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1.
Inger Schousboe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,543(3):373-382
After exposure to serum, an agglutination of mitochondria from yeast, liver, heart and kidney was observed. The degree of agglutination was dependent on the ratio between the amount of serum protein and mitochondrial protein. The serum protein which induced agglutination was bound irreversibly to the mitochondria, was heat stable and partly resistant to acidification. Maximal agglutination was observed at an ionic strength equal to 40 mM Tris, at pH 6.0–7.5. Preincubation of mitochondria with calcium ions at slightly acidic pH prevented the agglutination. Neuraminidase treatment of either serum or mitochondria had no effect upon the agglutination. 相似文献
2.
(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. 相似文献
3.
The uncoupler-induced inactivation of H+-ATPase in hepatoma 22a and mouse liver mitochondria has been studied. The dependence of this process on delta microH, and pH and ATP was established. The inactivated ATPase could be reactivated at alkaline pH values in the absence of ATP. These data indicate that the inactivation is apparently caused by the natural protein inhibitor. ATP- and pH-dependent decrease of ATPase activity is also observed after Lubrol-WX disruption of mitochondria. It can be proposed that practically all ATPase molecules in hepatoma mitochondria are in a catalytically active complex with the protein inhibitor. At low delta microH this complex is inactivated via reversible pH-dependent and irreversible ATP-dependent rearrangements. The pH-dependent rearrangement of the isolated protein inhibitor from hepatoma mitochondria is also observed. 相似文献
4.
Activation of NAD-linked malic enzyme in intact plant mitochondria by exogenous coenzyme A 总被引:3,自引:0,他引:3
O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation. 相似文献
5.
I Schousboe 《Biochimica et biophysica acta》1976,450(2):165-174
A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria. 相似文献
6.
1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates. 相似文献
7.
R J Pavlica C B Hesler L Lipfert I N Hirshfield D Haldar 《Biochimica et biophysica acta》1990,1022(1):115-125
The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein. 相似文献
8.
《Journal of Fermentation and Bioengineering》1992,73(6):435-439
Hen egg-white lysozyme (HEL), ovalbumin and bovine serum albumin (BSA) were covalently immobilized onto styrene/methacrylic acid [P(St/MAA)] copolymer latex particles by the carbodiimide method. The initial rates of the immunological agglutination of these particles initiated by the addition of antibodies were quantified by the absorbance changes at a wavelength of 680 nm. The sensitivity of the immunological agglutination of the particles with covalently immobilized antigens was higher than that with physically adsorbed ones. The immunological agglutination kinetics showed a similar tendency irrespective of antigen-antibody systems. That is, the initial agglutination rates (i) increased with increasing immobilized amount of antigens, (ii) were largest in the ionic strength range of 0.02 to 0.05 at pH 7 and (iii) decreased with increasing pH. These results indicate that the electrostatic interactions of particle-particle and particle-antibody are main factors which control the immunological agglutination. On the other hand, the sensitivity of the immunological agglutination increased with increasing molecular size of antigens. 相似文献
9.
Hypothyroidism in rats does not lower mitochondrial ADP/O and H+/O ratios. 总被引:3,自引:3,他引:0
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We investigated reports that mitochondria isolated from hypothyroid rats have decreased ADP/O and H+/O ratios. We observed no decrease in the H+/O ratio in mitochondria from hypothyroid rats, in the presence of either 2% (w/v) fatty-acid-free bovine serum albumin or 100 nM free Ca2+. The ADP/O ratio in mitochondria isolated from hypothyroid rats in the presence of 2% fatty-acid-free bovine serum albumin was measured. Under normal experimental conditions we found no decrease in the ADP/O ratio, relative to that measured for littermate controls. At the low concentrations of mitochondrial protein used in the previously reported studies, the ADP/O ratio of mitochondria from hypothyroid rats was decreased, whereas that for control rats was only slightly decreased. The difference between the ADP/O ratios measured for mitochondria form hypothyroid rats and from control rats under these conditions was eliminated by inhibition of endogenous adenylate kinase. We suggest that the lowering of the apparent ADP/O ratio in mitochondria from hypothyroid rats at low concentrations of mitochondrial protein is an experimental artefact resulting from the breakdown of ADP to AMP. 相似文献
10.
Chronic ethanol ingestion induced a 47% increase in the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (nucleoside-2':3'-cyclic-phosphate 2'-nucleotidohydrolase, EC 3.1.4.37) in whole mitochondria. Both inner and outer mitochondrial membranes showed increased (cyclic nucleotide)phosphohydrolase activity, but the inner was increased 94% compared to 67% for the outer. Techniques which disrupt membrane structure increased (cyclic nucleotide)phosphohydrolase activity. After these treatments, whole mitochondria from ethanol-treated animals still showed a 50% increase in activity. This increase may be related either to an inherent increase in the resistance of (cyclic nucleotide)phosphohydrolase to protein degradation or turnover, or to ethanol-induced membrane changes. An increase in (cyclic nucleotide)phosphohydrolase reaction medium pH was observed when freshly isolated, highly-coupled mitochondria were used. The total increase in pH was about 2-fold greater in the controls compared to the ethanol-treated mitochondria. It is suggested that the smaller initial increase in pH and the greater activity of (cyclic nucleotide)phosphohydrolase in the mitochondria from the ethanol-treated animals relate to previously observed changes in the lipid and protein composition of the mitochondrial membranes. In addition, (cyclic nucleotide)phosphohydrolase may represent an excellent marker for membrane integrity. 相似文献
11.
Claus-Jens Doersen Eric J. Stanbridge 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,698(1):62-69
The growth of HeLa cells in Hepes-buffered medium was significantly more sensitive to the inhibitory effects of erythromycin than in medium buffered by the more conventional bicarbonate-CO2 system. Since growth inhibition by erythromycin became more pronounced as the pH of the medium was increased the difference in erythromycin sensitivity between the Hepes-buffered medium vs. the bicarbonate-CO2-buffered medium is most likely due to pH effects. The relative growth sensitivity to erythromycin of ERY2301, an erythromycin-resistant mutant of HeLa, was also affected by elevated pH of the growth medium. However, ERY2301 cells were able to proliferate to a greater extent in the presence of erythromycin than HeLa cells grown under the same conditions. The selective growth advantage of ERY2301 (in the presence of erythromycin) is best seen in medium of pH 7.4, or in the Hepes-buffered medium. In vitro protein synthesis by intact mitochondria isolated from HeLa cells was relatively insensitive to erythromycin inhibition at pH 7.4 and 7.6, but at high pH values was inhibited approx. 50%. Although the erythromycin sensitivity of ERY2301 mitochondrial protein synthesis was also affected by increasing the pH, the incorporation of [3H]leucine was more resistant to erythromycin than that observed for HeLa mitochondria over the pH range tested. Increasing the concentration of erythromycin at a given pH did not result in a further increase in the inhibition of either HeLa or ERY2301 mitochondrial protein synthesis. When the mitochondrial membranes were disrupted by Triton X-100, erythromycin inhibition of HeLa mitochondrial protein synthesis was pH dependent and, at the lower pH values tested, greater inhibition was observed as the erythromycin concentration was increased. ERY2301 mitochondrial protein synthesis under the same conditions displayed a high level of erythromycin-resistant activity independent of both pH and erythromycin concentration. It is suggested that, as has been proposed for bacterial systems, only the non-protonated molecule of erythromycin is effective in inhibiting mitochondrial protein synthesis. The ability of erythromycin to permeate the mitochondrial membranes and the plasma membres may also be facilitated by a higher pH. 相似文献
12.
A G Tahiliani 《The Journal of biological chemistry》1989,264(31):18426-18432
Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH. 相似文献
13.
Jarmuszkiewicz W Hryniewiecka L Sluse FE 《Journal of bioenergetics and biomembranes》2002,34(3):221-226
Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence. 相似文献
14.
Calvin B. Coulter 《The Journal of general physiology》1922,4(4):403-409
1. The addition of blood serum displaces the optimum for agglutination of red blood cells in a salt-free medium to the reaction characteristic of flocculation of the serum euglobulin. 2. This effect is not due merely to a mechanical entanglement of the cells by the precipitating euglobulin, since at reactions at which the latter is soluble it protects the cells from the agglutination which occurs in its absence. 3. A combination of some sort appears therefore to take place between sheep cells and sheep, rabbit, and guinea pig serum euglobulin, and involves a condensation of the serum protein upon the surface of the red cell. 4. At the optimal point for agglutination of persensitized cells both mid- and end-piece of complement combine with the cells. 5. Agglutination is closely related to an optimal H ion concentration in the suspending fluid, and probably of the cell membrane, and not to a definite reaction in the interior of the cell. 相似文献
15.
Latex immunoassay of human serum Lp(a+) lipoprotein 总被引:1,自引:0,他引:1
A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108). 相似文献
16.
T A Link W R Hagen A J Pierik C Assmann G von Jagow 《European journal of biochemistry》1992,208(3):685-691
The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex. 相似文献
17.
Defence parameters of non-infected clams (Ruditapes decussatus) and clams heavily infected with Perkinsus atlanticus were assessed. Cellular (haemocyte density and phagocytic activity) and humoral (lysozyme and anti-bacterial activities, protein concentration and agglutination titre) parameters were measured in clams collected in an area enzootic for P. atlanticus. The infection intensity of each clam was assessed, and the immune parameters measured in the most infected clams were compared with those measured in the non-infected ones. Only the serum anti-bacterial activity and the agglutination titre were significantly different between infected and non-infected clams. The phagocytic rate, haemocyte density, lysozyme concentration and protein concentration were not statistically different but they showed the same trend in the two trials performed. Phagocytic rate, haemocyte concentration and anti-bacterial activity were higher in non-infected clams, while they had lower lysozyme concentration, serum protein concentration and agglutination titre than infected clams. Although infected and healthy clams were not different for every parameter measured, probably due to the high variability among individuals, P. atlanticus seems to affect the clam immune system, at least in advanced stages of the infection. 相似文献
18.
On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons 总被引:2,自引:0,他引:2
The inner membrane of mitochondria possesses a pH-regulated anion uniporter which is activated by depletion of matrix divalent cations with A23187 (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). It is now shown that Cl- transport through this pathway is inhibited by Mg2+ and Ca2+. There appear to be two sites for inhibition by Mg2+. One has an IC50 = 38 microM at pH 7.4 and appears to be on the inside since it is only observed in the presence of A23187 (10 nmol/mg). The other has an IC50 = 440 microM at pH 7.4 and appears to be on the outside since it is observed in mitochondria pretreated with very low doses of A23187 (0.25 nmol/mg or less) and in A23187-pretreated mitochondria washed to remove A23187. Ca2+ is found to inhibit anion uniport in the presence or absence of A23187 with an IC50 of about 17 microM. In contrast to these findings Cl- uniport, activated by addition of valinomycin to respiring mitochondria without depleting endogenous Mg2+ is found to be very insensitive to exogenous Mg2+, being inhibited with an IC50 of 3.2 mM. This is explained by examination of the pH dependence of the Mg2+ IC50 in non-respiring mitochondria. The internal IC50 is found to be pH-dependent, rising to about 250 microM at pH 8.4. The external IC50 is also pH-dependent, rising to 2.5 mM or above at pH 8.4. These data are consistent with a model in which Mg2+ can only bind to the protein when it is protonated at a site with a pK of about 6.8 located in the matrix. Thus, both the intrinsic activity of the uniporter and its inhibition by Mg2+ appear to be regulated by matrix protons. This makes the rate of anion uniport much more sensitive to changes in matrix pH which is physiologically advantageous for its proposed role in volume homeostasis. 相似文献
19.
31P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external Pi. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal Pi peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external Pi and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal Pi increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons. 相似文献
20.
六价铬污染对水车前叶片生理生化及细胞超微结构的影响 总被引:28,自引:3,他引:28
以培养在铬 ( VI)浓度梯度的污水中 6 d的水车前为实验材料 ,分析了叶绿素含量和抗氧化酶系统的变化 ,并用透射电镜观察了叶细胞超微结构的损伤。实验结果表明 :( 1 )叶绿素含量在 0 .1 mg/L浓度达到最高 ,而后下降。而可溶性蛋白则一直成下降趋势 ;( 2 ) SOD(超氧化物歧化酶 )、POD(过氧化物酶 )、CAT(过氧化氢酶 )活性影响明显 :三者分别在 0 .1 mg/L、1 0 mg/L、和 1 mg/L处理浓度有抗性峰出现 ,随处理浓度的继续增大则下降 ;( 3)对超微结构造成不可逆损伤 ,特别是对叶绿体、线粒体和细胞核 :叶绿体膨胀 ,被膜破裂和消失 ,叶绿体解体 ;线粒体脊突膨胀和空泡化 ;细胞核变形 ,染色质凝集和核质解体 ,核膜破裂。并根据实验结果初步讨论了重金属对植物的毒害机制 相似文献