The enzyme catalyzes reversible amination-deamination between mesaconic acid and (2 S ,3 S )-methylaspartic acid, which is the best substrate.  相似文献   

17.
Mg2+- and Ca2+-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg2+ was measured. Ca2+-uptake by the roots was mainly passive. Mg2+-uptake mainly active; the passive component of Mg2+-uptake was accompanied by Ca2+-efflux up to 60% of the Ca2+ present in the roots.
In general Mg2+ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg2+-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg2+-uptake was plotted against Mg2+-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg2+ J corresponded with active uptake of a single Mg2+ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg2+ ion. The results are discussed against the background of regulation of the Mg2+-level of the cytoplasm of root cells by transport of Mg2+ by a Mg2+-ATPase to the vacuole, to the xylem vessels, and possibly outwards.  相似文献   

18.
Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with Km values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg2+ concentration. pH had no effect on the Km value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg2+, although it was essential for maximum activity. Mg2+ could not be replaced by Cu2+, Ca2+, Ba2+, Co2+ or Ni2+. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.  相似文献   

19.
Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg2+- and Ca2+-ATPase activities were detected. Orthovanadate-sensitive Mg2+-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca2+, Ca2+-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg2+.
Mg2+-ATPase activity was inhibited by 68% and inhibition of Mg2+ activation by 0.3 m M Ca2+ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg2+-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg2+ activation was as high as without tentoxin. K+-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca2+-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg2+-ATPase are discussed in relation to its influence on K+ transport in wheat seedlings.  相似文献   

20.
The Mg2+ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg2+, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca2+, were used, the fertilization rate remained quite low in the absence of Mg2+. In Strongylo-centrotus intermedius , the lowest concentration of Mg2+ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca2+, whereas that of calcium was 3 mM in the presence of 49 mM Mg2+. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg2+ or Ca2+ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg2+ and/or Ca2+. These results indicate that Mg2+ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.  相似文献   

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