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1.
Addition of Pb 2+ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca 2+ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca 2+. When the incubation media are supplemented with ruthenium red, the effect of Pb 2+ on NAD(P)H oxidation, membrane , and Ca 2+ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb 2+ induces Ca 2+ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca 2+-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb 2+ through the calcium uniporter, since such effect is ruthenium red sensitive. 相似文献
2.
Objective: We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness. Methods: Catalase activity was evaluated by the permanganatometric and calorimetric assays. Results: This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide. Discussion: We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO+ group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds. 相似文献
3.
The effect of lipid peroxidation on the affinity of specific active sites of Na +, K +-ATPase for ATP (substrate), K + and Na + (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl 2 at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na +, K +-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K + and Na + was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na +, K +-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K + will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na +, K +-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process. 相似文献
4.
Summary An investigation has been undertaken to determine whether ionizing radiation might engender racemization of optically active amino acids, along with their usual radiolysis. As prototypes, crystalline D- and L-leucine, as well as aqueous solutions of their sodium salts were exposed to the radiation from a 3000 Ci 60Co -ray source. -ray doses which caused about 68% radiolysis of solid leucine left a residue which was about 5% racemized, while racemization proved even greater at lower doses for the dissolved sodium salts. In aqueous solution both percent degradation and percent racemization of the sodium salts were proportional to -ray dosage within the range employed (1–27 · 10 6 rads). Implications of these observations for the origin of molecular asymmetry by the -decay parity violation mechanism are discussed. 相似文献
5.
Formaldehyde (HCHO) and choline, trigonelline and N -trimethyl-L-lysine (TML) were detected and measured in tissue cultures of Datura innoxia Mill. by TLC and OPLC. The higher level of HCHO in young tissues appears to originate from the methylation reactions. This is in correlation with the high level of fully N-methylated substances. In spite of this the level of the fully N-methylated substances decreased with the aging of tissue. HCHO level also decreased with aging. However, in old tissues the amount of HCHO again increased. This HCHO originates mainly from demethylation reactions. It seems that there is a dynamic relationship between methylation/ demethylation processes based on HCHO reactions and cell proliferation.Abbreviations HCHO
formaldehyde
- HPTLC
high-performance thin-layer chromatography
- HS
heat shock
- OPLC
overpressured layer chromatography
- SAM
S-adenosyl-L-methionine
- TML
N -trimethyl-L-lysine
- TLC
thin-layer chromatography
This work was supported by National Science Foundation grant (OTKA T-14141, -17394, -17351 and ETT-119, AMFK-465). 相似文献
6.
Some of the characteristics of unisite hydrolysis of [ 32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [ 32P]ATP bound at the catalytic sites of F 1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [ 32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [ 32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [ 32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [ 32P]ATP/ 32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F 1 in the sense that in a fraction of F 1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [ 32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site. 相似文献
7.
The active species of CO 2 , i.e. CO 2 or HCO
3
–
–(H 2CO 3) utilized by enzymes catalyzing ferredoxin-linked carboxylation reactions was determined. The enzyme investigated was pyruvate synthase from Clostridium pasteurianum (EC 1.2.7.1; Pyruvate: ferredoxin oxidoreductase). Data were obtained which were compatible with those expected if CO 2 is the active species.The dissociation constant (K
S) of the enzyme-CO 2 complex was measured. At pH 7.2 K
Sfor CO 2 of pyruvate synthase was found to be approximately 5 mM.Abbreviations Fd
ferredoxin
No distinctions are made between CO 2, H 2CO 3, HCO
3
–
and CO
3
=
when the symbol CO 2 is used. 相似文献
8.
Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated from the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([] D = +30.5°) and highly viscous ([] = 1430 ml/g). An analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54 : 1). The main chain of this galactomannan consists of 1,4-- D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of - D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by 13C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man–Man units, monosubstituted Gal(Man–Man) and (Man–Man)Gal units taken together, and for the disubstituted Gal(Man–Man)Gal units. 相似文献
9.
This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC 5010 –9M, IC 10010 –6M), while the other compounds exhibited IC 50 values of 5·10 –10 M and IC 100 5·10 –9 M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation. 相似文献
10.
Galactomannan, a polysaccharide with a molecular weight of 660 kDa, was isolated for the first time from the seed of Gleditsia triacanthos f. inermis (yield, 15.4%). Its aqueous solutions were optically active ([]
D
= +31.0°) and highly viscous ([] = 578 ml/g). Analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR and 13C NMR spectroscopy, showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.42 : 1). The main chain of this galactomannan comprises 1,4-- D-mannopyranose residues, 41% of which are substituted at C6 with single residues of - D-galactopyranose. The probability of occurrence in the chain of mannobiose units substituted otherwise, determined experimentally, was 0.16 for the Man–Man unit, 0.50 for the Gal(Man–Man) and (Man–Man)Gal units, and 0.34 for the disubstituted Gal(Man–Man)Gal unit. 相似文献
11.
Summary To assess the active components of sodium flux across toad bladder as a function of transepithelial potential, unidirectional sodium fluxes between identical media were measured before and after adding sufficient ouabain (1.89×10 –3
m) to eliminate active transport, while clamping transepithelial potential to 0, 100 or 150 mV. Evidence was adduced that ouabain does not alter passive fluxes, and that fluxes remain constant if ouabain is not added. Hence, the ouabain-inhibitable fluxes represent fluxes through the active path. Results were analyzed by a set of equations, previously shown to describe adequately passive fluxes under electrical gradients in this tissue, here modified by the insertion of E, the potential at which bidirectional sodium fluxes (
E
and
E
) through the active pathway are equal. According to these equations,
E
and
E
are the logarithmic mean of bidirectional fluxes through the active path at any potential, and the flux ratio in this path is modified by a constant factor Q
ia, which represents the ratio of the bulk diffusion coefficient to the tracer diffusion coefficient in this pathway. The data are shown to conform closely to these equations. Q
ia averages 2.54. Hence, serosal-to-mucosal flux vanishes rapidly as potential falls below E. Mean E in these experiments was 158±1 mV. Thus, linear dependence of net flux in both active and passive pathways on potential is present, even though the sodium fluxes in both paths fail to conform to the Ussing flux ratio equation. Q
i p<1 in the passive path (qualitatively similar to exchange diffusion) and Q
ia>1 in the active path (as in single file pore diffusion). Both of these features tend to reduce the change in serosal-to-mucosal sodium flux induced by depolarization from spontaneous potential to zero potential (short-circuiting). 相似文献
12.
The period length of the leaf movement rhythm of Trifolium repens L. is lengthened by continuously offered cAMP (0.5–1.0 mol m -3) and theophylline (0.5–4 mol m -3). At the higher concentrations this effect is more pronounced and the rhythm damps out faster. Imidazole (0.5 and 1 mol m -3) has no effect on the period length; however, after 5 mol m -3 the rhythm is abolished. Offered as 4 h pulses the resulting phase response curves for cAMP and imidazole are similar and show delays of up to 4 h during the day position of the leaves. Theophylline pulses lead to delays of up to 5 h during closure and advances of up to 3 h during opening. No phase shift is brought about by 4-(3,4-dimethoxybenzyl)-2-imidazolidone. The results do not support the cAMP-model of the circadian clock which has been proposed by Cummings (J. theor. Biol. 55, 455–470; 1975). The effect of the substances tested could, however, be based upon influences on the transport of Ca 2+.Abbreviations ATP
adenosine triphosphate
- cAMP
cyclic adenosine 35 monophosphate
- AMP
adenosine 5 monophosphate
- AC
adenyl cyclase
- PDE
phosphodiesterase
- LL
continuous light 相似文献
13.
Summary The only known habitat of the astaxanthin-containing Phaffia rhodozyma is in slime fluxes of deciduous trees at high altitudes. In this habitat, the function of carotenoids in P. rhodozyma is probably to provide protection against photogenerated antifungal substances in the tree flux such as singlet oxygen ( 1O 2). To investigate the role of carotenoids in P. rhodozyma, genetic selections were employed to determine if carotenogenic yeast strains of P. rhodozyma have enhanced ability to quench 1O 2. Singlet oxygen was generated in liquid culture by the interaction of visible light (-550 nm) with the photosensitizer rose bengal or by the activation of -terthienyl with ultraviolet light (=366 nm). In each case the treatments selected for growth of pigmented strains of P. rhodozyma. Albino (carotenoid-less) or yellow (-carotene producing) strains grew less well in media containing 1O 2. Addition of the 1O 2 quencher sodium azide to the medium with -terthienyl allowed growth of non-pigmented strains. Since the ecological niche of P. rhodozyma is highly specific, we investigated whether extracts of birch trees ( Betula), the original source of P. rhodozyma, contained a compound that would select for pigmented populations of the yeast. When P. rhodozyma strains were exposed to ethyl acetate extracts of Betula papyrifera excited with 366 nm ultraviolet light, only pigmented cells were able to grow. These results suggest that carotenogenesis developed in P. rhodozyma in response to the presence of photoactivatable antifungal compounds produced by the host tree.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues. 相似文献
14.
In the freshwater Chlorophyceae Haematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co 2+ (at least 10 -5 M), Mn 2+ (at least 10 -6 M) and Ag 2+ (at least 10 -4 M), inhibited by Cu 2+ (at least 10 -5 M) and not affected by Zn 2+, Fe 2+ or Mg 2+. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits. 相似文献
15.
Enchytraeus albidus is able to absorb dissolved 14C-labeled neutral amino acids (glycine, L-alanine, L-valine, -aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded -aminoisobutyric acid against external glycine or -aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non- -amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to 14C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus, 14C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S). 相似文献
16.
It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the -amino groups were found to be either preferentially or selectively trimethylated. Reaction with 13C-labeled iodomethane permitted detection and identification of individual trimethylated -amino groups by 13C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free -amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated -amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential 13C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus. 相似文献
17.
Yeast cytochrome c peroxidase was used to construct a model for the reactions catalyzed by the second cycle of nitric oxide synthase. The R48A/W191F mutant introduced a binding site for N-hydroxyguanidine near the distal heme face and removed the redox active Trp-191 radical site. Both the R48A and R48A/W191F mutants catalyzed the H 2O 2 dependent conversion of N-hydroxyguanidine to N-nitrosoguanidine. It is proposed that these reactions proceed by direct one-electron oxidation of NHG by the Fe +4O center of either Compound I (Fe +4O, porph +) or Compound ES (Fe +4O, Trp +). R48A/W191F formed a Fe +2O 2 complex upon photolysis of Fe +2CO in the presence of O 2, and N-hydroxyguanidine was observed to react with this species to produce products, distinct from N-nitrosoguanidine, that gave a positive Griess reaction for nitrate + nitrite, a positive Berthelot reaction for urea, and no evidence for formation of NO . It is proposed that HNO and urea are produced in analogy with reactions of nitric oxide synthase in the pterin-free state. 相似文献
18.
Summary Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 µM EDTA or 10 µM DETAPAC yielding full protection. Ag +, Zn 2+ and Cd 2+ potentiated the redox inactivation promoted by NADPH alone, while Cr 3+, Fe 2+, Fe 3+, Cu +, and Cu 2+ protected the enzyme. The Zn 2+ and Cd 2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn 2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn 2+ or Cd 2+.The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP + also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.Abbreviations DETAPAC
diethylenetriaminepentaacetic acid
- 2,5-ADP-Sepharose-N 6-(6-aminohexyl) adenosine
2,5-bisphosphateSepharose 相似文献
19.
Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg 2+. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect. 相似文献
20.
It has been found that aqueous–ethanol extracts of brown seaweeds contain substances inhibiting endo-13--D-glucanases, the digestive enzymes of marine mollusks. The inhibitors were detected in 14 of 21 brown seaweeds investigated. An irreversible protein inhibitor possessing high specificity toward endo-13--D-glucanases of marine mollusks was isolated from the brown seaweed Laminaria cichorioides. As determined by gel filtration, the molecular mass of the inhibitor is 46 kD. The value of [I] 50 (10 –8 M) for the inhibitor is comparable with the corresponding value for natural inhibitors of amylases from terrestrial plants. The results of chemical modification indicated that tryptophan, glutamic acid, aspartic acid, histidine, and probably tyrosine residues are important for the interaction of the inhibitor with the enzyme.(subject: to Zvyagintseva) 相似文献
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