PubMed Health⌕ Search

Biomedical subjects

S Pikula

Publications and source records attributed to S Pikula.

At least 19 recordsLinked to original sources
PubMed2006

A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.

Animals↗
PubMed2001

N- and C-terminal halves of human annexin VI differ in ability to form low pH-induced ion channels.

Human recombinant annexin VI (AnxVI) or its N- (AnxVIA) and C-terminal (AnxVIB) fragments were expressed in E. coli. Their ability to form voltage-dependent ion channels in membranes, induced by low pH, was measured to verify the hypothesis that, upon acidification, the hydrophobicity of AnxVI at a specific domain significantly increases allowing the AnxVI interaction with lipids in a Ca(2+)-independent manner. By theoretically analyzing changes in protein hydrophobicity, we found that hydrophobicity of AnxVIA significantly differed from that of AnxVIB at low pH. These predictions were confirmed experimentally by using planar lipid bilayers and liposome pull-down assay. We found striking difference between AnxVIA and AnxVIB in the ion channel activity, as well as in the membrane binding, suggesting that the halves of AnxVI maybe functionally different. Moreover, we calculated and predicted that the ion channel activity at low pH should appear in other human annexins, as AnxII, AnxV (as known), AnxVIII, and AnxXIII. The possibility that AnxVI acts as cytosolic component of a transmembrane pH-sensing mechanism is proposed.

Annexin A6↗
PubMed2001

Conformational states of annexin VI in solution induced by acidic pH.

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.

Acids↗
PubMed2001

UDP hydrolase activity associated with the porcine liver annexin fraction.

In the crude fraction of porcine liver annexins, we identified annexin IV (AnxIV), AnxII and AnxVI of MW (molecular weight) of 32, 36 and 68 kDa, respectively, an albumin of MW of 61.5 kDa and an UDP hydrolase (UDPase) of MW of 62 kDa, related to the human UDPase from Golgi membranes. The latter enzyme exhibits its highest specificity towards UDP and GDP but not ADP and CDP, and it is stimulated by Mg(2+) and Ca(2+). AnxVI itself, although it binds purine nucleotides, does not exhibit hydrolytic activity towards nucleotides. Taken together, these results suggest that AnxVI may interact in vivo with a nucleotide-utilizing enzyme, UDPase. This is in line with observations made by other investigators that various annexins are able to interact with nucleotide-utilizing proteins, such as protein kinases, GTPases, cytoskeletal proteins and p120(GAP). Such interactions could be of particular importance in modulating the biological activities of these proteins in vivo.

Albumins↗
PubMed2001

Annexins as nucleotide-binding proteins: facts and speculations.

Annexins are ubiquitous multifunctional Ca2+ and phospholipid-binding proteins whose mechanism of function remains largely unknown. The accumulated in vitro experimental evidence indicates that ATP and GTP are functional ligands for nucleotide-sensitive annexin isoforms. Such nucleotide binding could modulate Ca2+ homeostasis, vesicular transport and/or signal transduction pathways and link them to cellular energy metabolism. Alternatively, since annexins are able to interact with other nucleotide-utilizing proteins, such as various kinases, GTPases and structural proteins, these proteins could influence the guanine nucleotide exchange metabolism and/or control the activity of various G proteins. The nucleotide-binding properties of annexins may affect the development or maintenance of some pathologies and diseases in which changes in physiological concentrations of purine nucleotides or disruption of Ca2+ homeostasis are crucial targets.

Acid Anhydride Hydrolases↗
PubMed2000

Lipid metabolism as a target for potassium channel effectors.

K(+) channel effectors are widely used in the treatment of various diseases, including diabetes mellitus type II, hypertension, and cardiac arrhythmia. In addition, a constantly growing body of literature reveals that some of these substances, despite their direct effect on K(+) channels, may influence cellular lipid metabolism. As a result, membrane lipid content and cellular concentrations of lipid messengers are changed. Due to the dependence of K(+) channel activity on membrane lipids, these observations seem to be of particular importance not only to characterize secondary effects of K(+) channel effectors but also to understand the long-term effects of these agents on K(+) channel activity.

Anti-Arrhythmia Agents↗
PubMed2000

Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin.

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.

ATP-Binding Cassette Transporters↗
PubMed1999

ATP-Binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.

Adenosine Triphosphate↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

Adenosine Triphosphatases↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity.

Purified dinitrophenyl S-glutathione (DNP-SG) ATPase was reconstituted into artificial liposomes prepared from soybean asolectin. Electron micrography confirmed the formation of unilamellar vesicles with an average radius of 0.25 micron. Intravesicular volume estimated by incorporation of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 microL/mL reconstitution solution. Accumulation of the glutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposomes was increased in the presence of ATP as compared to equimolar ADP or adenosine 5'-[beta,gamma-methylene]triphosphate tetralithium. ATP-dependent transmembrane movement of DOX and DNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive to the osmolarity of the assay medium, and temperature dependent. The energy of activation was found to be 12 and 15 kcal/mol for DNP-SG and DOX, respectively. Optimal temperature for transport was 37 degrees C. Saturable transport was demonstrated for DNP-SG (Vmax of 433 +/- 20 nmol/min/mg of protein, KmATP = 2.4 +/- 0. 3 mM and KmDNP-SG = 36 +/- 5 microM) as well as DOX (Vmax = 194 +/- 19 nmol/min/mg of protein, KmATP = 2.5 +/- 0.6 mM and KmDOX = 2.4 +/- 0.7 microM). The kinetic data for both DNP-SG and DOX transport were consistent with a random bi-bi sequential reaction mechanism. DOX was found to be a competitive inhibitor of DNP-SG transport with Kis of 1.2 +/- 0.2 microM and DNP-SG was found to be a competitive inhibitor of DOX transport with Kis of 13.3 +/- 2.6 microM.

Adenosine Triphosphatases↗
PubMed1997

Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.

We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.

Animals↗
PubMed1997

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomes.

Properties of Ca(2+)-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent K(m) values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their K(m) values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCl. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCl and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were also found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca(2+)-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.

Animals↗
PubMed1996

A glutathione S-transferases isozyme (bGST 5.8) involved in the metabolism of 4-hydroxy-2-trans-nonenal is localized in bovine lens epithelium.

Previous studies have suggested that a group of GST isozymes (bGST 5.8) with substrate preference for 4-hydroxy-2-trans-nonenal (4-HNE) were present in bovine retina, cornea, iris-ciliary body and sclera, but not in lens. The present studies demonstrate that bGST 5.8 is present in bovine lens epithelium and absent in the cortex and nucleus. Immunochemical studies demonstrated that the enzyme is selectively expressed in epithelium where it can be induced by about 2.5-fold when the lenses are cultured in Medium-199 for 24 hr in the presence of 10 microM BHT. bGST 5.8 was purified to homogeneity from the epithelium using immunoaffinity chromatography. Upon SDS-PAGE, the enzyme showed a single band corresponding to an M(r) value of 25 kDa and its CNBr-peptide maps in SDS-gels were identical to those of the isozymes of this group of GSTs reported previously. The enzyme exhibited high activity towards 4-HNE, and showed glutathione peroxidase activity towards phospholipid hydroperoxides. The Km values of the enzyme for 4-HNE (57 microM from control and 52 microM from BHT-treated) were in the same range as those reported for GSTs 5.8 of human ocular tissues. However, the Kcat value of the lens epithelium enzyme for 4-HNE (15.4 mol mol-1 sec-1 from control, and 20.2 mol mol-1 sec-1 from BHT treated) were considerably less than those reported for the human ocular GST 5.8. Results of these studies suggested that a GST isozyme involved in the detoxification of the electrophilic products of lipid peroxidation was localized in the epithelium of bovine lens.

Aldehydes↗
PubMed1994

Organic anion-transporting ATPase of rat liver. I. Purification, photoaffinity labeling, and regulation by phosphorylation.

An ATP-dependent transport system specific for non-bile acid organic anions such as S-(2,4-dinitrophenyl)-glutathione is present in the canalicular plasma membrane of hepatocytes. It has been shown recently that transport of these anions by isolated hepatocytes is modulated by the activity of cellular protein kinase C (Roelofsen, H., Ottenhoff, R., Oude Elferink, R. P., and Jansen, P.L. (1991) Biochem. J. 278, 637-641, 1991). Using a series of affinity chromatography steps, we have purified to apparent homogeneity a 90-kDa glycoprotein which has S-(2,4-dinitrophenyl)glutathione-dependent ATPase activity. As shown by binding to immobilized S-(2,4-dinitrophenyl)glutathione and by photoaffinity labeling with 8-azido-ATP, the binding sites for organic anions and for ATP of the 90-kDa protein are interdependent, a behavior that is consistent with an ATP-dependent transport function. The ATP binding site has been further characterized using a fluorescent ATP analog. The 90-kDa protein was phosphorylated by protein kinase C, and the Vmax (but not the Km) of the S-(2,4-dinitrophenyl)glutathione-dependent ATPase activity increased at least 2-fold upon phosphorylation. On the basis of its enzymatic properties, we propose that the 90-kDa protein is identical with the multispecific organic anion transporter (MOAT) of the hepatic canalicular plasma membrane. The size and oligosaccharide content of the 90-kDa protein indicate that it does not belong to the family of mammalian plasma membrane P-glycoproteins.

Adenosine Triphosphatases↗
PubMed1994

Organic anion-transporting ATPase of rat liver. II. Functional reconstitution of active transport and regulation by phosphorylation.

We have previously purified to homogeneity from rat liver plasma membranes a 90-kDa glycoprotein with S-(2,4-dinitrophenyl)glutathione-stimulated ATPase activity and other properties which identify it as the multispecific organic anion transporter (MOAT) specific for the transport into bile of non-bile acid organic anions (Pikula, S., Hayden, J. B., Awasthi, S., Awasthi, Y. C., and Zimniak, P. (1994) J. Biol. Chem. 269, 27566-27573). In the present communication, we report the functional reconstitution of this protein into artificial proteoliposomes. The reconstituted protein catalyzed time- and concentration-dependent uptake of S-(2,4-dinitrophenyl)glutathione into the vesicles. The transport required the presence of ATP. Phosphorylation of the 90-kDa protein by protein kinase C prior to reconstitution more than tripled the Vmax of transport but did not change the Km for S-(2,4-dinitrophenyl)glutathione. The protein created and, at steady state, maintained a more than 200-fold and 500-fold S-(2,4-dinitrophenyl)glutathione gradient across the membrane for the unphosphorylated and phosphorylated form, respectively. The transport activity of the 90-kDa protein is sufficient to account for the hepatic secretory maximum of non-bile acid organic anions in the rat.

Adenosine Triphosphatases↗
PubMed1990

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides.

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).

Animals↗