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Biomedical subjects

W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 55 records · Page 3Linked to original sources
PubMed1983

Reversal of decreased phosphorylation of sarcoplasmic reticulum calcium transport ATPase by 1,25-dihydroxycholecalciferol in experimental uremia.

When compared to that from sham-operated controls, sarcoplasmic reticulum isolated from skeletal muscle of uremic rabbits had a lower rate of calcium uptake and storing capacity. In vivo administration of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] restored the values in uremic animals toward normal. To obtain information about the mechanisms responsible for these differences, phosphorylation of the calcium transport ATPase was studied. The steady-state levels of phosphoprotein in uremic membranes were lower and returned to normal when the secosteroid was administered. Electrophoresis of the membranes phosphorylated with 32P-inosine triphosphate (32P-ITP) showed that the differences were related to a 100,000 dalton protein. The rate of phosphoprotein formation, determined with 32P-ITP and at 0 degrees C, was considerably lower in uremic than in control animals. Pretreatment with 1,25(OH)2D3 prevented this change. The hypothesis is advanced that the vitamin D metabolite affects the steady-state concentration and rate constant of formation of active sites in the Ca-ATPase. These results may partly explain the altered Ca transport function of the sarcoplasmic reticulum in experimental uremia.

Animals↗
PubMed1981

Preparation of sarcoplasmic reticulum with high calcium-sensitive ATPase and stable calcium transport function from rat skeletal muscle.

A method was developed for the isolation from rat skeletal muscle of sarcoplasmic reticulum vesicles in which the calcium transport function does not decay during storage. High initial and maximum uptake of calcium and calcium-dependent ATPase activity were obtained for membranes isolated from mixed muscles or pure red fibers. Unstable vesicles resulted when 2 mM EDTA was included in the isolation medium. The calcium uptake activity was lost upon ageing at 0 degrees C, probably due to conversion of the calcium-dependent ATPase to a calcium-independent form. Addition of Ca2+ counteracted the affects of EDTA, suggesting their involvement in maintaining the structure of the calcium transport system. This is supported by the fact that different structural states of the ATPase in stable and unstable vesicles were detected by DEAE-cellulose column chromatography.

Animals↗
PubMed1981

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle.

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.

Animals↗
PubMed1981

The formation of intravesicular calcium phosphate deposits in microsomes of smooth muscle. A comparison with sarcoplasmic reticulum of skeletal muscle.

The calcium uptake in the microsomial fraction isolated from the smooth muscle of the antrum of the pig stomach is stimulated by phosphate. The microsomial vesicles which are loaded with calcium phosphate can be purified by differential centrifugation. A purification of 36 times in terms of calcium content was reached. Electron microscopy of the freshly prepared material revealed calcium phosphate deposits in the form of needles of crystalline calcium phosphate. This structure differs from that of the deposits which appear in the fragmented sarcoplasmic reticulum of skeletal muscle. Their morphology is that of non-crystalline calcium phosphate. However, on standing these deposits convert slowly into crystalline calcium phosphate. This difference reflects different kinetics of crystallization of the precipitates in the two preparations. After negative staining of the calcium phosphate loaded microsomes of skeletal and of smooth muscle, only few deposits are preserved because a release of calcium occurs as a consequence of the action of the stain and also of the dilution and warming up of the suspension. Smooth muscle microsomes partially purified by loading with calcium phosphate were studied by freeze etching and rotary replication. Membrane fragments displaying subunit intramembrane particles similar to those observed in sarcoplasmic reticulum of skeletal muscle could be identified. However, in the smooth muscle microsomes the intramembrane particles were much less densely packed. Part of these particles could correspond to calcium transport sites.

Animals↗
PubMed1981

Variable Ca2+ transport:phosphoprotein ratios in the early part of the GTP-driven calcium-transport reaction of the sarcoplasmic reticulum.

Initial Ca2+ transport and phosphoprotein formation of the sarcoplasmic reticulum membrane with GTP were investigated in a comparative study. While saturation of the high-affinity sites for Ca2+ binding and transporting as well as for GTP binding on the external surface of the membrane resulted in Ca2+ transport and phosphoprotein formation in a molar ratio of 2, the variation of the concentrations of the two reactants yielded ratios between 1.7 and 5.7. The ratios varied with a similar dependence on the concentrations of Ca2+ and GTP, except at 500 microM Ca2+, if the reaction was started by Ca2+ instead of GTP but the overall rates decreased. 1 mM DL-propranolol in the preincubation medium selectively inhibited Ca2+ transport but had no effect on initial phosphoprotein formation. These observations indicate that:L (a) phosphorylation of one enzyme molecule induces Ca2+ transport by a variable but limited number of neighbouring molecules, (b) not all Ca2+ bound is essential for phosphorylation but can be transported in parallel, (c) Ca2+ bound to low-affinity sites occupied at 500 microM Ca2+ in the reaction medium is also transported initially, (d) the accessibility of the high-affinity Ca2+ binding sites for DL-propranolol differs, (e) DL-propranolol interacts with Ca2+ binding and transporting sites only in that conformation of the enzyme that can be phosphorylated by the nucleotide.

Adenosine Triphosphate↗
PubMed1981

Preparative isolation of Apo(Ca2+-ATPase) from sarcoplasmic reticulum and the reactivation by lysophosphatidylcholine of Ca2+-dependent ATP hydrolysis and partial-reaction steps of the enzyme.

1. A preparative method for the isolation of the lipid free apoprotein, the Ca2+-ATPase of sarcoplasmic reticulum, from the partially purified lipoprotein, Ca2+-ATPase vesicles, is presented. 2. By enzymatic hydrolysis of the phospholipids and removal of the splitting products and endogenous neutral lipids, the apoprotein was consistently delipidated to 0.02 mumol Pi/mg protein. 3. Reactivation of the splitting of ATP and the pseudo substrate, dinitrophenyl phosphate, was demonstrated with a variety of lipids and detergents. 4. A total reactivation of ATP splitting was achieved after a mild ultrasonication of the apoprotein with myristoylglycerophosphocholine which resulted in solubilization of the enzyme as an optically clear solution. 5. The stable resolubilized enzyme could be stored for several weeks maintaining full enzymatic activity. Gel chromatography suggested that under the assay conditions, the monomeric form of the enzyme predominated. 6. In comparison with the native enzyme, the resolubilized enzyme showed differences in the temperature dependence of the activation of ATP hydrolysis and a reduced apparent affinity for MgATP. 7. The phosphate-transferring activities of the resolubilized enzyme were only partially reactivated in the forward direction, and none of the reverse partial-reaction steps of the enzyme could be demonstrated.

Animals↗
PubMed1981

Synthetic monoacylphospholipids as reactivators of the calcium-dependent ATPase of enzymatically delipidated sarcoplasmic membranes.

1. The reactivating effect of synthetic analogues of natural lysophosphatidylcholines, acyldeoxyglycerophosphocholines, with acyl chain lengths between C10-C18 on enzymatically delipidated sarcoplasmic membranes has been analyzed. 2. The calcium-dependent ATPase is fully restored by myristoyldeoxyglycerophosphocholine. The restoring effect of deoxyglycerophosphocholines declines when the length of the saturated acyl chain becomes shorter or longer. 3. In contrast to the weakly effective palmitoyldeoxyglycerphosphocholine, its cis-9-mono unsaturated analogue oleyldeoxyglycerophosphocholine proved to be a highly effective reactivating compound. 4. The transfer of the terminal phosphate residue of ATP to the transport protein and the phosphate exchange between ADP and ATP displays the same dependence on the acyl chain length of the deoxyglycerophos-phocholines. In contrast to the ATPase activity the ATP-supported phosphoryltransfer reactions can only partially be restored. 5. A significant restoration of the phosphorylation of the protein by inorganic phosphate could be achieved with none of the deoxyglycerophosphocholines. 6. Below 23 degrees C the apparent activation energy of the calcium-dependent ATPase increases with increasing chain length of the deoxyglycerophosphoclines while above 23 degrees C the activation energies were identical for all restituted preparations.

Adenosine Diphosphate↗
PubMed1981

Magnesium dependence of sarcoplasmic reticulum calcium transport.

The activity of the calcium transport systems in the sarcoplasmic reticulum membranes operating in the forward and reverse modes depends on the presence of magnesium ions. When the system operates as an NTP-driven calcium pump, magnesium enters the reaction chain chelated with NTP. Magnesium is a component of the NDP-sensitive phosphoprotein species involved in the NDP-NTP exchange reaction. When the system operates in the reverse mode as a calcium efflux-driven NDP synthetase, magnesium together with inorganic phosphate rapidly forms the initial phosphorylated intermediate. The stability of the magnesium-phosphoprotein complex is considerably enhanced by an existing calcium gradient. Magnesium as well as calcium affects the partitioning of the energy in the reaction sequence.

Adenosine Diphosphate↗
PubMed1981

[Calcium transport in sarcoplasmic reticulum in the presence of AR-L 115 BS].

2-[(2-Methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS) is a substance with positive inotropic activity which does not influence the activity of the sarcoplasmic calcium pump. It can, therefore, be expected that AR-L 115 BS does not interfere with the distribution and movement of calcium in the resting and active muscle.

Animals↗
PubMed1980

Lack of involvement of sarcoplasmic reticulum in myopathy of acute phosphorous depletion.

Acute and chronic hypophosphatemia are known to cause metabolic myopathy. It has been proposed that impaired Ca transport in subcellular membranes is involved in its genesis. In the present study, calcium transport in the sarcoplasmic reticulum (SR), concentrations of ions or nucleotides and transmembrane potential were investigated in muscles of acutely hypophosphatemic rats, i.e. animals with chronic dietary phosphorous deprivation (PD) and superimposed acute hypophosphatemia resulting from the administration of insulin and glucose. Despite hypophosphatemia and low muscle phosphorous concentration, no significant change of the initial rate of Ca uptake or Ca concentrating ability was observed in the SR of PD rats. Storing capacity was decreased; this may result from altered vesicle geometry. Water content, Na concentration, the concentration of several nucleotides and transmembrane potential of muscle were unchanged in PD rats. The findings document that no intrinsic abnormality of vectorial Ca transport is present in the SR of acutely hypophosphatemic PD animals.

Animals↗
PubMed1980

Electron cytochemistry of oxalate-stimulated calcium uptake in microsomes from the smooth muscle of the pig stomach.

A microsomal fraction was isolated from the smooth muscle of the antrum of the pig stomach by differential centrifugation. Electron microscopy of the negatively stained material showed that this fraction is heterogeneous in composition. The microsomes accumulated calcium in the presence of ATP, magnesium and oxalate. The amount of calcium taken up per mg protein was in the same range as observed for other smooth muscle microsomal preparations. Although this amount is much smaller than that in the microsomal fraction of skeletal muscle, calcium oxalate crystals were formed in some vesicles, as occurs in the skeletal muscle fragmented sarcoplasmic reticulum. Through the presence of the calcium oxalate crystals, many of these vesicles acquired sufficient mass and density to allow them to be isolated by centrifugation. A purification of about 40 fold in terms of calcium content was reached.

Adenosine Triphosphate↗
PubMed1979

Effects of phospholipase A2 and albumin on the calcium-dependent ATPase and the lipid composition of sarcoplasmic membranes.

1. The calcium-dependent ATPase activity of phospholipase-A2-digested sarcoplasmic vesicles decreases concomitantly with the contents of residual lysophospholipids and fatty acids when increasing albumin concentrations are applied. 2. Delipidated albumin preferentially removes unsaturated fatty acids and lysophosphatidylcholine. A complete removal of the phospholipids by albumin does not occur. 3. The membrane-bound lysophospholipids were analysed with respect to type of phospholipid, plasmalogen content and fatty acid chains by means of thin-layer chromatography and gas chromatography. 4. While the fatty acid composition of the lysophospholipids is independent of the degree of delipidation, the composition of the residual free fatty acids is found to change with the albumin concentration. 5. Reactivation of the Ca2+-ATPase by oleate leads to reasonable activities at room temperature as long as a minimum of about 30 lysophospholipid molec-les per ATPase is left. The course of the residual Ca2+-ATPase activity with the degree of delipidation is related to the presence of unsaturated fatty acids. 6. No specific role of either sphingomyelin or the plasmalogens has been found.

Animals↗
PubMed1979

Comparison between ATP-supported and GTP-supported phosphate turnover of the calcium-transporting sarcoplasmic reticulum membranes.

The study deals with the interrelationship of the phosphate-transferring activities of the calcium-transporting sarcoplasmic reticulum membrane vesicles: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being approximately equal to 3 mumol . mg-1 . min-1 for ATP-ADP and only approximately equal to 0.3 mumol . mg-1 . min-1 (22 degrees C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.

Adenosine Diphosphate↗