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1.
In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme. 相似文献
2.
Y Blouquit M D Rhoda J Delanoe-Garin R Rosa J C Prome C Poyart G Puzo J M Bernassau J Rosa 《The Journal of biological chemistry》1986,261(15):6758-6764
A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined. 相似文献
3.
Compartmentalization of Ca2+ in sickle cells 总被引:2,自引:0,他引:2
Control (AA) and sickle cell anemia (SS) erythrocytes were loaded with Ca-chelator (Quin2 or Benz2) to increase the cellular exchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Ca]i) (Lew et al., 1982, Nature, 298, 478). The chelator incorporation induced a decrease in the ATP content which was smaller in SS than in AA cells and partially reversible upon reincubation in a chelator-free medium. The amount of trapped chelator was determined by two methods: 45Ca binding to the chelator in Ca-ionophore treated cells in Ca-EGTA buffers and [3H]Quin2 incorporation. A slight over-estimation of the chelator content was found with the second method but incorporation was the same in both types of cells. The kinetics of 45Ca equilibration and 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenated chelator-loaded cells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75%) but [Ca]i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not available for the Ca2+ pump or the K+ channel a conclusion in agreement with that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeable Ca2+ behaved as one compartment. In SS cells, the existence of a second slowly-exchangeable Ca2+ compartment was demonstrated. This latter (3-5 mumol/l cells) was independent of the concentration of the chelator and thus could represent exchangeable Ca2+ enclosed within the intracellular inside-out vesicles recently observed in SS cells (Williamson et al., 1984, J. Cell. Biol., 99, 430a). Alternatively, these two kinetic pools could reflect heterogeneity of the SS cell population. 相似文献
4.
Na+-ATPase of high-K+ and low-K+ sheep red cells was examined with respect to the sidedness of Na+ and K+ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na+ in the medium, i.e., at the cytoplasmic surface, Nacyt+, the activation curves show that high-K+ Na+-ATPase has a higher affinity for Nacyt+ compared to low-K+. The apparent affinity for Nacyt+ is also increased by increasing the ATP concentrations in high-K+ but not low-K+. With Nacyt+ present, Na+-ATPase is stimulated by intravesicular Na+, i.e., Na+ at the originally external surface, Naext+, to a greater extent in low-K+ than high-K+. Intravesicular K+ (Kext+) activates Na+-ATPase in high-K+ but not in low-K+ vesicles and extravesicular K+ (Kcyt+) inhibits low-K+ but not high-K+ Na+-ATPase. Thus, the genetic difference between high-K+ and low-K+ is expressed as differences in apparent affinities for both Na+ and K+ and these differences are evident at both cytoplasmic and external membrane surfaces. 相似文献
5.
Rhoda Feldman Amelita Luncsford Robert L. Heinrikson John Westley Joseph Jarabak 《Archives of biochemistry and biophysics》1981,211(1):375-381
Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (KiGSSG = 26 μM, KiGSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (KiGSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A1-glutathione adduct (Ki = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function. 相似文献
6.
The kinetics of active K+ transport were studied in immature red blood cells cells from high-K+ and low-K+ sheep, particularly with respect to the effects of varying intracellular K+ concentration, [K]i. Comparison was made with active transport, or pump, activity in mature high-K+ and low-K+ red cells. Reticulocytes from both types of sheep had much higher maximal active K+ influxes than did mature cells. In both types of reticulocytes, and in mature high-K+ cells as well, the pump was relatively insensitive to increasing [K]i. In contrast, intracellular K+ markedly inhibited the pump in mature low-K+ cells. Active K+ transport in low-K+ reticulocytes, however, as in mature low-K+ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K+ pumps of high-K+ and low-K+ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K+ and low-K+ cells with K+ pumps of very different kinetic characteristics. 相似文献
7.
8.
Xing De Su Baofeng Li Shangjia Bangs Max Creamer David Coogan Michael Wang Jinhai Simora Rhoda Ma Xiaoli Hettiarachchi Darshika Alston Veronica Wang Wenwen Johnson Andrew Lu Cuiyu Hasin Tasnuba Qin Zhenkui Dunham Rex 《Marine biotechnology (New York, N.Y.)》2022,24(3):513-523
Marine Biotechnology - Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in... 相似文献
9.
Tissue-distinct interactions of theNa+-K+-ATPasewith Na+ andK+, independent ofisoform-specific properties, were reported previously (A. G. Therien,N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol.Chem. 271: 7104-7112, 1996). In this paper, wedescribe a detailed analysis of tissue-specific kinetics particularlyrelevant to regulation of pump activity by intracellularK+, namelyK+ inhibition at cytoplasmicNa+ sites. Our results show thatthe order of susceptibilities of 1 pumps of various rat tissuestoK+/Na+antagonism, represented by the ratio of the apparent affinity forNa+ binding at cytoplasmicactivation sites in the absence ofK+ to the affinity constant forK+ as a competitive inhibitor ofNa+ binding at cytoplasmic sites,is red blood cell < axolemma rat1-transfected HeLa cells < small intestine < kidney < heart. In addition, we havecarried out an extensive analysis of the kinetics ofK+ binding and occlusion to thecytoplasmic cation binding site and find that, for most tissues, thereis a relationship between the rate ofK+ binding/occlusion and theapparent affinity for K+ as acompetitive inhibitor of Na+activation, the order for both parameters being heart kidney > small intestine rat1-transfected HeLa cells. Thenotion that modulations in cytoplasmicK+/Na+antagonism are a potential mode of pump regulation is underscored byevidence of its reversibility. Thus the relatively highK+/Na+antagonism characteristic of kidney pumps was reduced when rat kidneymicrosomal membranes were fused into the dog red blood cell. 相似文献
10.
The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer. 相似文献