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Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 217 records · Page 12Linked to original sources
PubMed1983

A high-affinity, calmodulin-dependent Ca2+ pump in the basal-lateral plasma membranes of kidney cortex.

A purified preparation of kidney basolateral membrane vesicles is capable of ATP-dependent Ca2+ uptake. The reaction has high affinity for Ca2+ (Km about 0.1 microM) and a V of 5.8 nmol Ca2+ X mg-1 protein X min-1 in the predominantly right-side-out vesicular preparation used. It is inhibited by vanadate (K0.5 about 5 microM) and by anti-calmodulin drugs. A stimulatory effect of calmodulin is visible in membranes depleted of the activator. Exposure of basolateral membranes to 125I-azido-modified calmodulin results in the specific labeling of a membrane protein of Mr 141 000, which is tentatively suggested to be the Ca2+-pumping ATPase.

Adenosine Triphosphate↗
PubMed1983

Phospholamban of cardiac sarcoplasmic reticulum consists of two functionally distinct proteolipids.

Phosphorylation of phospholamban by either a cAMP-dependent or a calmodulin-dependent kinase stimulates the Ca2+ transporting activity of cardiac sarcoplasmic reticulum membranes. It has now been found that phospholamban consists of 2 distinct proteins; one is the specific substrate for the cAMP-dependent phosphorylation, and the other for the calmodulin-dependent kinase. In spite of functional diversity, the 2 polypeptides share a number of properties. Among them, the proteolipid character, Mr, resistance to trypsinization, and subunit composition.

Adenosine Triphosphatases↗
PubMed1983

The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin.

The phosphate carrier has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin vesicles. It has been found that 100 microM doxorubicin and 100 microM Br-daunomycin inhibit the unidirectional phosphate uptake in the reconstituted liposomes to the same extent as N-ethylmaleimide. The inhibition by Br-daunomycin is not due to covalent interaction with the carrier. The specific interaction between doxorubicin and cardiolipin is responsible for the inhibition of the phosphate carrier. Br-daunomycin interacts with 3 mitochondrial proteins of apparent Mr approximately 45 000, approximately 35 000 and approximately 30 000.

Animals↗
PubMed1983

Dicyclohexylcarbodiimide does not inhibit proton pumping by cytochrome c oxidase of Paracoccus denitrificans.

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.

Animals↗
PubMed1983

Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma.

The Ca2+-pumping ATPase has been isolated from calf heart sarcolemma by calmodulin affinity chromatography (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 3263-3270) as a polypeptide of Mr about 140,000. The purified enzyme has high affinity for Ca2+ in the presence of calmodulin (Km about 0.4 microM) but shifts to a low affinity state (Km about 20 microM) in its absence. Calmodulin increases also the Vmax of the enzyme. The effects of calmodulin are mimicked by phosphatidylserine and by a limited proteolytic treatment of the enzyme with trypsin. The purified ATPase can be reconstituted in asolectin liposomes, where it pumps Ca2+ with an approximate stoichiometry to ATP of 1. The purified (and reconstituted) enzyme is not phosphorylated by added ATP and cAMP-dependent protein kinase under conditions where the enzyme in situ is stimulated concomitant with the phosphorylation of the sarcolemmal membrane (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 9371-9373). Hence, the target of the regulatory phosphorylation system is not the ATPase molecule. The purified ATPase cross-reacts with an antibody raised against the erythrocyte Ca2+-pumping ATPase. Under the same conditions, the purified sarcoplasmic reticulum Ca2+-ATPase does not react. The proteolytic splitting pattern of the purified heart sarcolemma and erythrocyte enzymes are similar but not identical.

Animals↗
PubMed1983

The regulation of the Na+ -Ca2+ exchanger of heart sarcolemma.

The Na+/Ca2+-exchange of calf-heart sarcolemma is activated by a treatment with ATP, Mg2+, and Ca2+, and deactivated by a treatment with phosphorylase phosphatase. The effect of the latter can be substituted by a treatment with Mg2+, Ca2+, and calmodulin. The activating treatment does not require added calmodulin, but is inhibited by calmodulin antagonists. Evidently, endogenous calmodulin is required and sufficient. Activation is half-maximal at about 2 microM Ca2+. Added calmodulin, however, decreases the Km (Ca2+) of the activating process to about 0.8 microM. Deactivation is half-maximal, at optimal calmodulin concentrations, at about 1.5 microM Ca2+. Experiments with adenosine 5'-[gamma-thio]triphosphate have shown that the activating treatment is mediated by a kinase and the deactivating treatment by a phosphatase. The concerted operation of the two enzymes is made possible by their different Ca2+ affinity. At saturating Ca2+ concentrations, the level of ATP may also influence the balance of the two enzymes.

Animals↗
PubMed1983

Role of calmodulin in skeletal muscle sarcoplasmic reticulum.

Three proteins having Mr of 20 000, 35 000, and 57 000 were phosphorylated by a calmodulin-dependent system in fast skeletal muscle sarcoplasmic reticulum (SR). The 20 000-dalton phosphoprotein was an acidic proteolipid distinct from phospholamban, which was present in cardiac SR preparations. The 57 000-dalton phosphoprotein became phosphorylated very rapidly (t1/2 = 5-10 s at 0 degrees C) and was distinct from calsequestrin and the 53 000-dalton glycoprotein, as judged from the electrophoretic mobility on neutral Laemmli gels and from its detergent-extraction characteristics. None of the three phosphoproteins interacted directly with calmodulin, implying that they were not the regulatory subunit of the calmodulin-dependent kinase. The calmodulin-dependent kinase(s) responsible for the phosphorylation of the three protein substrates was (were) membrane bound. Its Km(ATP) was about 200 microM, and at the probable physiological calmodulin concentration of 1-2 microM, its Km (Ca) was 0.7 microM. The 57 000-dalton phosphoprotein was dephosphorylated by an endogenous phosphatase activity, which was also activated by the Ca-calmodulin complex.

Animals↗
PubMed1983

The stoichiometry of the Ca2+-pumping ATPase of erythrocytes.

The stoichiometry of the erythrocyte Mg2+ dependent Ca2+-stimulated ATPase has been determined in a reconstituted system. Purified Ca2+ ATPase was incorporated into calcium impermeable liposomes and the ATP dependent calcium uptake was determined simultaneously with the hydrolysis of ATP. The results indicate that 1 gram atom of calcium is transported for each gram molecule of ATP hydrolysed, i.e., an ATP/Ca2+-stoichiometry of 1.

Adenosine Triphosphate↗
PubMed1982

Molecular properties of calcium-pumping ATPase from human erythrocytes.

The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.

Amino Acids↗
PubMed1982

Vanadate-induced movements of Ca2+ and K+ in human red blood cells.

Fresh human red blood cells become highly labeled with 45Ca2+ when exposed to 0.5 mM vanadate. The effect of vanadate requires its penetration into the cell, and is attributed to the inhibition of the outwardly directed Ca2+-pumping ATPase which would otherwise "mask" the uptake of 45Ca2+. Since the inhibition of the CA2+ pump by vanadate is not complete, a transmembrane Ca2+-Ca2+ exchange can be detected. The influx leg of the exchange is inhibited by verapamil, quinidine, and Co2+. This, as well as additional (kinetic) evidence, indicates that the influx of Ca2+ is a carrier-mediated process. Experiments in which the transmembrane K+ gradient has been abolished or decreased with ionophores, or by increasing the K+ concentration in the medium, suggest that the K+ gradient may play a role in the influx of Ca2+. The vanadate-induced accumulation of Ca2+ by red cells promotes a massive efflux of K+, indicating the activation of a Ca2+-sensitive K+-channel. The results indicate the occurrence of a slow cycling of Ca2+ across the red cell membrane. The influx leg of the cycle occurs through a verapamil-sensitive channel, and is possibly driven by the discharge of the transmembrane K+ gradient. The efflux leg of the cycle consists of the Ca2+-pumping ATPase.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system.

The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6. With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium. When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed. This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin. Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c. We thus conclude that cytochrome c oxidase of P. denitrificans is a proton pump. Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers.

Animals↗
PubMed1982

The regulation of Ca2+ transport by fast skeletal muscle sarcoplasmic reticulum. Role of calmodulin and of the 53,000-dalton glycoprotein.

Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine (TFP). Inhibition, which is Ca2+-dependent, is 90% with 14 microM TFP and 0.2 microM Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000-dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2+-Mg2+)-ATPase from a high to a low affinity form. Calmodulin-dependent phosphorylation of three proteins (Mr = 57,000, 35,000, and 20,000) of the SR membrane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Ca2+-uptake reaction.

Animals↗