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S Pikuła

Publications and source records attributed to S Pikuła.

At least 19 recordsLinked to original sources
PubMed2001

Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.

ATP-Binding Cassette Transporters↗
PubMed2001

GTP-binding properties of the membrane-bound form of porcine liver annexin VI.

Annexin VI (AnxVI) of molecular mass 68-70 kDa belongs to a multigenic family of ubiquitous Ca2+- and phospholipid-binding proteins. In this report, we describe the GTP-binding properties of porcine liver AnxVI, determined with a fluorescent GTP analogue, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP). The optimal binding of TNP-GTP to AnxVI was observed in the presence of Ca2+ and asolectin liposomes, as evidenced by a 5.5-fold increase of TNP-GTP fluorescence and a concomitant blue shift (by 17 nm) of its maximal emission wavelength. Titration of AnxVI with TNP-GTP resulted in the determination of the dissociation constant (Kd) and binding stoichiometry that amounted to 1.3 microM and 1:1 TNP-GTP/AnxVI, mole/mole, respectively. In addition, the intrinsic fluorescence of the membrane-bound form of AnxVI was quenched by TNP-GTP and this was accompanied by fluorescence resonance energy transfer (FRET) from AnxVI Trp residues to TNP-GTP. This indicates that the GTP-binding site within the AnxVI molecule is probably located in the vicinity of a Trp-containing domain of the protein. By controlled proteolysis of human recombinant AnxVI, followed by purification of the proteolytic fragments by affinity chromatography on GTP-agarose, we isolated a 35 kDa fragment corresponding to the N-terminal half of AnxVI containing Trp192. On the basis of these results, we suggest that AnxVI is a GTP-binding protein and the binding of the nucleotide may have a regulatory impact on the interaction of annexin with membranes, e.g. formation of ion channels by the protein.

Animals↗
PubMed2000

Alzheimer's disease: its origin at the membrane, evidence and questions.

Numerous results on membrane lipid composition from different regions of autopsied Alzheimer's disease brains in comparison with corresponding fractions isolated from control brains revealed significant differences in serine- and ethanolamine-containing glycerophospholipid as well as in glycosphingolipid content. Changes in membrane lipid composition are frequently accompanied by alterations in membrane fluidity, hydrophobic mismatch, lipid signaling pathways, transient formation and disappearance of lipid microdomains, changes in membrane permeability to cations and variations of other membrane properties. In this review we focus on possible implications of altered membrane composition on beta-amyloid precursor protein (APP) and on proteolysis of APP leading eventually to the formation of neurotoxic beta-amyloid (A beta) peptides, the major proteinaceous component of extracellular senile plaques, directly involved in Alzheimer's disease pathogenesis.

Alzheimer Disease↗
PubMed1999

Annexin VI interacts with adenine nucleotides and their analogs.

Annexin VI (AnxVI), a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to interact in vitro with adenine nucleotides. Furthermore, it has been proposed that within the AnxVI molecule a nucleotidde-binding domain exists, which is located in the C-terminal half of the protein, in the vicinity of Trp343. By comparison of exposure of tryptophan and multiple tyrosine residues upon nucleotide binding, as revealed by quenching of intrinsic fluorescence of AnxVI by ATP, ADP or cAMP, it can be concluded that the binding of nucleotides evokes changes in the protein tertiary structure. Moreover, in the course of present study we have found that AnxVI binds to a non-hydrolysable analog of ATP, the triazine dye Cibacron blue 3GA (CB3GA), immobilized on agarose. Binding reveals negative cooperativity with respect to protein concentration and is Ca2+-dependent. Binding is prevented by ATP. CB3GA binds to AnxVI also in solution, evoking the formation of annexin multimers. On the basis of this observation it can be suggested that interaction of CB3GA with AnxVI is useful to examine, with some limitations, the self-association of annexin molecules implying to play a role in interacting of AnxVI with biological membranes.

Adenine Nucleotides↗
PubMed1999

10-Undecynoic acid, an inhibitor of cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of cytochrome P450 4A1.

Animals↗
PubMed1999

Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.

Adenosine Triphosphate↗
PubMed1999

Annexin VI: an intracellular target for ATP.

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.

Adenosine Triphosphate↗
PubMed1998

Positive feedback between ethanolamine-specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid.

The results of the present investigation relate the effects of the nutritional state and administration of clofibric acid (CLA), a hypolipidaemic drug and peroxisomal proliferator, on phosphatidylethanolamine (PE) synthesis in rat liver and fatty acid metabolism. Fasting and CLA treatment of animals causes an increase in the amount of PE in endoplasmic reticulum (ER) membranes and mitochondria, as well as in the PE/phosphatidylcholine (PC) ratio. Moreover, the activity of the ethanolamine-specific phospholipid base exchange (PLBE) enzyme in liver ER membranes of fasted animals was enhanced by 75% in comparison to that of animals fed ad libitum. The effect of CLA treatment was additive to that of starvation; PE synthesis tested in vitro via the Ca2+-sensitive PLBE reaction increased 3-fold in comparison to rats fed ad libitum. This is confirmed by an increased Vmax for the reaction, but the affinity of the enzyme for ethanolamine was not significantly changed. These effects were accompanied by an enhanced expression of cytochrome P450 CYP4A1 isoform and elevated activity of the enzyme upon CLA administration. The stimulatory effect of CLA administration on the efficiency of the ethanolamine-specific PLBE reaction can be explained by elimination of lauric acid, a known inhibitor of de novo PE synthesis, during the course of omega-hydroxylation catalysed by CYP4A1, and by increased expression of the PLBE enzyme. The products of omega-hydroxylation of lauric acid, which are then converted by dehydrogenase to 1,12-dodecanedioic acid, did not significantly affect the in vitro synthesis of PE.

Animals↗
PubMed1998

Modulation of annexin VI--driven aggregation of phosphatidylserine liposomes by ATP.

Annexin (Anx) VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. In addition, AnxVI of porcine liver is an ATP-binding protein, and ATP in vitro modulates its interaction with membranes and cytoskeletal elements (Bandorowicz-Pikuła and Awasthi, FEBS Lett. 409 (1997) 300-306). In this study, we examined the effect of ATP on phosphatidylserine (PtdSer) aggregation in the presence of annexin and on calcium-dependent binding of protein to liposomes, and found that ATP stimulates the former process, although it increases the calcium concentration necessary for half-maximal binding of AnxVI to membranes. These results were corroborated by the experiments with fluorescent analog of ATP, in which binding of ATP to AnxVI was affected by binding of Ca2+ and/or phospholipids to the protein. Taken together they favour an idea of ATP being a functional ligand for AnxVI, which even in the relative absence of Ca2+ may modulate interaction of AnxVI with PtdSer-enriched membranes.

Adenosine Triphosphate↗
PubMed1998

Is the glutathione conjugate of trans-4-hydroxy-2-nonenal transported by the multispecific organic anion transporting-ATPase of human erythrocytes?

Trans-4-hydroxy-2-nonenal (4-HNE), a cytotoxic end product of lipid peroxidation, is present in normal human blood plasma at concentrations of 0.1-1.0 microM. It can be, however, further metabolized within a cell, and one of the main products is 4-HNE glutathione conjugate (HNE-SG). In human erythrocyte membrane the system for active extrusion of glutathione (GSH) conjugates of various endo- and xenobiotics has been described; it exhibits either a low (Km at submillimolar concentration range) or a high (Km at low micromolar range) affinity for the transported substrates, such as for example S-(2,4-dinitrophenyl)glutathione (Dnp-SG). In the present study it has been shown that the high affinity transport system for Dnp-SG is competitively inhibited by HNE-SG with Ki of 0.2 microM, while 4-HNE inhibits non-competitively the activity of the transport system for Dnp-SG with Ki of 220microM. These observations point to the possibility that HNE-SG shares the same transport system with GSH conjugates of other endo- and xenobiotics in erythrocytes. This may be of importance for overall detoxification of the organism under oxidative stress.

Adenosine Triphosphatases↗
PubMed1998

The induction of cytochrome P450 isoform, CYP4A1, by clofibrate coincides with activation of ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

Administration of a hypolipidaemic drug, clofibrate, to rats resulted, 24 h after a single intraperitoneal injection (250 mg/kg body weight), in pronounced enhancement of the rate of phosphatidylethanolamine (PE) synthesis via the PE-specific base exchange (PEBE) reaction in liver microsomes. This was accompanied by 3.4-fold activation of microsomal omega-hydroxylation of lauric acid by cytochrome P450 4A1 isoform (CYP4A1) and an increase in the protein content of this isoform in endoplasmic reticulum (ER) membranes. Since PE represents a class of phospholipids (PL) prerequisite for proper functioning of CYP4A1, and the PEBE reaction is an inducible pathway of PL synthesis in hepatocytes under metabolic stress, one may speculate that this reaction is switched on when extensive remodelling of PL molecular species or/and massive synthesis of lipid bilayer components for membrane assembly is required.

Animals↗
PubMed1998

Annexins and ATP in membrane traffic: a comparison with membrane fusion machinery.

Annexins, calcium- and membrane-binding multifunctional proteins, have been implicated in N-ethylmaleimide (NEM)-independent fusion of vesicular structures involved in membrane traffic. This view is based on intracellular localization of annexins, which are frequently associated with endosomes, chromaffin granules, caveolae, clathrin-coated pits, and other membrane compartments, engaged in endo- and exocytosis. Moreover, annexins were found to modulate budding and aggregation of vesicle membranes, to interact with cytoskeletal proteins, and, upon binding to membranes, to change the structure of lipid bilayer, leading to membrane fusion. In addition, some annexins are substrates for various protein kinases and, in membrane-bound form, reveal calcium channel activity. Recently, annexins were observed to interact in vitro and in vivo with nucleotides, ATP, GTP or cAMP, which are potent mediators of membrane traffic processes. In addition, annexin VII showed hydrolytic activity towards GTP, and similarities in the mechanism of action to that of small GTP-binding proteins were found. The aim of the present review is to summarize the observations implying annexins as possible effectors in endo- and exocytosis and to compare them with well known complexes of cytosolic and membrane proteins forming the true membrane fusion machinery within a cell, conserved from yeast to the neurons of humans.

Adenosine Triphosphate↗
PubMed1998

Adenosine 5'-triphosphate--a new regulator of annexin function. A hypothesis.

The paradigm of annexins as phospholipid-binding proteins interacting with membranes in a calcium-dependent manner has been recently questioned in light of observations that some annexin isoforms may behave like membrane integral proteins or remain associated with their target membranes at low, resting, concentrations of Ca2+ in the cytoplasm. In addition, an evidence has been presented that some annexins (annexins I, VI and VII) bind in vitro ATP and GTP, and upon binding the nucleotide the in vitro activity of these proteins is modified. However, annexins do not contain Walker A and B consensus sequences for ATP/GTP binding. This review presents the hypothesis that a new ATP-binding motif exists within the annexin molecules and that ATP may play a role of functional ligand for annexins also in vivo.

Adenosine Triphosphate↗
PubMed1997

Fluorescence spectroscopic studies on interactions between liver annexin VI and nucleotides--a possible role for a tryptophan residue.

Annexin VI is a 68-kDa calcium-, phospholipid-, and cytoskeletal-element-binding protein, which has been implicated in various processes, including calcium release and sequestration in calcifying cartilage, in a receptor-mediated endocytosis in human fibroblasts, and in secretion from chromaffin granules. In these processes it was found that, in addition to Ca2+ and annexin, the presence of ATP is also a prerequisite. In the present report we show that annexin VI binds ATP and the binding of nucleotide to protein is accompanied by quenching of an intrinsic fluorescence of annexin VI, which was found to be specific for 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, GTP and ATP, and dependent on the annexin conformation. The nucleotide-binding site within an annexin VI molecule is likely to be close to the tryptophan-containing domain of annexin VI. We propose that ATP plays the role of a physiological ligand for annexin VI, and its binding to annexin VI may represent an alternative cellular mechanism for the regulation of annexin-membrane interactions coupled to overall energy transitions in the cell.

Acrylamide↗
PubMed1997

An antagonist of ATP-regulated potassium channels, the guanidine derivative U-37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes.

The guanidine derivative U-37883A has been found to stimulate in vitro synthesis of phosphatidylserine in endoplasmic reticulum membranes, catalyzed exclusively by a serine-specific base exchange enzyme. The stimulation of the enzyme activity by the drug was concentration-dependent, with EC50 of 54 microM, while the biologically inactive analog of U-37883A, U-42069, was without effect. The stimulation caused by U-37883A was enhanced under the conditions when active transport of Ca2+ into the lumen of microsomal vesicles was induced, whereas it was inhibited by a calcium ionophore, A23187, and by a specific inhibitor of Ca2+-ATPase, thapsigargin. On the other hand, a potassium ionophore, valinomycin, had no effect on phosphatidylserine synthesis. U-37883A did not affect the Km of the base exchange enzyme for serine, but greatly reduced the EC50 value of the enzyme for calcium. Furthermore, Ca2+ uptake by endoplasmic reticulum vesicles has been found to increase in the presence of U-37883A. These observations suggest that U-37883A enhances phosphatidylserine synthesis indirectly by acting on calcium transport, thus affecting calcium concentration within the lumen of endoplasmic reticulum membranes. Alternatively, the effect of the drug could be propagated via the mechanism by which phospholipid flip-flop movement, known to regulate the serine-specific base exchange reaction, is modulated.

Adamantane↗