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Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 199 records · Page 11Linked to original sources
PubMed1984

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin.

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.

Amino Acids↗
PubMed1984

Properties of the Ca-pumping ATPase of sarcoplasmic reticulum from vascular smooth muscle.

A microsomal fraction enriched in sarcoplasmic reticulum membranes has been isolated from bovine aorta smooth muscle. The properties of the Ca-pumping ATPase were compared to those of the enzymes of skeletal and cardiac sarcoplasmic reticulum. The kinetic (Km and turnover rate) and structural (tryptic digestion pattern) properties of the three ATPases were strikingly similar. The three enzymes, however, displayed (almost) no immunological cross-reactivity. Skeletal muscle sarcoplasmic reticulum and aorta microsomes did not contain phospholamban: their Ca-pumping activity was not regulated by either a cAMP-dependent or a calmodulin-dependent pathway.

Animals↗
PubMed1984

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin.

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.

Animals↗
PubMed1984

Different conformational states of the purified Ca2+-ATPase of the erythrocyte plasma membrane revealed by controlled trypsin proteolysis.

The purified Ca2+-pumping ATPase of the erythrocyte membrane has been exposed to trypsin at 37 degrees C, in the presence of different effectors of its activity. The control proteolytic pattern is characterized by a number of transient and of limit polypeptides (Zurini, M., Krebs, J., Penniston, J. T., and Carafoli, E. (1984) J. Biol. Chem. 259, 618-627). The effectors influence the pattern in the Mr region 90,000-76,000, which contains the calmodulin binding domain and the active site of the enzyme. In this region, polypeptides of 90, 85, 81, and 76 kDa are clearly visible in the controls. 1) Calmodulin plus Ca2+ induces the faster disappearance of the 90-kDa product and the relative accumulation of the 85-kDa with respect to the 81-kDa polypeptide. 2) Vanadate plus Mg2+ also accelerates the disappearance of the 90-kDa product. However, they induce the relative accumulation of the 81-kDa polypeptide. 3) Linoleic acid, which stimulates the activity of the enzyme to the same levels obtained with calmodulin, greatly accelerates the rate of trypsin proteolysis, causing the virtual disappearance of all polypeptides in the 90-76-kDa region. 4) The 81-kDa polypeptide has maximal ATPase activity and is insensitive to calmodulin; the 85-kDa polypeptide has lower ATPase activity and binds calmodulin, but is not stimulated (or is stimulated only negligibly) by the activator.

Calcium-Transporting ATPases↗
PubMed1984

ATP synthesis catalyzed by the purified erythrocyte Ca-ATPase in the absence of calcium gradients.

The Ca2+-transporting ATPase of erythrocytes was isolated by calmodulin affinity chromatography. The backward reaction of the ATPase was investigated. The phosphorylation of the solubilized enzyme by Pi required Mg and was inhibited by Ca and vanadate in the micromolar concentration range. Significant amounts of phosphoenzyme could be obtained only in a medium containing high dimethyl sulfoxide concentrations (greater than 25%) in order to diminish water activity at the phosphorylation site. The phosphoenzyme formed in this way could not phosphorylate ADP. However, upon addition of Ca2+ ions and dilution of dimethyl sulfoxide in the phosphorylated preparation (water activity jump), a highly reactive phosphoenzyme species was obtained which could transfer phosphate in nearly stoichiometric amounts to ADP to form ATP.

Adenosine Triphosphate↗
PubMed1984

Calmodulin-dependent protein phosphorylation and calcium uptake in rat-liver microsomes.

A 20-kDa protein becomes phosphorylated in a process stimulated by Ca2+ and calmodulin in the light microsomal fraction of rat liver homogenate. The uptake of Ca2+ in light microsomal fraction is also calmodulin-stimulated. The stimulation of Ca2+ transport is associated with the operation of a protein phosphorylation system dependent on Ca2+ and calmodulin. Transport is inhibited by a protein phosphatase which is stimulated by Ca2+ and calmodulin. It is proposed that the phosphorylation of the 20-kDa protein, which is stimulated by Ca2+ and calmodulin, plays a role in the regulation of the microsomal Ca2+, Mg2+-ATPase.

Animals↗
PubMed1984

Doxorubicin inhibits the phosphate-transport protein reconstituted in liposomes. A study on the mechanism of the inhibition.

The phosphate transport protein (PTP) has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin and phosphatidylcholine vesicles. As expected, the activity of the reconstituted PTP is inhibited by N-ethylmaleimide and mersalyl. It is also inhibited by adriamycin and Br-daunomycin. Storage of isolated mitochondria at -80 degrees C prior to the isolation of the PTP decreases the maximal activity of the unidirectional transport of phosphate (Pi) in reconstituted vesicles. The sensitivity of the system to the four inhibitors remains the same. The inhibition of the PTP by adriamycin is reversed by KCl and prevented by MgSO4. Since the interaction between adriamycin and cardiolipin is of ionic type, KCl apparently replaces adriamycin on the negative charges of cardiolipin. By contrast, MgSO4 complexes adriamycin directly, eliminating its inhibitory effect on the PTP. No PTP activity is found after reconstitution in phosphatidylcholine vesicles in the absence of cardiolipin. Addition of buffer-dispersed cardiolipin to the vesicles restores the Pi-transport activity. The addition of adriamycin to the vesicles together with cardiolipin removes the reactivation. Succinylation of the PTP at pH 8.0 eliminates the inhibitory effect of adriamycin in the reconstituted system. The effects of N-ethylmaleimide and mersalyl are not modified. The succinylated preparation reconstituted in phosphatidylcholine vesicles is reactivated by cardiolipin, but in this case the reactivation is not counteracted by adriamycin. Succinylation of the PTP at pH 9.0 results in its complete inhibition. The results indicate, (a) that the sites of interaction of MalNEt /mersalyl and adriamycin with the PTP X cardiolipin complex are different, and (b) that the adriamycin-binding site in the complex is probably on cardiolipin molecules essential to the PTP activity. It is postulated that succinylation of the PTP prevents the interaction of adriamycin with these molecules.

Animals↗
PubMed1984

Calmodulin binding proteins in rat liver mitochondria.

Calmodulin binding proteins have been found in submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contains two major calmodulin binding proteins: one, having Mr of 145,000, apparently is carbamoyl-phosphate synthetase. Another has a Mr of 58,000 and has not been associated with enzyme activities. A major calmodulin binding protein of unknown function and having Mr of 32,000 has been found in the Triton X-100 solubilizate of the inner membrane. Minor amounts of two calmodulin binding proteins having Mr of about 37,000 and 56,000 have been found in the outer membrane.

Animals↗
PubMed1984

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a hydrophobic, photoreactive probe, labels calmodulin and calmodulin fragments in a Ca2+-dependent way.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means to characterize calmodulin sites that play a role in the interaction with targets.

Amino Acids↗
PubMed1984

Controlled proteolysis of the purified Ca2+-ATPase of the erythrocyte membrane. A correlation between the structure and the function of the enzyme.

The purified Ca2+-pumping ATPase of the erythrocyte plasma membrane has been subjected to a controlled proteolytic treatment with trypsin. The treatment has been previously shown to shift the enzyme from low to high Ca2+ affinity in the absence of calmodulin. The treatment leads to the fragmentation of the ATPase molecule into a number of products and to the accumulation of major limit polypeptides having Mr of 14,000, 28,000, 33,500, 48,000, and 76,000. The 33,500 Mr fragment reacts with 3(trifluoromethyl)-3 (m-[125I]iodophenyl)-diazirine, suggesting that it contains intramembrane regions of the enzyme. A number of minor fragmentation products were also formed, among them a polypeptide of Mr = 90,000 which is rapidly degraded further and a limit polypeptide of Mr about 25,000. [125I]Iodoazidocalmodulin cross-linked only to the original ATPase and to the fragments with Mr = 90,000 and Mr about 25,000. The Mr = 90,000 fragment can be isolated on calmodulin-Sepharose and on phenothiazine affinity columns. It retains the functional properties of the intact pump; it has a calmodulin-responsive ATPase activity and it actively accumulates Ca2+ upon incorporation into liposomes. Further proteolysis of the Mr = 90,000 fragment produces a fragment of Mr = 81,000-76,000 which forms an acylphosphate intermediate, which does not bind to calmodulin-Sepharose, and which seems to be responsible for the calmodulin-insensitive activity which appears as it is produced. A model is presented which postulates a major and a minor degradation pattern and rationalizes the pattern of proteolysis and of the [125I]iodoazidocalmodulin binding observed.

Azirines↗
PubMed1984

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma.

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10 microns Ca++, free, but does not change the Km for Ca++. A possible physiological role for the activator is discussed.

Animals↗
PubMed1984

Calmodulin-sensitive calcium-pumping ATPase of plasma membranes: isolation, reconstitution, and regulation.

The calmodulin-sensitive Ca2+ -pumping ATPase was purified to virtual homogeneity from erythrocytes. The purified enzyme exists in two functional states, having low and high Ca2+ affinity. Transition from low to high affinity is induced by 1) calmodulin; 2) acidic phospholipids, long-chain polyunsaturated fatty acids, polyphosphoinositides; and 3) a controlled proteolytic treatment with trypsin or chymotrypsin. The ATPase can be reconstituted into liposomes, where it pumps Ca2+ in exchange for H+ with a stoichiometry to ATP approaching 1. The purified enzyme can be fragmented by trypsin into a number of transient and of limit polypeptides, of which the most interesting from the functional standpoint are the following: 1) a limit polypeptide of Mr 76,000 that contains the active site (i.e., the sequence where the acyl-phosphate is formed); 2) a limit polypeptide of Mr 33,500 that binds the hydrophobic photoactivable label 3-trifluoromethyl-3-(m-(125I-iodophenyl]-diazirine, and is thus presumably the most hydrophobic portion of the molecule; and 3) a transient polypeptide of Mr 90,000 and a limit polypeptide of Mr 25,000-28,000, which specifically bind azido-modified, 125 I-labeled calmodulin. The transient 90,000-dalton calmodulin receptor is rapidly degraded to the 81,000-76,000 limit polypeptide. It can be isolated from the other proteolysis products on calmodulin affinity chromatography columns. The isolated 90,000-dalton fragment is a fully competent, calmodulin-sensitive ATPase that pumps Ca2+ into reconstituted liposomes.

Biological Transport, Active↗