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S Pikuła

Publications and source records attributed to S Pikuła.

At least 37 records · Page 2Linked to original sources
PubMed1996

Glibenclamide inhibits mitochondrial K+ and Na+ uniports induced by magnesium depletion.

Magnesium depletion induces K+ and Na+ uniports in rat liver mitochondria. The purpose of the present study was to investigate the effects exerted by the antidiabetic sulfonylurea, glibenclamide, a well known blocker of ATP-sensitive potassium channels, on mitochondrial K+ and Na+ uniports. The K+ and Na+ uniport activities were monitored indirectly, in energized mitochondria, by following K+ and Na+ influxes as measured by light scattering. The membrane potential of the mitochondria was determined using a TPP+ selective electrode. Equilibrium binding measurements of glibenclamide to the inner mitochondrial membrane was performed with [3H]glibenclamide. Mitochondrial K+ and Na+ uniports were found to be inhibited by glibenclamide in a concentration-dependent manner, with IC50 of 20 +/- 7 and 15 +/- 8 microM, respectively. On lowering of the pH value, the potency of glibenclamide to inhibit the uniports activity was increased. Binding studies revealed the presence of a single class of low affinity binding sites for glibenclamide in the inner mitochondrial membrane, with a Kd of 4 +/- 2 microM and a BMAX of 148 +/- 50 pmoles/mg of protein. The present study provides evidence that both mitochondrial K+ and Na+ uniport activities are sensitive to the antidiabetic sulfonylurea, glibenclamide.

Animals↗
PubMed1996

Effects of inhibitors and activators of ATP-regulated K+ channel on mitochondrial potassium uniport.

The effect of inhibitors and activators (channel openers) of ATP-regulated potassium channel on potassium ion transport was studied in isolated rat liver mitochondria. In order to follow the potassium transport into mitochondrial matrix light scattering measurements have been employed. The membrane potential of rat liver mitochondria, upon addition of potassium channel openers, was measured with the use of TPP-ion selective electrode. The results show that some of the potassium channel openers like HOE 234 and Y27152 are able to activate potassium transport into mitochondria. On the other hand, mitochondrial potassium uniport found was inhibited by the antidiabetic sulfonylureas and was not affected by other potassium channel inhibitors like 4-amino pyridine.

Adenosine Triphosphate↗
PubMed1994

The relationship between phospholipid content and Ca2+-ATPase activity in the sarcoplasmic reticulum.

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction. Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity. Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects. The phosphatidylinositol (PI)-specific phospholipases of B. cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content. The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8). These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis. Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation. These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).

Animals↗
PubMed1994

Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties.

Glutathione S-transferase P1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398-408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S-transferase P1-1 was expressed and isolated as described for [I104]glutathione S-transferase P1-1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for [I-104]glutathione S-transferase P1-1 and [V-104]glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half-lives at 45 degrees C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding.

Base Sequence↗
PubMed1992

Annexins IV (p32) and VI (p68) interact with erythrocyte membrane in a calcium-dependent manner.

Purification of annexin IV and VI from porcine liver was achieved by Mono Q ion exchange chromatography at pH 8.9 and pH 7.5, respectively. The isolated proteins interacted with erythrocyte membrane as function of calcium ion and the protein concentration. Half-maximal binding of annexin VI to erythrocyte membrane was found to occur at 8 microM Ca2+. The maximal binding was estimated as 2 micrograms of annexin VI per 1 microgram or erythrocyte membrane protein, in the presence of 100 microM Ca2+. The property of erythrocyte membrane to interact with annexins was utilized in preparation of a affinity-column with polyacrylamide-immobilized erythrocyte membrane.

Animals↗
PubMed1991

Thyroid hormones control lipid composition and membrane fluidity of skeletal muscle sarcolemma.

Sarcolemma membrane lipid phase of skeletal muscles of hyperthyroid animals was compared to that of control (euthyroid) ones. Hyperthyroidism caused 15% decrease in cholesterol and 70% increase in the phospholipid content of the membrane. This was accompanied by the alterations in proportions between individual phospholipid classes, and was followed by changes in the composition of phospholipid fatty acids. The calculated fatty acid unsaturation index was higher for membrane lipid phase of hyperthyroid animals than of euthyroid ones. Thyroxine-induced alterations in the lipid composition of sarcolemma caused changes in the membrane fluidity and the activity of calmodulin-stimulated (Ca(2+)-Mg(2+)-ATPase. Measurements of the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the lipid phase transition of membrane vesicles occurred at 25.9 degrees C and at 28.9 degrees C for preparations isolated from hyperthyroid and euthyroid rabbits, respectively. Arrhenius plot break-point temperature for CaM-stimulated (Ca(2+)-Mg(2+)-ATPase activity was lower in membrane preparations isolated from hyperthyroid (26.9 degrees C) than from euthyroid ones (30.0 degrees C). Thus, the increase of the membrane fluidity presumably caused that the enzyme was characterized by the lower activation energy value. This phenomenon may be viewed as a supplementary mechanism for activation of the enzyme by thyroid hormones to previously reported elevation of the amount of (Ca(2+)-Mg(2+)-ATPase protein exerted by hyperthyroidism (Famulski et al. (1988) Eur. J. Biochem., 171, 363-368; Famulski and Wrzosek (1988) in The Ion Pumps-Structure, Function and Regulation (Stein, W.D., ed.), pp. 355-360, Alan R. Liss, New York).

Animals↗
PubMed1991

Effect of hyper- and hypothyroidism on phospholipid fatty acid composition and phospholipases activity in sarcolemma of rabbit cardiac muscle.

Lipid content and composition of fatty acids esterified to phospholipids of cardiac sarcolemma isolated from hyperthyroid, hypothyroid and control rabbits were analysed. Hyperthyroidism resulted in a significant reduction of the cholesterol to phospholipid molar ratio as compared to control animals, while hypothyroidism exerted the opposite effect. Complex changes in composition of phospholipid fatty acids observed in hyperthyroid state led to an elevation of the fatty acid unsaturation index over the control value. The unsaturation index value was, however, not affected in the hypothyroid state. Thyroxine hormone administration increased phospholipase A1 and decreased phospholipase A2 activity. The opposite effect was observed in thyreodectomized animals. The effect of changes in sarcolemmal bulk phospholipids upon thyroxine administration or deficiency on regulation of activity of membrane-bound enzymes is discussed.

Animals↗
PubMed1991

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Pikułla, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.

Animals↗
PubMed1990

Circular dichroism and fluorescence studies on interaction of calmodulin (CaM) with purified (Ca2(+)-Mg2+)ATPase of erythrocyte ghosts.

It was found, using circular dichroism spectroscopy, that CaM, in the presence of Ca2+, decreases the alpha-helix content of (Ca2(+)-Mg2+)ATPase of porcine erythrocytes from 66% to 55%. In the absence of Ca2+ the enzyme showed 46% of alpha-helix. Moreover, quenching of the ATPase intrinsic fluorescence by acrylamide indicated that, depending on the enzyme conformational status, the accessibility of its tryptophan residues is influenced by direct interaction with CaM at micromolar Ca2+ concentration. This was also confirmed by the observation that fluorescence energy transfer occurred from tryptophan residues of (Ca2(+)-Mg2+)ATPase to dansylated CaM. The presented results may indicate that binding of CaM gives rise to a novel conformational state of the enzyme, distinct from E1 and E2 forms of the Ca2+ pump.

Animals↗
PubMed1989

Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies.

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.

Acrylamide↗
PubMed1987

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum.

1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles. 2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor--S-adenosyl-L-methionine (SAM). 3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane. 4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

Animals↗
PubMed1986

Transbilayer distribution of phospholipid synthesizing enzymes in the sarcoplasmic reticulum membrane.

The topology of enzymes involved in phospholipid synthesis in the membrane of sarcoplasmic reticulum vesicles was investigated by means of limited proteolysis. The digestion of sarcoplasmic reticulum proteins with different proteases caused the considerable decrease of the activities of choline- and ethanolaminephosphotransferases without significant inactivation of glycerophosphate and lysophosphatidylcholine acyltransferases. On the basis of these results we concluded that the active sites of phosphotransferases are located on the cytoplasmic side, whereas the active sites of acyltransferase are present either on the extracytoplasmic side or burried into the sarcoplasmic reticulum membrane.

1-Acylglycerophosphocholine O-Acyltransferase↗