PubMed Health⌕ Search

Biomedical subjects

J P Andersen

Publications and source records attributed to J P Andersen.

At least 73 records · Page 4Linked to original sources
PubMed1986

Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions.

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.

Adenosine Triphosphate↗
PubMed1986

Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography.

Sarcoplasmic reticulum Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca2+-ATPase occurred within a few hours in the presence of less than or equal to 50 microM Ca2+. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high Ca2+ concentration (500 microM), monomeric Ca2+-ATPase was stable for several hours. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca2+-ATPase was found to be 10(5)-10(6) M-1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca2+-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca2+ and vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. These results suggest that formation of Ca2+-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit.

Animals↗
PubMed1986

Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase.

Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units-does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K)-ATPase in both soluble and membranous states.

Adenosine Diphosphate↗
PubMed1986

The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: a caution and a list of membrane proteins suitable as standards.

We propose a list of 15 water-soluble globular proteins and 13 forms of detergent-soluble membrane proteins of known Stokes radii for the calibration of high-performance liquid chromatography columns. It is shown that it is advisable to use different sets of standards for these two types of proteins as the detergent-solubilized membrane proteins may behave differently, being excluded or retarded, depending upon the gel support. A smooth, although nonlinear, relationship between Stokes radii and erf-1(1--KD) is observed while a large scatter of points exists if the calibration is expressed as the molecular weight as a function of KD.

Calibration↗
PubMed1986

Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase.

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.

Adenosine Triphosphate↗
PubMed1986

Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase.

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.

Animals↗
PubMed1986

Localization of E1-E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling.

Tryptic peptides of Ca-ATPase in E1 and E2 conformational states (Andersen, J. P., Jørgensen, P. L., J. Membrane Biol. 88:187-198 (1985] have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E1-E2 transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E2 state, both in membranous and in soluble monomeric Ca-ATPase. Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[125I]iodophenyl-diazirine indicated that E2 and E2V states are more exposed to the membrane phase than E1 and E1P (Ca2+-occluded) states. The preferential hydrophobic labeling in E2 forms was found to be localized in the A1 tryptic fragment.

Amino Acid Sequence↗
PubMed1985

Equilibrium between monomers and oligomers of soluble Ca2+-ATPase during the functional cycle.

Molecular sieve HPLC shows that soluble sarcoplasmic reticulum Ca2+-ATPase at low concentrations of the non-ionic detergent octaethylene glycol monododecyl ether exists as monomers in equilibrium with dimers and higher oligomers. Binding of vanadate or ATP as well as phosphoenzyme turnover shifts the equilibrium towards the monomer. This suggests that the Ca2+-pump cycle can occur without transient self-association of Ca2+-ATPase peptides.

Adenosine Triphosphate↗
PubMed1985

The role of Mg2+ and Ca2+ in the simultaneous binding of vanadate and ATP at the phosphorylation site of sarcoplasmic reticulum Ca2+-ATPase.

The sarcoplasmic reticulum Ca2+-ATPase was reacted with vanadate in the presence of Mg2+ and EGTA, and the effect of Ca2+, Mg2+ and ATP on the kinetics of vanadate release from the enzyme vanadate complex was studied after dilution with vanadate-free media. Ca2+ increased, whereas ATP decreased the rate of vanadate release. In absence of free Mg2+ in the release media ATP was bound to the vanadate-reacted Ca2+-ATPase with high affinity (Kd 4-5 microM), and full saturation with ATP resulted in complete inhibition of vanadate release. In media containing free Mg2+, where ATP predominantly was present as MgATP, binding of the nucleotide to vanadate-reacted Ca2+-ATPase occurred with low apparent affinity. Mg2+ alone did not affect the rate of vanadate release. At saturating ATP concentrations the release rate in the presence of free Mg2+ was less inhibited than in its absence. These results indicate that uncomplexed ATP interacts with the same Mg2+ at the catalytic site, which is involved in formation of the enzyme-vanadate complex (EMgV), and thereby hinders dissociation of vanadate. Destabilization of the complex by free Mg2+ may be caused by the presence of an additional magnesium ion in the catalytic site together with ATP.

Adenosine Triphosphate↗
PubMed1985

Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum.

Changes in Ca2+ binding after phosphorylation of membranous or detergent-solubilized preparations of sarcoplasmic reticulum Ca2+-ATPase with ATP were followed spectrophotometrically by the use of murexide. Distinct Ca2+ release from the two high-affinity translocation sites was observed, particularly at alkaline pH and at low Ca2+/Mg2+ concentration ratios. Phosphorylation also induced additional binding of Ca2+ at a third site in competition with Mg2+. Ca2+ release was increased after solubilization of Ca2+-ATPase in predominantly monomeric form with the nonionic detergent octaethyleneglycol monododecyl ether. At 0 degree C, chemical-quench studies with [32P]ATP indicated that release of Ca2+ is correlated with the level of ADP-insensitive phosphoenzyme (2 mol of Ca2+ released per mol of E2P formed), both for membranous and detergent solubilized Ca2+-ATPase. Ca2+ release was also found to be accompanied by changes in intrinsic fluorescence. Analysis of the data at 20 degrees C, pH 8.0, showed that binding of Ca2+ to transport sites on E2P occurs with a half-saturation constant of 0.7 mM and a Hill coefficient of 1.8. This is consistent with a drastic decrease in Ca2+ affinity following conversion of ADP-sensitive E1P to ADP-insensitive E2P. The similarity between membranous and detergent-solubilized Ca2+-ATPase supports the view that not more than a single Ca2+-ATPase polypeptide chain is required to complete the conformational transitions which are the basis for active transport of Ca2+.

Adenosine Triphosphate↗
PubMed1985

Conformational states of sarcoplasmic reticulum Ca2+-ATPase as studied by proteolytic cleavage.

Conformational states in sarcoplasmic reticulum Ca2+-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca2+ binding (E1 state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca2+ (E2 state) exposes bonds in the B fragment, which are protected by binding of Mg2+ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E1P and ADP-insensitive E2P have cleavage patterns identical to those of unphosphorylated E1 and E2, respectively, indicating that two major conformational states are involved in Ca2+ translocation. The transition from E1P to E2P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process. The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca2+-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca2+ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca2+-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca2+-ATPase in membranous form.

Animals↗
PubMed1985

Direct demonstration of structural changes in soluble, monomeric Ca2+-ATPase associated with Ca2+ release during the transport cycle.

The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypeptide chain is the functional unit in energy transduction.

Adenosine Diphosphate↗
PubMed1983

Perturbation of the structure and function of a membranous Ca2+-ATPase by non-solubilizing concentrations of a non-ionic detergent.

The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca2+-ATPase membranes, prepared from sarcoplasmic reticulum (SR). At low concentrations C12E8 is incorporated into the membrane (less than or equal to 0.2 g/g protein), without any solubilization or appreciable morphological changes of freeze-fracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2 degrees C) phospholipid is extracted somewhat more easily than ATPase. Electron-spin resonance (ESR) spectra of appropriate spin labels, incorporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+-ATPase in a characteristic way: V is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of the protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+-depleted conformation) of the enzyme. Furthermore, stopped-flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady-state ATP hydrolysis. It is concluded that C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.

Animals↗
PubMed1983

Reconstitution of sarcoplasmic reticulum Ca2+-ATPase with excess lipid dispersion of the pump units.

Sarcoplasmic reticulum Ca2+-ATPase has been reconstituted with excess lipid (25-150 g egg phosphatidylcholine per g sarcoplasmic reticulum protein) by a procedure combining the use of a non-ionic detergent with cholate dialysis. The reconstituted vesicles were analyzed by sucrose density fractionation and freeze-fracture electron microscopy. At the lowest lipid to protein ratios some vesicles containing aggregated protein were observed. At a lipid to protein ratio of 150:1 (w/w) only 30-40% of the reconstituted protein sedimented through 7% (w/v) sucrose. The remainder of the latter preparation was characterized by a high Ca2+-uptake capacity and a coupling ratio of 1.6 mol Ca2+ transported per mol ATP hydrolyzed. Intramembranous particles in this preparation occurred isolated in the membrane. In most cases only one particle could be seen on a fracture face. Cross-linking with cupric phenanthroline indicated that protein-protein contacts were drastically reduced by reconstitution. It is concluded that aggregation of intramembranous particles is not required for optimal Ca2+-transport function. The dispersed preparation obtained by a combined reconstitution and sucrose density fractionation procedure is useful for further characterization of the Ca2+ pump.

Animals↗
PubMed1983

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins.

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.

Animals↗
PubMed1982

The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements.

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.

Animals↗