Search PubMed⌕ Search

Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 235 records · Page 13Linked to original sources
PubMed1982

Ca2+ transporting activity of membrane fractions isolated from the post-mitochondrial supernatant of rat liver.

The post-mitochondrial supernatant of rat liver contains two vesicular fractions which transport Ca2+ actively. The heavier fraction, sedimenting at 17.500 xg, 20 min, is enriched in plasma membrane markers and apparently contains both a Ca2+ pumping ATPase and a Na+/Ca2+ exchanger. These activities have been attributed to the plasma membrane vesicles. The lighter fraction, sedimenting at 100.000 xg, 60 min, is enriched in endoplasmic reticulum markers, and contains only a Ca2+ pumping ATPase, which can be differentiated from that of the heavier fraction on the basis of the sensitivity to vanadate. The Ca2+ pumping activity of endoplasmic reticulum appears to be regulated by both a cAMP-dependent, and a calmodulin-dependent system. The former system involves a heat-stable protein fraction from the cytosol. The regulation by the cAMP and the calmodulin-dependent systems involves the phosphorylation of several proteins in the endoplasmic reticulum membrane.

Animals↗
PubMed1982

The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme.

The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adunyah, J.T. Penniston and E. Carafoli, 1981, J. Biol, Chem. 256, 395 - 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that ot the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+/ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.

Adenosine Triphosphatases↗
PubMed1982

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin.

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.

Calcium-Binding Proteins↗
PubMed1982

Modulation by calcium of the potassium permeability of dog heart sarcolemmal vesicles.

The movement of K+ in heart sarcolemmal vesicles has been followed through the opposing movement of the tetraphenylphosphonium ion. Ca2+ (5-50 microM) stimulates the efflux of K+ from K+-loaded vesicles [Km(Ca2+) approximately equal to 10 microM]. and the activation requires that Ca2+ be present inside the vesicles together with K+. The efflux of 86Rb+ from K+-, Rb+-loaded vesicles is similarly stimulated by 5-50 microM Ca2+ [Km(Ca2+) approximately equal to 10 microM]. The Ca2+-induced increase of K+ permeability does not become spontaneously inactivated. The effects of some inhibitors have been tested under conditions in which Ca2+ promotes the entry of K+ into vesicles. In this system, direct interaction of the drug with the Ca2+ and K+ membrane binding site(s) was therefore studied. Tetraethylammonium ion, which inhibits the potential-dependent K+ channel, does not interfere with the effect of Ca2+ whereas quinidine (IC50 = 12 microM) and trifluoperazine (IC50 = 8 microM at 50 micrograms of sarcolemmal protein per ml) inhibit.

Animals↗
PubMed1982

Influence of Ca2+ and trifluoperazine on the structure of calmodulin. A 1H-nuclear magnetic resonance study.

Ca2+-induced conformational changes of calmodulin under a variety of different experimental conditions have been studied by 1H-nuclear magnetic resonance techniques. The assignment for Tyr-99 has been corrected. Ca2+ titration performed at pH 7.5 and greater than 9.5 apparently induces a different sequence of the protein folding process as can be monitored by the resonances of His-107. These two structural forms cannot be interconverted. The phenylalanine residue(s) responsible for the resonances at 6.47 ppm (Ca2+-free form) and 6.64 ppm (Ca2+-saturated form) respectively, are apparently located close to Ca2+-binding sites III and IV. This can be recognized from nuclear Overhauser enhancement and Gd3+-broadening techniques. Gd3+-broadening experiments classify Ca2+-binding site IV as the site with the highest Gd3+/ca2+-exchange rate. The antipsychotic drug trifluoperazine, which is known to bind to calmodulin in a calcium-dependent way [Levin, R. M. and Weiss, B. (1977) Mol. Pharmacol. 13, 690-697], has been found to induce a conformational change of the Ca2+-saturated form of calmodulin. The methionine and phenylalanine residues were especially affected. Possible binding site(s) for trifluoperazine are discussed.

Animals↗
PubMed1982

Calcium uptake in isolated hepatic plasma-membrane vesicles.

A liver plasma-membrane fraction capable of Ca2+ uptake was isolated. The fraction exhibited high Na+, K+-ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent, and has an acid pH optimum. The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 X 10(-8) M and a Vmax of transport of 30 nmol X mg protein-1 X min-1. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na+, and incubation of the vesicles in a high Na+ medium (as compared to high K+ medium) resulted in lower (about 25%) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H+. The releasing effect of H+, taken together with the absence of Ca2+ uptake at acid pH, suggests the possibility of a H+/Ca2+ exchange.

Animals↗
PubMed1981

Regulation of Ca2+-pumping ATPase of heart sarcolemma by a phosphorylation-dephosphorylation Process.

The Ca2+-ATPase of dog heart sarcolemma (1, 2) is affected by phosphorylation. As normally prepared, sarcolemmal vesicles are phosphorylated to a high degree, resulting in a relatively low additional incorporation of hydroxylamine resistant [32P]phosphate from [gamma-32P]ATP. The 32P incorporation is increased up to 20-fold by pretreating the vesicles with phosphorylase phosphatase and is inhibited by an inhibitor of cAMP-dependent protein kinases. The phosphatase treatment inhibits markedly the Ca2+-ATPase and the ATP-dependent Ca2+ uptake. The inhibition is more evident at relatively higher levels of free Ca2+ and is reversed by preincubation with ATP. The Ca2+-pumping activity is stimulated markedly by phosphorylase b kinase and inhibited by the (cAMP-dependent) protein kinase inhibitor. Both the protein kinase inhibitor and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevent the rephosphorylation of sarcolemmal vesicles, but the effects are not additive. The Ca2+ dependence curve of the Ca2+ uptake in phospho- and dephosphorylated vesicles suggests that the phosphorylation might affect the efficiency of the enzyme (turnover rate) rather than its affinity for Ca2+.

Animals↗
PubMed1981

Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase.

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.

Calcium-Binding Proteins↗
PubMed1981

Rotation of cytochrome oxidase in phospholipid vesicles. Investigations of interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex.

Cytochrome oxidase was incorporated into lipid vesicles composed of phosphatidylethanolamine-phosphatidylcholine-cardiolipin. Large proteoliposomes of 1,000-15,000 A diameter were prepared by calcium-induced fusion of small vesicles. Rotational diffusion of cytochrome oxidase was measured by detecting the decay of the absorption anisotropy, r(t), after photolysis of the heme a3.CO complex by a vertically polarized laser flash. Because of the large size of the proteoliposomes, there was no contribution of vesicle-tumbling to r(t) over the experimental time range of 5 ms for samples in 60% sucrose. Analysis of r(t) curves was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex co-reconstituted in the above lipid vesicles. In vesicles of a high lipid to protein ratio (congruent to 27), nearly all cytochrome oxidase molecules are rotating with an approximate rotational relaxation time, phi 1, on the order of 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. The immobilized fraction is presumably due to nonspecific self-aggregation of cytochrome oxidase. The presence of cytochrome bc1 complex does not change r(t) curves significantly, either in the presence or absence of cytochrome c. Previously, we have observed the co-existence of mobile and immobile populations of cytochrome oxidase in bovine heart and rat heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510). The present results suggest that the immobile population of about one-half of cytochrome oxidase could be simply due to nonspecific protein aggregation resulting from the high concentration of enzymes in the inner mitochondrial membrane (lipid to protein ratio, less than or equal to 0.5). We also conclude that there is no specific interaction between cytochrome oxidase and cytochrome bc1 complex in the above large lipid vesicles. A lateral collision-controlled model for electron transfer from cytochrome bc1 complex to cytochrome oxidase through cytochrome c is discussed based on the above results.

Animals↗
PubMed1981

The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification.

The (Ca2+ + Mg2+) ATPase of dog heart sarcolemma (Caroni, P., and Carafoli, E. (1980) Nature 283, 765-767) has been characterized. The enzyme possesses an apparent Km (Ca2+) of 0.3 +/- 02 microM, a Vmax of Ca2+ transport of 31 nmol of Ca2+/mg of protein/min, and an apparent Km (ATP) of 30 microM. It is only slightly influenced by monovalent cations and is highly sensitive to orthovanadate (Ki = 0.5 +/- 0.1 microM). The high vanadate sensitivity has been used to distinguish the sarcolemmal and the contaminating sarcoplasmic reticulum Ca2+-dependent ATPase in heart microsomal fractions. Calmodulin has been shown to be present in heart sarcolemma. Its depletion results in the transition of the Ca2+-pumping ATPase to a low Ca2+ affinity; readdition of calmodulin reverses this effect. The Na+/Ca2+ exchange system was not affected by calmodulin. The results of calmodulin extraction can be duplicated by using the calmodulin antagonist trifluoperazine. The calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column. One major (Mr = 150,000) and 3 minor protein bands could be eluted from the column with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The major protein band (72%) has Ca2+-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+-dependent reaction.

Animals↗
PubMed1981

Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids.

The Ca2+-pumping ATPase from human erythrocyte membranes, purified nearly to homogeneity (Niggli, V., Penniston, J. T., and Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958), can be reconstituted into phospholipid vesicles. The purified and the reconstituted forms of the enzyme displayed the properties expected of the intact Ca2+ pump; they had an appropriate (Ca2+-Mg2+)-ATPase activity which displayed a relatively low affinity for Ca2+. Added calmodulin increased both the maximum rate and the affinity for Ca2+ of the enzyme. Mg2+ alone caused no significant ATP hydrolysis in the purified enzyme, indicating that the Mg2+-ATPase is a separate enzyme. Vesicles of the reconstituted enzyme accumulated Ca2+ with a ratio of Ca2+ accumulated to ATP hydrolyzed of approximately 1. Ca2+ accumulation and ATPase of the reconstituted enzyme were inhibited concurrently by vanadate ion, with a K 1/2 for inhibition which was indistinguishable from that observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts. While the above properties were all consistent with those observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts, the purified enzyme displayed an unexpected response to acidic phospholipids. Enzyme reconstituted with or prepared in phosphatidylserine acted as if calmodulin were already present, and added calmodulin caused no effect beyond that due to phosphatidylserine. This mimicry of the calmodulin effect by acidic phospholipids is similar to that reported for cyclic nucleotide phosphodiesterase (Wolff, D. J., and Brostrom, C. O. (1976) Arch. Biochem. Biophys., 173, 720-723).

Biological Transport, Active↗