Biomedical subjects

W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 145 records · Page 8Linked to original sources

PubMedDate not supplied

The biphasic Ca2+-uptake by the fragmented sarcoplasmic reticulum.

The non-equilibrium dialysis has been used for kinetic studies of ATP dependent calcium uptake by the sarcoplasmic reticulum. The uptake displays two phases, which are defined as fast and slow uptake. The former is an exponential function of time, with a half-life time of approximately 15--20 sec, the latter presents the characteristics of an autocatalytic reaction. The fast uptake is characterized by its amplitude, the slow uptake by its rate. Compared with the fast uptake, the slow uptake requires higher amounts of Mg2+ or ATP, and is more sensitive to pH variations and aging. The reasons which formerly prevented the resolution of the second phase from the first are discussed. It is concluded that the fast uptake is not a simple binding reaction, and that the slow uptake is more sensitive to changes by the vesicular membrane.

Adenosine Triphosphatases↗

PubMedDate not supplied

The location of the calcium precipitating protein in the sarcoplasmic membrane.

Fluram, a fast reacting reagent for primary amino groups does not label proteins enclosed in reconstituted sarcoplasmic vesicles (SR vesicles) when applied at low reagent/protein ratios. Hence, Fluram does not penetrate through SR membranes under these conditions. Likewise, the membrane of erythrocytes prevent the reagent from reacting with hemoglobin. However, at high reagent/protein ratios the membranes of the SR vesicles disintegrate. In contrast, liposomal membranes prepared from SR lipids are not affected even at a high degree of labeling. Disintegration of SR membranes starts to occur when the phosphatidylethanolamine fraction is completely substituted by Fluram and the transport protein is labeled with 3-4 mol of Fluram per 100000 d. When closed SR vesicles are labeled at low Fluram/protein ratios, the calcium precipitating protein is four times more intensely labeled than the transport protein. The labeling of the calcium transport protein in closed vesicles excludes its location in the intravesicular space. In SR vesicles solubilized with deoxycholate the relative degree of labeling of the calcium precipitating protein remains unchanged while the transport ATPase is more intensely labeled at the expense of the labeling of the amino lipids. The relative degree of labeling of the protein components depends not only on the number of labable groups but also on the rates with which these groups react with Fluram. Therefore, the experimental data do not give quantitative information concerning the distribution of the protein components in the SR membranes.

Adenosine Triphosphatases↗

PubMedDate not supplied

Arrangement of proteins and lipids in the sarcoplasmic membrane.

The number of amino residues present in the proteins of the sarcoplasmic reticulum which can react with Fluram has been determined in native and sonicated SR vesicles. Sonication increases the number of amino groups accessible to Fluram from 0.57 to 0.87 mumol-mg prot.-1. This increase indicates that 66% of the amino residues are present in the external and 34% in the internal membrane leaflet. The distribution of the amino phospholipids is computed from the distribution of Fluram in the membrane proteins in comjunction with the relative distribution of Fluram between protein and lipid in native and sonicated vesicles. The distribution of the calcium transport protein has been approximated under different assumptions concerning the distribution of the residual protein and taking into account that 15% of the membranes of the SR vesicles might have changed their sideness during preparation.

Amines↗

PubMedDate not supplied

The effect of ethylene glycol and DMSO on fusion of isolated sarcoplasmic reticulum membranes.

Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit skeletal muscle were suspended in 5-10% ethylene glycol (EG) or in 5, 10, or 15% dimethylsulfoxide (DMSO) and were pelleted onto flat aluminum foil disks. No vesicle fusion o-curs with either 5 or 10% EG treatment and 2-21/2 hours drying. After 4-5 hours drying, 5% EG-treated vesicles have established more areas of close contact and individual vesicles have begun to flatten when compared with the 10% EG vesicles remained round as in the 2-21/2 hour samples of all treatments. Overnight drying also causes extended sheets of bilayer pairs to form in the 5% DMSO-treated samples but, with 10 and 25% treated vesicles, destroys the double bilayers and only occasional dense regions of membrane whorls remain. Both EG and DMSO promote morerapid fusion of FSR vesicles than does glycerol but overnight drying after treatment with 10 or 25% DMSO DESTROYS THE FUSED MEMBRANE.

Animals↗

PubMedDate not supplied

Calcium gradient dependent pyrophosphate formation by sarcoplasmic vesicles.

The vesicles of the sarcoplasmic membranes synthesize pyrophosphate from inorganic phosphate. Pyrophosphate synthesis proceeds as long as a calcium gradient is maintained across the vesicular membranes. Pyrophosphate synthesis is inhibited by low concentrations of nucleoside triphosphates.

Adenine Nucleotides↗

PubMedDate not supplied

Competition between oxalate and phosphate during active calcium accumulation by sarcoplasmic vesicles.

1. During ATP supported active calcium uptake oxalate as well as phosphate are accumulated with calcium. The uptake of calcium exceeds that of both anions by a small quantity--accounting for calcium binding to vesicular proteins and lipids. 2. From assay media containing phosphate and oxalate--nearly exclusively either oxalate or phosphate are taken up together with calcium by the sarcoplasmic reticulum vesicles. The mutual exclusion occurs in a very narrow concentration range of the anions. 3. In solutions containing phosphate and oxalate, calcium phosphate or calcium oxalate precipitates are formed according to their solubility properties. 4. When phosphate prevents oxalate from being taken up, calcium transport is inhibited. Inhibition occurs, because the concentration of ionized calcium inside the vesicles rises approximately 100-fold when oxalate is replaced by phosphate. The activity of the calcium dependent ATPase parallels the calcium uptake activity. 5. It is excluded that the inhibition of calcium uptake produced by phosphate is caused by an enhanced permeability of the sarcoplasmic reticulum membranes for calcium in the presence of phosphate.

Animals↗

PubMedDate not supplied

Phosphorylation by inorganic phosphate of sarcoplasmic membranes.

The calcium transport protein of the sarcoplasmic reticulum accepts inorganic phosphate rapidly when phosphorylation is initiated either by the addition of phosphate or magnesium ions to the calcium free protein. Phosphorylation proceeds much more slowly when it is initiated by the addition of the calcium chelatro ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the phosphate and magnesium containing assay. The time course of phosphorylation following immediately calcium removal is monophasic at all temperatures between 20 degrees and 37 degrees C. In contrast, biphasic time course doses not only apply to net formation of phosphoprotein but also to its exchange with medium phosphate. On addition of calcium, the phosphoprotein decays in a biphasic process the time constants of which are much longer than those observed for phosphoprotein formation. The temperature dependence of the rate as well as of the extent of phosphoprotein formation indicate a discontinuity in the reactivity of the protein.

Animals↗

PubMedDate not supplied

Effect of lipid modification on fusion of sarcoplasmic reticulum vesicles.

Freeze fracture technique ascertains that sarcoplasmic reticulum vesicles fuse during anisodiametric dehydration which leads to the formation of sheetlike structure of a mean extension of 5000 A. Modification of the membrane lipids by phospholipase A2 digestion or the incorporation of deoxycholate facilitates the coalescence of the vesicles, while it is completely prevented by lipid removal. Membrane fusion during anisodiametric dehydration is considered as resulting from the close contact of highly curved edges from which the membrane proteins have been excluded.

Animals↗

PubMedDate not supplied

The collapse of the sarcoplasmic reticulum in skeletal muscle.

When various cations, including Ca2+, are in the fixative, both sarcoplasmic reticulum (SR) of whole skeletal muscle and isolated SR vesicles collapse to form pentalaminate "compound membranes" that result from the apparent fusion of the lumenal lamellae of the membranous envelope of the SR. The process may be reversed by subsequently soaking the tissue in 1 M NaCl. An identical morphological phenomenon is observed in unfixed quickly frozen isolated frog skeletal muscle fibers, the cation in that case coming from endogenous sources. The hypothesis is advanced that the collapse is an in vivo process mediated by the sequestration of Ca2+ after contraction. The resulting obliteration of the SR lumen would have the effect of displacing the SR contents into the junctional SR, as well as electrically isolating the free SR from the junctional SR during relaxation. As a consequence, resistive coupling between the plasmalemma and the junctional SR becomes a plausible mechanism for the translation of the action potential into Ca2+ release, since the bulk of the SR membrane capacitance would now remain separated from the plasmalemma during relaxation.

Animals↗

PubMedDate not supplied

The inhibition of the calcium transport ATPase of the sarcomplasmic reticulum by fluorescamine: evidence for an oligomeric functional unit of the calcium transport system.

The labeling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluorescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphorylation by [gamma-32P]ATP and [32P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase, it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules.

4-Nitrophenylphosphatase↗

PubMedDate not supplied

Low affinity calcium binding sites of the calcium transport ATPase of sarcoplasmic reticulum membranes.

Calcium binding sites having low affinity constants of less than 10(3) M-1 were titrated in native sarcomplasmic reticulum vesicles as well as in lipid deprived membranes and in the isolated calcium transport ATPase. Short time calcium binding measurements and the determination of the calcium binding heat allow to distinguish low affinity calcium binding sites located on the external surface of th sarcoplasmic reticulum membranes from those present in the section of the transport molecule directed to the vesicular space. The same number of internal binding sites was found for preparations deprived of their lipid content as well as of preparations depleted of their lipids and of their accessorial proteins. Magnesium interferes with calcium binding to the external as well as to the internal low affinity calcium binding sites. The implications of the existence of the low affinity calcium binding sites in the internal section of the calcium transport ATPase are discussed.

Animals↗

PubMedDate not supplied

Fluorescence studies on N-(3-pyrene)maleinimide-labeled sarcoplasmic reticulum ATPase in native and solubilized membranes.

Fluorescence polarization and formation of excimers were studied in N-(3-pyrene)maleinimide-labeled sarcoplasmic reticulum vesicles. 1. The polarization of pyrenemaleinimide labeled vesicles does not change with temperature and shows a pronounced decrease at labeling concentrations larger than 1 mol pyrenemaleinimide per 10 mol ATPase. 2. Solubilization of the membrane with myristoylglycerophosphocholine renders the polarization temperature dependent, but does not affect the concentration dependent depolarization observed in native vesicles. 3. The polarization of labeled vesicles is much smaller than to be expected from the temperature independent polarization indicating that the pyrenemaleinimide polarization did not monitor the rotation of the entire ATPase. Thus segmental motion occurs. 4. Pyrene excimers are observed at label concentrations larger than 1 mol label per 2.5 mol ATPase. 5. The amount of excimers was critically dependent on added detergents. From the fact that non-solubilizing amounts of myristoylglycerophosphocholine strongly reduced the amount of pyrene excimers it is concluded that in the native sarcoplasmic reticulum vesicles at least two ATPase molecules must be in close contact.

Animals↗

PubMedDate not supplied

Inactivation of the sarcoplasmic reticulum calcium-transport-ATPase by Lasolocid in combination with Triton X-100.

The calcium-transport-ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately 0.2 mg . ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. The enzyme is more rapidly inactivated in the active than in the inactive state.

Animals↗

PubMedDate not supplied

Bile salt delipidation, residual phospholipids and reactivation of the Ca2+-ATPase from sarcoplasmic reticulum.

1. Delipidation of the Ca2+-ATPase of sarcoplasmic reticulum membranes by gel chromatography employing ionic detergents (cholate, deoxycholate and mixtures of both) in the presence of glycerol has been studied with respect to residual phospholipids and ATPase activities. 2. The extent of delipidation depends on the detergent chosen and on the ionic strength of the elution buffer. Increasing ionic strength favours a more effective removal of phospholipids, down to about 1 phospholipid molecule per ATPase molecule. 3. The residual ATPase activities of the delipidated preparations are negligibly low. Extensive restoration of the Ca2+-dependent ATPase activity has been achieved by oleic acid, a lysolecithin (myristoylglycerophosphocholine) and a lecithin (dimyristoylglycerophosphocholine). The percentage of reactivation by oleate depends linearly on the amount of residual phospholipids and on the detergent employed. 4. After gel filtration through an Ultrogel or Sepharose column containing 1% cholate in the elution buffer the delipidated ATPase is eluted as a reactivatable high molecular aggregate, whereas 1% deoxycholate favours the formation of completely lipid-free monomeric units which cannot be reactivated, however. A high molecular aggregate is also formed in deoxycholate, the ratio of monomer to polymer depending on the solubilizing and elution conditions. 5. The residual lipids are always composed of a mixture of all different lipid classes present in the native sarcoplasmic vesicles, even at high degrees of delipidation. Specific changes with varying extent of delipidation were not detected.

Animals↗

PubMedDate not supplied

The influence of detergents on the Ca2+- and Mg2+-dependent adenosine triphosphatase of the sarcoplasmic reticulum.

During the stepwise solubilization of sarcoplasmic reticulum vesicles with detergents, the following changes in the structural and enzymatic properties of the preparation are observed: 1. The viscosity of the vesicular suspension initially rises. This change is accompanied by the formation of elongated tubules. Subsequently the membranes are completely disintegrated, resulting in a considerable reduction of the viscosity. 2. A decrease in the activity of the Ca2+-dependent ATPase, which is restored after complete solubilization. 3. A decrease in the change of intrinsic tryptophan-fluorescence on removal of calcium ions, which is also restored after complete solubilization. 4. A decrease of the calcium affinity of the ATPase. 5. A decrease in the amount of phosphorylated protein formed by the incorporation of inorganic phosphate. On the other hand, the amount of phosphoprotein formed from ATP is not affected during solubilization. 6. The dependence of the initial rates of phosphoprotein formation from inorganic phosphate on either magnesium or inorganic phosphate at low concentrations of the respective ligand changes from an S-shape profile to a normal hyperbolic profile after solubilization.

Animals↗

PubMedDate not supplied

A conformational transition of the sarcoplasmic reticulum calcium transport ATPase induced by vanadate.

Vanadate binding to sarcoplasmic reticulum vesicles results in the loss of the externally located high affinity calcium binding sites of the calcium transport ATPase. Conversely the occupation by calcium of the internally located low affinity sites in the vanadate enzyme complex leads to the release of vanadate. Since the total number of calcium binding sites is not diminished by vanadate binding but slightly increases we conclude that vanadate binding induces a transition of the enzymes external high to internal low affinity calcium binding sites. The transposition of external to internal calcium binding sites is accompanied by a definite change in the structure of the sarcoplasmic reticulum membranes. On vanadate binding the asymmetrically arranged electron dense protein particles become symmetrically distributed.

Animals↗

PubMedDate not supplied

Transient activation of the Ca2+-ATPase from sarcoplasmic reticulum during thiol modification by 5,5'-dithiobis(2-nitrobenzoate).

In the reaction of sarcoplasmic reticulum membranes with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) some new features were observed: The Ca2+-dependent ATPase activities of increasingly modified preparations were considerably enhanced during the initial stage of thiol blockage. A maximum (130-160% of the control activity) was reached when about 1.5-2 mol thiol groups per 10(5) g vesicular protein had reacted, in the absence of ATP and detergent. At higher extents of modification inactivation occurred. Purified ATPase behaved principally similar to native sarcoplasmic vesicles. In the presence of Mg2+ and ATP the activity maximum (up to 180% of control) was broadened and shifted towards a higher degree of thiol blockage. Concomitantly the modification and inactivation rates were considerably reduced. Glycerol (10-30%, v/v) slightly enhanced the ATPase activity maximum and reduced the rate of inactivation essentially only by decreasing the DTNB modification rate. In the presence of sufficient myristoylglycerophosphocholine for solubilization no activation was observed. The steady state level of phosphoprotein from ATP was raised to about 150% of the control level 10 s after addition of DTNB (about 1/2 thiol blocked), followed by a linear decrease with the number of thiols labeled, while the Ca2+-dependent ATPase activity of preparations modified under equivalent conditions (10(-4) M Ca2+ and 2 X 10(-3) M Mg2+ present) showed a broader maximum corresponding to 1.5 thiols blocked.

Adenosine Triphosphate↗

PubMedDate not supplied

Dependence on membrane lipids of the effect of vanadate on calcium and ATP binding to sarcoplasmic reticulum ATPase.

The affinity of the sarcoplasmic reticulum transport ATPase for calcium and ATP is not affected by lipid deprivation while vanadate binding is completely abolished. Lipid substitution restores vanadate binding as well as the vanadate induced disappearance of the enzyme's high affinity calcium and nucleotide binding sites. Nucleotide binding is simultaneously restored with the displacement of vanadate from the enzyme following the occupation of its low affinity calcium binding sites.

Adenosine Diphosphate↗