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M Zurini

Publications and source records attributed to M Zurini.

29 records · Page 2Linked to original sources
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

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↗
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

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↗
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↗
PubMed1981

Activity of phospholipase A2 in the inner membrane of rat-liver mitochondria.

The inner membrane of rat liver mitochondria contains a highly active phospholipase A2 which has alkaline pH optimum and requires Ca2+ in the micromolar range. The phospholipase is particularly active on the endogenous phosphatidylethanolamine and release relatively high amounts of docosahexanoic acid. The phospholipase A2 of mitochondria or mitoplasts is not dependent on calmodulin. Using fluorescamine-labelled mitoplasts there are indications that the enzyme is localized on both sides of the inner membrane.

Animals↗