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1.
Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed. 相似文献
2.
The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed. 相似文献
3.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,736(1):73-78
(1) Contrary to what has usually been assumed, (Na+ + K+)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na+ + Mg2+ to ADP-NH2 and Pi. The activity is ouabain-sensitive and is not detected in the absence of either Mg2+ or Na2+. The specific activity of the Na+ + Mg2+ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K+. The activity is not stimulated by K+, nor can K+ replace Na+ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na+ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The Km value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH2]P are not detectably hydrolysed by (Na+ + K+)-ATPase in the presence of Na+ + Mg2+. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg2+ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na+ + Mg2+ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na+ + K+)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C. 相似文献
4.
Exposure of canine myocardial tissue homogenates to Me2SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na+-K+ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me2SO provides a similar enhancement of Na+-K+ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me2SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me2SO at 0 °C. The results suggest that disruption of the CPK-Na+-K+ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations. 相似文献
5.
Reisin I.L. and Rotunno C.A. 1981. Water and electrolyte balance in protoscoleces of Echimcoccus granulosus incubated in vitro: General procedures for the determination of water, sodium, potassium and chloride in protoscoleces. International Journal for Parasitology11: 399–404. Protoscoleces of E. granulosus (sheep strain) were incubated in vitro at 37°C in Ringer Krebs solution (RKS) for up to 3 h. When they were briefly washed in sucrose 0.3 M at 4°C, the water and electrolyte contents were: 1.768 ± 0.034 mlg?1 d.w. for water content and 123 ± 2, 209 ± 2 and 78 ± 2 μmolg?1 d.w. for Na+, K+ and Cl? respectively. When protoscoleces were not washed in sucrose solution but were spun down from RKS, the K+ content suffered a very small change but larger values for Na+ and Cl+ contents were obtained. These higher Na+ and Cl? contents are attributed to the RKS ions retained in the trapping space. The steady state distribution of Na+ and K+ in the protoscoleces incubated at 37°C indicates the activity of an active transport mechanism. 相似文献
6.
Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme ??45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70°C, respectively. The metal ions Na+, K+, Ca2+, and Co2+ showed partial inhibition of the activity, while Mn2+ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55?C70°C. It presented the highest stability after incubation at 55°C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60°C for 60 min as well as at 65 and 70°C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli. 相似文献
7.
《Anaerobe》2002,8(5):259-267
In the present study, factors influencing the synthesis and activity of β-galactosidase of two strains of Propionibacterium acidipropionici with some probiotic properties are described for the first time. The enzyme 6-phospho-β-D-galactosidase of the PEP-PTS system was not detected, suggesting that P. acidipropionici metabolize lactose only by using β-galactosidase. The highest enzymatic activities were obtained from cultures developed in a basal broth medium containing 1.0% sodium lactate or 0.25% lactose. Maximum β-galactosidase activity from cell-free extracts of the strains was obtained at pH 7.0 and 50°C, but a high activity was even detected at 37°C. The enzyme was competitively inhibited by lactose and activated by glucose and sodium lactate. The remaining activities after heating cell-free extracts up to 20 min at 60°C were 70% and 25% of untreated control activities for P. acidipropionici Q4 and CRL 1198, respectively. Cations like Mg2+, Mn2+, Li+, Na+, and K+ acted as stimulators of the β-galactosidase activity whereas Ca2+, Co2+, Ni2+, Hg2+ and Cu2+ showed inhibitory effect in different extent. These results suggest that the environmental conditions commonly present in the human's intestine may be adequate for the synthesis and activity of β-galactosidase from these strains of Propionibacterium. The enzyme resist the cooking temperature of Swiss-type cheeses in different extent depending on the strain tested and most of the cations present in milk stimulate the enzymatic activity. Our results suggest that a cheese would be an appropriate vehicle for delivery of β-galactosidase from propionibacteria to the host and efforts to develop a Swiss-type probiotic cheese for lactose intolerant persons should be done. 相似文献
8.
SYLVIA LINDBERG 《Physiologia plantarum》1976,36(2):139-144
Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na+ and K+ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na+ and K+ are suggested. 2) In the absence of K+, Na+ acts as an uncompetitive modifier raising the apparent Km and Vmax for MgATP. 3) In the absence of Na+, K+ activates non-competitively with respect to MgATP. Thus K+ increases Vmax but does not affect the apparent affinity constant. 4) K+ and Na+ double the rate constants. 5) In the presence of Na+ or K+, Mg2+ in excess acts as a weak inhibitor to Na+ and/or K+ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C. 相似文献
9.
Moreira K.A. Albuquerque B.F. Teixeira M.F.S. Porto A.L.F. Lima Filho J.L. 《World journal of microbiology & biotechnology》2002,18(4):309-315
The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K+, Na+, and Mg++ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg). 相似文献
10.
The lesser mealworm, Alphitobius diaperinus (Panzer), is the main insect pest in the poultry industry, thus causing serious damage to production. In this work, the properties of midgut α-amylase from larvae of A. diaperinus were characterized, and its in vitro activity to proteinaceous preparations from different cultivars of common bean (Phaseolus vulgaris) was determined, as well as the amylolitic activity of insects reared on different types of poultry diet. In order to establish some assay conditions, time course and enzyme concentration upon the reaction rate were determined. Product proceeded linearly with time, and the activity was directly proportional to the enzyme concentration. Banding patterns in mildly denaturing electrophoresis showed a single band with apparent molecular weight of 42 kDa. α-Amylase reached optimal temperature at 45°C and pH 5.0 as the optimal one. It maintained 34.6% of the activity after being kept at 60°C for 5 min, and 23%, after 60 min. However, at 80°C, only 14 and 6% remained after 5 and 60 min, respectively. The presence of Ca2+ and Na+ ions decreased the enzyme activity at concentrations higher than 2 and 100 mM, respectively. The activity was significantly inhibited by some proteinaceous extracts from common bean cultivars, and it declined with increasing proteinaceous concentration. No significant difference was observed when the amylolytic activity was determined in A. diaperinus reared on different poultry diets, offered to broilers in the starter, grower, finisher, and layer phases. 相似文献
11.
The activity of the soluble enzyme CPK and the membrane bound enzyme Na+-K+ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na+-K+ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer. 相似文献
12.
H.K. Kimelberg 《生物化学与生物物理学报:生物膜》1975,413(1):143-156
The (Na++K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na++K+)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg2+: ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (Ea) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the Ea below this was very high (> 100 kcal/mol).Incresed Mg2+ concentrations at Mg2+: ATP ratios in excess of 1:1 inhibited the (Na++K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na++K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and Ea values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km for ATP.Since both of cholesterol and Mg2+ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context. 相似文献
13.
The specific activity of (Na+ + Mg2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na+ + K+ + Mg2+)-dependent ATPase in both cases.(Na+ + Mg2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na+ + Mg2+)-dependent ATPase activity of gills arises from the presence of a (Na+ + K+ + Mg2+)-dependent ATPase. 相似文献
14.
Reisin I.L., Rabito C.A. and Cantiello H.F. 1981. Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: effect of metabolic inhibitors. International Journal for Parasitology 11: 405–410. The effects of metabolic inhibitors on the Na+, K+, Cl? and water balance of protoscoleces of Echinococcus granulosus (sheep strain) were studied in vitro. The protoscoleces were incubated at 37°C in Ringer Krebs solution for 3 h in the presence of iodoacetate, 3 mM (IA); potassium cyanide, 3 mm (KCN); 2?4 dinitrophenol, 0.2 mm (DNP); ouabain, 10?M or ethacrynic acid 0.5 mm. The effects of IA and/or KCN on the water and electrolyte balance were tested at high (0.95 × 105Pa) and low (0.05 × 105 Pa) oxygen tensions. Inhibitors produced a decrease in K+ as well as an increase in Na+ contents. At both high and low O2 tensions the Na+ balance was greatly altered by IA, the action of which could be already observed during the first hour of treatment. The cations did not reach a steady state balance during 3 h of incubation. At high oxygen tension Na+ and K+ balance was also altered by KCN or DNP though their actions were not as marked as that of IA. Ouabain affected the Na+ and K+ contents that reached new steady state distribution between 1.5 and 3 h of treatment while water and electrolyte contents were not modified by ethacrynic acid. In all the experiments no changes in Cl? and water contents were observed. It is concluded that the energy required to maintain the Na?K balance mechanisms within protoscoleces is largely provided by the anaerobic glycolytic pathway and that the aerobic oxidative pathway contribution to the energy balance is only accessory. 相似文献
15.
G.Rodríguez de Lores Arnaiz M.Antonelli de Gómez de Lima 《Neurochemistry international》1983,5(1):117-124
The activity of Na+K+-ATPase in the membranes of nerve endings isolated from rat cerebral cortex was inhibited by dopamine. On the contrary, when the soluble fraction from cortical homogenates was added, dopamine stimulated enzyme activity. By varying the concentration of the soluble fraction present in the incubation medium for Na+K+-ATPase assay, it was possible to establish that this fraction modulates those effects of dopamine on Na+K+-ATPase.The preincubation of the membranes with N-ethylmaleimide under conditions in which the Na+K+-ATPase activity was not inhibited (5 × 10?5 M for 10 min at 37°C), prevented both the inhibitory and the stimulatory effects of dopamine observed without or with the soluble fraction from brain respectively.These results suggest that dopamine probably acts on regions of the protein containing -SH groups, different from those sites responsible for the catalytic activity of the enzyme. 相似文献
16.
《Enzyme and microbial technology》2008,42(6-7):828-834
An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The kcat/Km value for alginate (heterotype) was 1.7 × 106 s−1 M−1. The enzyme activity was completely lost by dialysis and restored by addition of Na+ or K+. The optimal activity exhibited in 0.1 M of Na+ or K+. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA). 相似文献
17.
This paper describes a partially heat-labile, naturally occurring bactericidal factor in cell-free hemolymph preparations obtained from Limulus polyphemus. This bactericidal activity has been shown to be directed against two Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae, whereas it had no effect on the Gram-positive bacteria tested, Micrococcus lysodeikticus and Staphylococcus aureus. Maximal bactericidal activity of this factor was observed at 30°C and pH 6.0. Since complement and antibody are required for antimicrobial activity in vertebrate sera, the activity of this factor in the presence of various complement inhibitors was assayed. The bactericidal activity of Limulus hemolymph is abolished by treatment with endotoxin; however, other anticomplementary substances were without effect. Limulus amebocyte lysate is known to contain protein which may be precipitated by endotoxin; it is possible that the reduction of bactericidal activity produced by endotoxin treatment may be caused by the denaturation of a bactericidal protein moiety produced by the hemocytes. 相似文献
18.
Xiawei Wei Bin Shao Zhiyao He Tinghong Ye Min Luo Yaxiong Sang Xiao Liang Wei Wang Shuntao Luo Shengyong Yang Shuang Zhang Changyang Gong Maling Gou Hongxing Deng Yinglan Zhao Hanshuo Yang Senyi Deng Chengjian Zhao Li Yang Zhiyong Qian Jiong Li Xun Sun Jiahuai Han Chengyu Jiang Min Wu Zhirong Zhang 《Cell research》2015,25(2):237-253
Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na+ overload was found to accompany the cell death. Depletion of Na+ in culture medium or pretreatment of cells with the Na+/K+-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na+/K+-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na+/K+-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na+/K+-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery. 相似文献
19.
Coelomic fluid from the common British sea urchin Echinus esculentus was shown to kill in vitro a black-colonied, agar-digesting marine Pseudomonas sp. (strain No. 111). The bactericidal reaction, which was monitored by viability counting on marine agar plates, took 48 hr to reach full expression. An incubation temperature of 4°C gave the most complete and consistent killing. Clotting of the coelomocytes did not influence bactericidal activity. The vast majority of nearly 200 specimens of E. esculentus from the Clyde Sea area of Scotland yielded coelomic fluid with bactericidal activity, and there was no apparent seasonal variation. Activity was localized in the coelomocytes and could be obtained in cell-free extracts by ultrasonic disruption of these cells. The bactericidal activity in these extracts was lost during dialysis but was only partially destroyed by heating for 30 min at 56° or 100°C. 相似文献