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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Changes in the calcium levels under the influence of estradiol were investigated in rat vaginal epithelial cells (VEC). After single estradiol injection, the immature rats showed 1.5-fold increase in Ca2+ levels within 15 min when compared to control animals. Progesterone priming brought calcium levels well below control values throughout the experimental period (up to 12 h). Ca2+ levels in serum did not show any appreciable change. Localization of calcium in VEC with electron microscopy showed aggregates of calcium oxalate on the inner nuclear membrane, nucleolus, mitochondria and keratohyaline granules. After 15 min of estradiol priming, maximum electron density was seen on all these cell organelles mentioned above, however, by 30 min the electron density was reduced considerably and did not increase during the experimental period (up to 12 h).
J Steroid Biochem Mol Biol 1990 Nov 30
PMID:Keratinization of rat vaginal epithelium--V. Modulation of intracellular calcium by estradiol. 170 74

A new visualization principle for the detection of cerium phosphate reaction product of phosphatases in light microscopy is described. The new mode is based on the conversion of cerium phosphate into cerium oxalate. The latter is able to react with OsO4. In this way osmium black is formed, staining enzymatic activity sites grey or greyish-black. Utilizing the argyrophilia of osmium black, the staining contrast could be remarkably intensified by posttreatment with Ag-proteinate (Ce-Ox-Os-Ag procedure). This procedure is of similar sensitiveness in comparison to the DAB-Ni method, proposed earlier. The advantages are a very pale background staining and a complete suppression of the cerophilia. Moreover, it substitutes to DAB, a probable potent carcinogen. The method is time-saving when the processing of the reactions was stimulated in a microwave oven.
Cell Mol Biol 1991
PMID:A new visualization principle of cerium phosphate reaction product of phosphatases in cryotome sections for light microscopy--the cerium-oxalate-osmium-silver (Ce-Ox-Os-Ag) method. 171 3

Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.
Mol Biochem Parasitol 1991 Jul
PMID:Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism. 185 83

The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.
J Mol Cell Cardiol 1991 Mar
PMID:Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding. 188 Aug 10

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.
J Mol Biol 1991 May 20
PMID:Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin. 203 52

The Ca2+ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca2+ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0 degrees or 37 degrees, but the Ca2+ uptake activity was relatively stable at 25 degrees. The decay of Ca2+ uptake activity at 0 degrees could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca2+ uptake activity of homogenates kept on ice but not of homogenates kept at 37 degrees. We also found that release of the CSR from the cellular debris required homogenization in high KCl. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mumol/min-mg in the absence of CSR calcium channel blockers and 0.67 mumol/min/mg in the presence of 10 microM ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca2(+)-ATPase rates averaged 1.07 mumol/min/mg and 0.88 mumol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a 'crude' preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca2(+)-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function.
Mol Cell Biochem 1990 Dec 03
PMID:Stabilization of rat cardiac sacroplasmic reticulum Ca2+ uptake activity and isolation of vesicles with improved calcium uptake activity. 214 64

We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1-20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl-beta-D-glucopyranoside (octylglucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram-negative cells to recover both inorganic phosphate (Pi)-linked antiport and oxalate:formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non-leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild-type and mutant.
Mol Microbiol 1990 Aug
PMID:A rapid method for reconstitution of bacterial membrane proteins. 228 Jun 90

This study demonstrates a simple, rapid, and reproducible microassay for real-time monitoring of Ca2(+)-sequestration by isolated sarcoplasmic reticulum (SR) using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. The SR membranes were isolated by differential centrifugation and suspended in a medium including Ca2+, indo-1, ATP and oxalate. As Ca2+ was sequestered by SR, Ca2(+)-bound indo-1 fluorescence decreased equivalently but reciprocally to the increase in Ca2(+)-free indo-1 fluorescence. The kinetic and thermodynamic properties of Ca2(+)-transport measured fluorometrically were similar to those measured radiometrically by 45Ca2+, with the exception that the former monitors changes in free Ca2+ whereas the latter monitors total Ca2+. An estimate of the maximal rate of change in total Ca2+ could be made by multiplying the maximal rate of change in free Ca2+ by the ratio of initial total Ca2+ to free Ca2+ concentration.
Mol Cell Biochem 1990 May 10
PMID:Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. 237 46

Transient ischemia does not induce myocardial necrosis but may be associated with prolonged contractile dysfunction ("stunned" myocardium). It has been suggested that alteration of the excitation-contraction coupling system (sarcoplasmic reticulum) could be responsible for this phenomenon. We tested this hypothesis by characterizing sarcoplasmic reticulum (SR) function in an isolated rat heart model of "stunned" myocardium (hearts reperfused after 10 min of normothermic global ischemia). At the end of the ischemic period oxalate-supported Ca-uptake was depressed either in the whole homogenate or in isolated SR (to 47% and 22% of control values, respectively). During reperfusion Ca-uptake of the whole heart homogenate recovered almost completely whereas slight but significant depression persisted in isolated SR (48 +/- 2 vs 67 +/- 4 nmol/min x mg, P less than 0.01). In the presence of ruthenium red or ryanodine, two inhibitors of SR Ca-release channels, Ca-uptake was stimulated. Both in the whole heart homogenate and in isolated SR, such stimulation was remarkably smaller after reperfusion than in control conditions (P less than 0.001) suggesting reduced conductivity state of the SR Ca-release channels. Ca-stimulated, magnesium-dependent ATPase activity was remarkably reduced during ischemia and postischemic reperfusion induced only incomplete recovery (93 +/- 18 vs 169 +/- 14 nmol ATP/min x mg protein, P less than 0.05). We conclude that complex modifications of SR function occur in the "stunned" myocardium and could contribute to the contractile impairment found in this condition.
J Mol Cell Cardiol 1989 Oct
PMID:Sarcoplasmic reticulum function in the "stunned" myocardium. 247 59

We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.
 ...
PMID:Guanosine triphosphate utilization by canine cardiac muscle sarcoplasmic reticulum. 253 46


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