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1.
The ATP/ADP exchange is shown to be a partial reaction of the by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to , -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration () required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+. 相似文献
2.
The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with . This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the enzyme and may reflect a protein-directed phospholipid specificity for this enzyme. 相似文献
3.
Background
Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets. 相似文献4.
(1) ( from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C12E8) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K+-stimulated Na+-influx is found to be about 35 μmol Na+ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na+ transported per ATP molecule hydrolyzed. 相似文献
5.
A.C. Simmonds J.M. East O.T. Jones E.K. Rooney J. McWhirter A.G. Lee 《生物化学与生物物理学报:生物膜》1982,693(2):398-406
Quenching of the fluorescence of the purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems. 相似文献
6.
7.
(1) Eosin bound to the in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site ( 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity ) for Na+ activation of hydrolysis and for Na+ protection against inactivation by . (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of the system reacts with two different ATP molecules. 相似文献
8.
Kristin K Nicodemus 《BMC bioinformatics》2008,9(1):130
Background
Risk for complex disease is thought to be controlled by multiple genetic risk factors, each with small individual effects. Meta-analyses of several independent studies may be helpful to increase the ability to detect association when effect sizes are modest. Although many software options are available for meta-analysis of genetic case-control data, no currently available software implements the method described by Kazeem and Farrall (2005), which combines data from independent family-based and case-control studies. 相似文献9.
P. K. Golovatenko-Abramov E. A. Zhirkova E. G. Kolokolchikova V. S. Bocharova E. S. Platonov 《Russian Journal of Developmental Biology》2010,41(4):240-246
One of the phenotypic effects of mutation in the Hr gene in mice is disintegration of hair follicles and their degeneration into open funnel-shaped structures (utricles) opened
on skin surface and cysts located in the depth of the dermis. The aim of the current study consists in analysis of the process
of reparative regeneration of skin in homozygotous mice with one of the mutant alleles of the Hr gene—Hr
hr
. It is shown that epithelial cells that constitute the inner pavement of cysts take part in the process of epithelization
of deep skin wounds. This indicates that the competence of ectodermal cells in relation to inductive signals from injured
skin remains in Hr
hr
homozygote mice, in spite of the significant anatomic abnormalities of the hair follicles. 相似文献
10.
Approximate formulas for the mean and variance of the FSTor GST statistic in a finite number of isolated populations are developed under the effect of random genetic drift. Computer simulation has shown that the approximate formulas give a fairly accurate result unless the initial frequency of one of the alleles involved is close to 1 and t/2N is large, where N is the effective size of a subpopulation and t is the number of generations. It is shown that when the number of subpopulations (s) is small, the mean of FSTor GST depends on the initial gene frequencies as well as on s. When the initial frequencies of all alleles are nearly equal to each other and the number of subpopulations is large, the distribution of FST in the early generations is bell-shaped. In this case Lewontin and Krakauer's k parameter is approximately 2 or less. However, if one of the initial allele frequencies is close to 1, the distribution is skewed and leptokurtic, and the k parameter often becomes larger than 2 in later generations. Thus, even under pure random genetic drift, Lewontin and Krakauer's test of selective neutrality of polymorphic genes in terms of FST is not always valid. It is also shown that Jacquard's approximate formula for k generally gives an overestimate. 相似文献
11.
Diketocoriolin B, a sesquiterpene antitumor antibiotic, inhibits particulate (ATP phosphohydrolase, EC 3.6.1.3) of Yoshida sarcoma cells competitively, with respect to ATP, and uncompetitively with respect to Na+ and K+. The inhibition is reduced by the addition of phosphatidylserine.Rat brain , which is solubilized by deoxycholate and requires phosphatidylserine for its activity, is also inhibited by diketocoriolin B competitively with respect to ATP and the inhibition was reversed by increasing the concentration of phosphatidylserine.However, several differences are found between the solubilized and particulate systems: (a) 2 moles of diketocoriolin B interact with the former, while only one mole interacts with the latter, (b) K+-dependent phosphatase activity of the former requires phospholipid and is sensitive to diketocoriolin B while the reverse is true with the latter.Based on these kinetic studies, it is supported that has two binding sites for phospholipid, one being essential for K+-dependent phosphatase activity and when these two sites are filled with the appropriate phospholipids, ATP can bind to the enzyme. 相似文献
12.
Yoshio Ushijima Minoru Nakano 《Biochemical and biophysical research communications》1980,93(4):1232-1237
The HOCl in chlorine-water oxidizes DPF to cis-DBE in parallel to the HOCl concentration. The addition of H2O2 produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis-DBE is initiated by a 1,2-position attack of OH? and Cl+, followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H2O2 solution, on the pH range 6.0 to 8.0 where both HOCl and OCl? are present. 相似文献
13.
Ehrlich cells actively accumulate neutral amino acids even if both the Na+ and K+ gradients are inverted. The seeming contradiction of this observation to the gradient hypothesis is, however, explained by the presence of a powerful electrogenic Na+ pump, which stongly raises the electrochemical potential gradient of Na+ under these conditions. Since the evidence of this pump has so far been found only during abnormal concentrations of alkali ions (low K+, high Na+) in these cells, the question arises whether the pump is equally powerful with completely normal cells, when the pump is not ‘needed’ for amino acid transport. Using the initial rate of uptake of the test amino acid (2-aminoisobutyrate) as a sensitive monitor of the electrical potential at constant cation distribution between cell and medium, a procedure has been devised to split the overall electrical potential into the diffusional and the pump component. With this procedure it could be shown that the electrogenic pump per se is most powerful in K+-depleted and Na+-rich cells but declines to a lower ‘resting’ value according as the electrolyte content of the cell approaches normality. A strong positive correlation between cellular Na+ content and the electrogenic pumping activity suggests that the intracellular activity of this ion regulates the rate of the electrogenic pump. The low activity of the pump under normal conditions may explain why the existance of this pump has rarely come to attention previously. 相似文献
14.
目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009(CA7)与A/California/4/2009(CA4)两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。 相似文献
15.
The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na+,+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a ; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of while ATP binds with an apparent for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is . In the presence of ATP a reduction in size is observed. 相似文献
16.
17.
Chana Vinkler 《Biochemical and biophysical research communications》1981,99(4):1095-1100
The of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K+) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold in the of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K+ are employed, the rate of photophosphorylation is decreased but a several-fold in the of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient. 相似文献
18.
W.H.M. Peters A.M.M. Fleuren-Jakobs J.J. Schrijen J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1982,690(2):251-260
(1) A preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, ) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, ) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, ) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits. 相似文献
19.
We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface receptor activity. 相似文献
20.
Structural changes in the purified accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of and activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either or activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid. 相似文献