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J V Møller

Publications and source records attributed to J V Møller.

At least 19 recordsLinked to original sources
PubMed2003

Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.

We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca(2+) to the same extent (1-2 Ca(2+) per ATPase monomer) as wild- type ATPase. The inherent ability of the synthetic L6-7 loop peptide to bind Ca(2+) was demonstrated with murexide and mass spectrometry. NMR analysis indicated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum with coordination by all three aspartate residues D813/D815/D818 that resulted in an altered conformation of the peptide chain. Overall our observations suggest that, in addition to mediating contact between the intramembranous Ca(2+) binding sites and the cytosolic phosphorylation site as previously suggested, the L6-7 loop, in a preceding step, participates in the formation of an entrance port important for lodging Ca(2+) at a high-affinity binding site inside the membrane.

Adenosine Triphosphate↗
PubMed2001

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

Allosteric Regulation↗
PubMed1999

NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase.

In current topological models, the sarcoplasmic reticulum Ca2+-ATPase contains 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and probably M8 participating in the formation of the membranous calcium-binding sites. We describe here the conformational properties of a synthetic peptide fragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+-ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of the six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively labeled with 15N and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical structure in its N-terminal part, between residues I788 and T799, while its C-terminal part (G801-N810) remains disordered. Addition of 20% trifluoroethanol stabilizes the alpha-helical N-terminal segment of the peptide, and reveals the propensity of the C-terminal segment (G801-L807) to form also a helix. This second helix is located at the interface or in the aqueous environment outside the micelles, while the N-terminal helix is buried in the hydrophobic core of the micelles. Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800. These conformational features may be related to the transient formation of a Schellman motif (L797VTDGL802) encoded in the M6 sequence, which probably acts as a C-cap of the N-terminal helix and induces a bend with respect to the helix axis. We propose a model illustrating two conformations of M6 and its insertion in the membrane. The presence of a flexible region within M6 would greatly facilitate concomitant participation of all three residues (N796, T799, and D800) believed to be involved in calcium complexation.

Amino Acid Sequence↗
PubMed1998

Construction and use of a detergent-sensitive electrode to measure dodecyl sulfate activity and binding.

We describe the construction and use of a dodecyl sulfate-sensitive electrode cell to measure the activity of the detergent in biological samples. The electrode is based on the incorporation of a cetyltrimethylammonium/dodecyl sulfate complex in a siloxane polymer membrane. The cell records changes in the activity of SDS from 10(-6) to 10(-5) M SDS up to the critical micellar concentration. In aqueous solutions the cell follows Nernst' law with an electrometric response which is not affected by protein per se, but is modified by supporting electrolytes like NaCl. We demonstrate by comparison with equilibrium dialysis that the electrode can be used both to detect the high-affinity binding sites of serum albumin for SDS and to follow cooperative binding of the detergent to serum albumin, ovalbumin, and beta-lactoglobulin in the concentration interval 10(-4)-10(-3) M of unbound SDS. We conclude that the electrode has properties which should enable its use to monitor changes in SDS activity during interaction with biological material. The electrode may also be used to measure the activity of other detergents which, like SDS, form a sparingly soluble complex with cetyltrimethylammonium bromide.

Calibration↗
PubMed1998

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog.

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.

Amino Acid Sequence↗
PubMed1998

The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase.

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.

Biological Transport↗
PubMed1998

Mechanisms of Ca2+ sensitization of force production by noradrenaline in rat mesenteric small arteries.

1. Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and alpha-toxin-permeabilized rat mesenteric small arteries. 2. The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K-PSS (125 mM K+) and NA-K-PSS (K-PSS + 10 microM noradrenaline) were titrated to 30 and 75%, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA-K-PSS than with K-PSS. With K-PSS, the proportion of MLC phosphorylated (approximately 30%) was similar at 30 and 75% of control force; with NA-K-PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34%). 3. In alpha-toxin-permeabilized arteries, the force response to 1 microM Ca2+ was increased by 10 microM noradrenaline, and MLC phosphorylation was increased from 35 to 45%. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline-induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATP gamma S in the presence of the MLC kinase inhibitor ML-9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4. The present results show that that in rat mesenteric small arteries noradrenaline-induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

Animals↗
PubMed1998

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.

Animals↗
PubMed1995

Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K?

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.

Amino Acid Sequence↗
PubMed1994

Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Animals↗
PubMed1993

Topology of Na,K-ATPase alpha subunit epitopes analyzed with oligopeptide-specific antibodies and double-labeling immunoelectron microscopy.

Using four oligopeptide-specific polyclonal antibodies, we mapped the alpha subunit of Na,K-ATPase by double-labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N-terminus (Gly1-His13), C-terminus (Ile1002-Tyr1016) and Leu815-Gln828 are located on the same face of crystallized Na,K-ATPase membranes from pig kidney, whereas the epitope Asn889-Gln903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C-terminus and that Leu815-Gln828 is exposed on the cytoplasmic and Asn889-Gln903 on the extracellular side. The results are consistent with an eight- or ten-segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu815-Gln828 and Asn889-Gln903.

Amino Acid Sequence↗
PubMed1993

Detergent binding as a measure of hydrophobic surface area of integral membrane proteins.

The mode by which four nonionic detergents interact with integral membrane proteins was studied by relating detergent binding to characteristic detergent monolayer and micellar dimensions and to the surface area of the hydrophobic sector of the proteins. The detergents used (C12E8, Triton X-100, dodecyl-beta-D-maltoside, and dodecyldimethylamineoxide) are efficient membrane solubilizers, which, according to our hydrodynamic analysis, form small approximately spherical micelles. Their binding by protomeric forms of bacteriorhodopsin, photosynthetic reaction center, sarcoplasmic reticulum Ca(2+)-ATPase, and cytochrome oxidase was measured after equilibration and delipidation, both by the use of successive chromatographies on silica gel and the use of agarose gel columns in combination with DEAE-cellulose chromatography. It was found that, despite detergent binding by silica gel, both chromatographic procedures gave equivalent and consistent results. The data obtained show systematic trends; thus dodecyldimethylamineoxide exhibited the highest binding levels, followed by dodecylmaltoside, whereas the polyethylene glycol detergents C12E8 and Triton X-100 had the lowest degree of binding. This ranking order is inversely related to the cross-sectional areas of these detergents in monolayers at an air-water interface and in micelles. Binding was reduced by aggregation of protomers, whereas the presence of strongly bound residual lipid slightly increased it. Among the membrane proteins with a high molecular mass (reaction center, Ca(2+)-ATPase, and cytochrome oxidase), relative binding could be related to the size of their hydrophobic sectors. On the other hand, bacteriorhodopsin bound somewhat more detergent per transmembrane helix, probably because of less steric hindrance, caused by the absence of a sizable hydrophilic domain in this protein. Theoretical calculations indicated monolayer binding of detergent to the hydrophobic surface of membrane proteins to be a better model for interaction with membrane proteins than binding of the detergents in micellar form.

Animals↗
PubMed1993

Transitional steps in the solubilization of protein-containing membranes and liposomes by nonionic detergent.

Membrane solubilization by dodecyl maltoside was studied, using Ca(2+)-ATPase membranes and liposome preparations as prototypes of biological membranes. In equilibrium dialysis experiments, transition from saturable incorporation of monomeric detergent into the membrane to cooperative binding already occurred at a free detergent concentration about 50% of the cmc. This transition was discontinuous for unilamellar liposomes of dioleoylphosphatidylcholine, but gradual for Ca(2+)-ATPase membranes and multilayered liposomes of sarcoplasmic reticulum lipid. Equilibrium detergent binding by Ca(2+)-ATPase membranes (expressed on the basis of lipid content) was the same as for detergent binding by multilamellar liposomes of sarcoplasmic reticulum lipid. Equilibration involving cooperative binding was considerably delayed (for many days) if detergent was presented gradually to the membranous preparations in nonmicellar form by diffusion across the dialysis membrane, while equilibration of detergent occurred rapidly if detergent in micellar form was added directly to the membrane preparations. In contrast, equilibration was rapid in both directions if detergent was added at levels below that required to initiate cooperative binding. Detergent interaction resulted in a biphasic decrease in light scattering of Ca(2+)-ATPase membranes. The first of these decreases coincided with the onset of cooperative binding, while the second one was associated with a decreased sedimentability during ultracentrifugation, i.e., with usual criteria of solubilization. The concentration at which this occurred corresponded to the level of free detergent at which lipid, after detergent solubilization, segregated from detergent after gel chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Sedimentation equilibrium of detergent-solubilized membrane proteins in the preparative ultracentrifuge.

Working with detergent-solubilized bacteriorhodopsin we have used a table top preparative centrifuge for determination of M(r) of membrane proteins by sedimentation equilibrium. We demonstrate the use of two new methods to measure protein concentration as a function of distance from rotor axis: (i) peak integration after HPLC on silica gel, and (ii) microdensitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. These methods, although somewhat lengthier than conventional spectrophotometric methods, are more reliable, especially in the presence of a large amount of detergent and small amount of protein. In addition they provide independent information on the status of the protein after sedimentation equilibrium, the association of the solubilized units being readily detected by gel chromatography and proteolytic cleavage by SDS-PAGE.

Bacteriorhodopsins↗