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Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin 总被引:6,自引:0,他引:6
The human erythrocyte membrane ATPase responded to the presence of an acidic phospholipase A2 and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane. 相似文献
3.
Diploid embryos which are homozygous for the t12 mutation die at the morula stage. In the current studies, ova from heterozygous () females were fertilized in vitro with spermatozoa from males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t12, +/t12/t12, and t12/t12/t12 embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, , and embryos developing from ova which were also fertilized in vitro with spermatozoa from males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid () counterparts. Like embryos, t12/t12/t12 embryos die at the morula stage, prior to blastocoelic cavity formation. 相似文献
4.
The correlation between the ATP-dependent Ca2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca2+ binding was measured by using 45CaCl2, and the phosphorylation by [γ-32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250 000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca2+-binding activity, and lost 30% of activity. The Ca2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes.These results suggest that the formation of triphosphoinositide rather than the of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes. 相似文献
5.
Thomas Nagylaki 《Theoretical population biology》1976,10(1):70-82
The ultimate rate of approach to equilibrium in the infinite stepping-stone model is calculated. The analysis is restricted to a single locus in the absence of selection, and every mutant is assumed to be new to the population. Let f(t, x) be the probability that two homologous genes separated by the vector x in generation t are the same allele. It is supposed that . In the absence of mutation, f(t, x) tends to unity at the rate in one dimension and (ln t)?1 in two dimensions. Thus, the loss of genetic variability in two dimensions is so slow that evolutionary forces not considered in this model would supervene long before a two-dimensional natural population became completely homogeneous. If the mutation rate, u, is not zero f(t, x) asymptotically approaches equilibrium at the rate in one dimension and (1 ? u)2tt?1(lnt)?2 in two dimensions. Integral formulas are presented for the spatial dependence of the deviation of f(t, x) from its stationary value as t → ∞, and for large separations this dependence is shown to be (const + x) in one dimension and (const + ln x) in two dimensions. All the results are the same for the Malécot model of a continuously distributed population provided the number of individuals per colony is replaced by the population density. The relatively slow algebraic and logarithmic rates of convergence for the infinite habitat contrast sharply with the exponential one for a finite habitat. 相似文献
6.
(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H2O2 at pH 8.0 emit chemiluminescence with quantum yield and absolute maximum intensity per cm3 (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H2O2, a weak fluorescence in the region 400–620 nm with λmax = 465–510 nm (λexc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λexc = 280 nm, λem = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λmax = 530 nm (λexc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen is not involved in either peroxidation or chemiluminescence. (6) The haem Fe3+ group and H2O2 appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds. 相似文献
7.
The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (ν, in μm2/min) by the concentration of fibrinogen (, in g/dl) and the sialic acid content (, in μmol/ml red blood cells), log , was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes. 相似文献
8.
J E Ekholm S D Shukla D J Hanahan 《Biochemical and biophysical research communications》1981,103(2):407-413
The membrane ATPase of density (age) separated human erythrocytes was examined for its stimulation by the cytosols of these cell groups. On the assumption that the stimulatory activity in the cytosol is only calmodulin, it was consistently observed that the young cytosol had a significantly higher activity towards the membrane ATPase activity (from any age group) than did the old cell cytosol. The data clearly demonstrates decided differences in the expression of calmodulin activity in cytosols from young and old erythrocytes and would support the conclusion that calmodulin activity is altered during aging of these cells. Possible mechanisms for these alterations are discussed. 相似文献
9.
86Rb and 42K loss from non-illuminated crayfish retinas is described by a sum of two exponential functions with rate constants λ1 ≈ 0.12 min?1 and λ2 ≈ 0.006 min?1. 86Rb and 42K movements distinguish mainly by the λ2 rate constants, .Ouabain causes a delayed increase in the rate of isotope loss from non-illuminated retinas (λ ≈ 0.009 min?1 after 40 min). Light stimuli evoke a temporary increase in the rate of both 86Rb and 42K loss by the same factor (2.7 under these conditions). In the presence of ouabain the light-induced isotope loss is abolished after 40 min if the retina is periodically illuminated but not if the retina is kept in the dark. 相似文献
10.
Thallium inhibition of ouabain-sensitive sodium transport and of the ()-ATPase in human erythrocytes
The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1–1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5–10 mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the ()-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the ()-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations. 相似文献
11.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte . In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal . In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes. 相似文献
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13.
G.D. Holman 《生物化学与生物物理学报:生物膜》1978,508(1):174-183
10?5 M cyclic AMP has high permeability in human erythrocyte ghosts (). Saturation of influx and efflux occurs. . . . at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ().Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments. 相似文献
14.
The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity had an apparent half saturation constant of for free calcium, a maximum reaction velocity of ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol . Since the kinetic properties of the high-affinity showed a close resemblance to those of erythrocyte plasma membrane , the high-affinity of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells. 相似文献
15.
The cost of assays using one or two coupling enzymes is optimized by using equations to calculate the minimum amount(s) of enzyme(s) which should be used to obtain a given time (t99) in which 99% of the rate V0 of the first reaction is obtained. Using two coupling enzymes and given a value of t99, the induction period L = L1 + L2 fulfills the requirement , allowing one to choose a cost lower than that derived from the until-now generally applied assumption of t99 = 4.6L. Being , in optimized assays the values α, t99, and L are related by , thus allowing (graphical) calculation of the amounts of coupling enzymes which will minimize the cost for every chosen t99 or L. Maximum practical rates, allowed in some supposed interesting cases, have been calculated. 相似文献
16.
Studies were carried out to determine the Hill coefficients for the inhibition by F? of the erythrocyte membrane-bound Mg2+-ATPase, and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of for the inhibition by F? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the and acetylcholinesterase, whereas the Mg2+-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon. 相似文献
17.
Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein 总被引:1,自引:0,他引:1
Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[] glucosamine into N-acetyl[]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl []chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[]glucosamine-labeled glycoprotein reveals two N-acetyl[]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[]chitobiose. The linkage between the -labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled -labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the -labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated []disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000. 相似文献
18.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
19.
In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes . A minimal concentration of about 6 · 1011 dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method is compared with the concentrations obtained from simultaneous electrical relaxation measurements. and agreed at low dipicrylamine concentrations (10?8–10?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10?6 M). In the saturation range was maximally four times higher than . The significance of this difference is discussed together with general aspects of the saturation phenomenon. 相似文献
20.
A series of recessive mutations which arrest embryonic development are located within the T/t region of chromosome 17 in the mouse. To assess whether these mutations cause death in specific differentiating cells or in all cells of the embryo, we removed the embryonic cells from normal developmental constraints and attempted to grow them ectopically in vivo and in vitro. We have succeeded in producing teratomas and teratocarcinomas by transplantation of inner cell masses from blastocysts of and genotypes. The ability of embryonic cells to grow as tumors was not affected by their genotype; 7 of the 17 tumors were homozygous for tw12, 7 were heterozygous, and 3 could not be analyzed. Virtually all the tumors of both genotypes contained derivatives of all three germ layers. Neuroepithelial and mature nervous tissue was present in all homozygous tumors and all except one heterozygous tumor. However, no cartilage or bone was found in 5 of 5 tw12 homozygous tumors, while both tissues were present in 3 of 4 tw12 heterozygous tumors. This observation is compatible with the abnormalities characteristic of embryos, which show very localized effects in nervous tissue and more general effects on bone and cartilage formation. Cells derived from homozygous tumors were capable of at least limited growth in culture and a cell line has been derived from one of them. The p63/6.9a marker protein was used to determine the presence of the tw12 haplotype in the tumor and cultured cells. We conclude that the lethality associated with the tw12 haplotype is due to lethality of specific cells, and not all cell types. 相似文献