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1.
A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATPase, an established inner membrane maker. 相似文献
2.
Marjorie Jones Roy W. Keenan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,678(3):403-407
A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATPase, an established inner membrane maker. 相似文献
3.
Randall L. Kincaid Tag E. Mansour 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,588(3):342-350
The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10−2 M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed. 相似文献
4.
Viktor Stolc 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(3):236-239
The content of adenosine 3′,5′-monophosphate in human mononuclear leukocytes was enhanced 3–5-times by venoms obtained from African toad (Bufo africanus), American toad (Bufo americanus), Colorado river toad (Bufo arenarum) and Marine toad (Bufo marinus) at 25 μg/ml for 5 min of incubation at 37°C. The maximum stimulation was observed after 1–5 min of incubation. The half-maximal stimulation was observed at 0.1 μg/ml venom obtained from Colorado river toad (Bufo arenarum). The increased content of adenosine 3′,5′-monophosphate in the mononuclear leukocytes persisted without significant change for at least 30 min of incubation at 37°C. 相似文献
5.
The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane. 相似文献
6.
Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of in the rapid phase and in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 102. Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells. 相似文献
7.
Differential enthalpic analysis of a series of 3′,5′-cyclic nucleotides indicates that homolytic cleavage of the six-membered phosphate ring occurs either immediately prior to or concurrent with decomposition of the crystal lattice. Homolysis is followed by a rapid polymerization to yield oligonucleotides. The enthalpies of these reactions have, with the exception of guanosine 3′,5′-cyclic phosphate, almost identical values of 25 kcal/mole. The anomalous case is attributed to a more stabilized phosphate ring as a result of hydrogen bonding through the two position of the purine ring. The pair of overlapping exothermic peaks observed in each of the thermograms for cAMP and cIMP is related to the presence of two conformational arrangements within the unit cell of each compound. 相似文献
8.
G.D. Holman 《生物化学与生物物理学报:生物膜》1979,553(3):489-494
Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis at 30°C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments.Lowering the temperature reduces from 0.69 mM at 30°C to 0.478 mM at 20°C, 0.108 mM at 10°C and 0.072 mM at 4°C (). The for activation of influx permeability of 10?5 M cyclic AMP is 1.55. 相似文献
9.
T. Kanamori C.R. Creveling J.W. Daly 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(3):434-447
The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in . In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity. 相似文献
10.
An earlier report (1a) has shown the utility of 8-N3cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N3cAMP stimulate the phosphorylation by [γ-32P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-32P]-ATP is stimulated by cAMP and 8-N3cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [32P]-8-N3cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of 32P-8-N3cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein. 相似文献
11.
Piotr Kaczmarek 《Inorganica chimica acta》2005,358(6):2073-2076
The metal binding ability of 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) molecule using copper(II) ion, as an example of biologically available divalent metal ion, was investigated by potentiometry, EPR and differential spectroscopy (UV-Vis, CD). One complex with stoichiometry Cu(3′,5′-cAMP)+ was found, where Cu(II) ion is bound by N-7 nitrogen of adenine moiety. 相似文献
12.
Ruben Ortega Perez Diederik Van Tuinen Dieter Marmé Gilbert Turian 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,758(1):84-87
Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine. 相似文献
13.
A. Krayevsky M. Kukhanova L. Alexandrova N. Belyakova V. Krutyakov 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(3):216-220
2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis. 相似文献
14.
Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained. 相似文献
15.
Adenosine 3′,5′-cyclic monophosphate (cAMP) binds to high-molecular substances which are probably proteins, in homogenates of sea urchin eggs and embryos. The bound cAMP is exchangeable. Optimal pH for the binding capacity of the proteins with cAMP is 4.0, and is shifted to 5.0 in the presence of 5 mM caffeine. The level of bound cAMP increases steeply during 10 minutes of incubation. This is then followed by a less steep increase. The level of bound cAMP decreases in the presence of NaCl. The dissociation onstant between cAMP and the proteins in homogenates of unfertilized and ertilized eggs is about 10 nM, and the value in the embryos at the gastrula stage is lower than that in the unfertilized egg homogenate. 相似文献
16.
M. Kukhanova A. Krayevsky N. Terentyeva V. Rasskazov 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(3):221-226
2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates have been shown to be inhibitors of replicative DNA synthesis in isolated nuclei of sea urchin embryo. These compounds inhibit the Okazaki fragment synthesis. The effect of 2′,3′-dideoxy-3′-aminothymidine 5′-triphosphate and arabinothymidine 5′-triphosphate is reversible when adding the corresponding substrate for DNA synthesis, 2′-deoxythymidine 5′-triphosphate. 相似文献
17.
Klaas A. Zachariasse Winchil L.C. Vaz Carlos Sotomayor Wolfgang Kühnle 《生物化学与生物物理学报:生物膜》1982,688(2):323-332
Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37°C to 570 cP at 5°C, as reported for di(1-pyrenyl)propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37°C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40°C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol () results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol (), whereas a polarity comparable to that of methanol () is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced. 相似文献
18.
A water-soluble Mg2+-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The value for ATP was 1 mM and apparent for Mg2+ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd2+ and Zn2+ salts, and , known inhibitors of membrane endocytosis. 相似文献
19.
Abstract: The binding of 2′,3′-cyclic nucleotide 3′-phosphodiesterase isoform 1 (CNP1) to myelin and its association with cytoskeletal elements of the sheath have been characterized with in vitro synthesized polypeptides and purified myelin. We have previously shown that the cysteine residue present in the carboxy-terminal CXXX box of CNP1 is isoprenylated, and that both C15 farnesyl and C20 geranylgeranyl isoprenoids can serve as substrates for the modification. Here, we have mutated the CXXX box to obtain selectively farnesylated CNP1 or geranyl-geranylated CNP1 and found that these two modified forms of CNP1 behave identically in all of the assays performed. Isoprenylation is essential but not sufficient for the binding of in vitro synthesized CNP1 to purified myelin, because a control nonmyelin protein is isoprenylated, yet unable to bind to myelin. In our assay, membrane-bound CNP1 partitions quantitatively into the non-ionic detergent-insoluble phase of myelin, suggesting that CNP1 binds to cytoskeletal elements within myelin. However, isoprenylated CNP1 fails to bind to the cytoskeletal matrix isolated from myelin by detergent treatment, implying that both detergent-soluble and insoluble myelin components are involved in the binding of CNP1. A model for the interactions between CNP1 and myelin is presented, consistent with models proposed for other isoprenylated proteins. 相似文献
20.
The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes
The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at ?70°C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high d-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific d-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extrac decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day. 相似文献