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R Klinger

Publications and source records attributed to R Klinger.

At least 37 records · Page 2Linked to original sources
PubMed1991

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

[Seborrheic keratoses. Intact epidermal differentiation despite disordered morphogenesis].

Seborrheic keratoses were investigated by means of histo- and biochemical techniques in order to define the epidermal differentiation and proliferation of this most common epithelial tumor in adults. The following markers have been used in our study: cytokeratins, epidermal proteins binding zinc, insulin receptors, tissue plasminogen activator (tPA), as well as calmodulin (immunohistochemical and quantitative evaluation). Immunohistochemical investigation of seborrheic keratoses revealed a decreased tPA expression; their epidermal concentration of calmodulin was doubled. The differentiation markers failed to show any significant deviation from normal skin. Thus, seborrheic keratoses are associated with normal epidermal differentiation in spite of morphogenic alterations. Pathogenetically, we have to consider epidermaldermal interactions.

Calmodulin↗
PubMed1989

Calmodulin inhibition by anthralin? Investigations in vitro and in vivo.

Increased epidermal calmodulin (CaM) levels have been reported in psoriatic lesions. It has been suggested that CaM inhibition might be of relevance in the treatment of psoriasis vulgaris. Therefore we investigated the possible CaM inhibition by the antipsoriatic drug anthralin in vitro and in vivo. For in vitro studies, anthralin (0.44 mM) effects on the CaM-dependent Ca++-ATPase of CaM-depleted erythrocyte ghosts were assessed. At 100 microM Ca++, no enzyme inhibition was measured either in the presence or absence of CaM. At 2 microM Ca++ anthralin inhibited the CaM stimulation of membrane-bound ATPase for 50% in presence of CaM. For in vivo studies, skin biopsies were taken from anthralin-treated psoriatic lesions without scaling but still palpable infiltration during the first 3 weeks of therapy. Lesions with anthralin-induced irritation were excluded. The epidermal CaM level was determined by measuring the ability of soluble epidermal protein to activate the Ca++-ATPase in CaM-depleted erythrocyte ghosts. Epidermal CaM was 7.07 +/- 4.57 (mean +/- SD) microgram CaM/mg soluble epidermal protein. This is a 6-fold increase compared to normal human epidermis and a 3-fold increase compared to nontreated lesional psoriatic epidermis (p less than 0.01 and 0.02, respectively). The results argue against CaM inhibition of psoriatic epidermal keratinocytes as the primary event in the antipsoriatic action of anthralin.

Anthralin↗
PubMed1987

Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Fast activation of Ca2+-ATPases in plasma membranes from cardiac muscle and from ascites carcinoma cells: a possible function of endogenous calmodulin.

Content of endogenous calmodulin, binding of calmodulin to, and Ca2+-ATPase activity in plasma membranes of cardiac muscle. Ehrlich ascites carcinoma (EAC) cells and erythrocytes were examined. The content of endogenous calmodulin in cardiac and EAC cells was shown to be considerably higher than in erythrocyte membranes. Ca2+-independent binding of calmodulin to cardiac and EAC cell membranes was found to be realized by some low molecular weight proteins. Ca2+-ATPases in cardiac and EAC cell membranes differ from those in erythrocytes with respect to their activation by Ca2+ and calmodulin. The erythrocyte enzyme is strongly stimulated by exogenous calmodulin and reaches its maximum activity about 2 min after Ca2+-addition. In contrast, the Ca2+-ATPases in cardiac and EAC cell plasma membranes cannot be considerably stimulated by exogenous calmodulin and are instantaneously activated by Ca2+.

Animals↗
PubMed1986

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells.

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.

Animals↗
PubMed1986

Calmodulin binding proteins in human erythrocyte membranes.

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.

Calcium-Transporting ATPases↗
PubMed1986

Membrane skeleton and diminution of transmembrane proteins and calmodulin in erythrocytic vesicles.

During preservation of erythrocytes vesicles are formed, the membranes of which do not contain membrane skeleton components (spectrin, actin). In comparison to the normal erythrocyte membrane (100%) the vesicle membrane exhibits significantly lowered amounts in band 3 protein (14-17%), sialic acid (30-40%), (Ca2+ + Mg2+)ATPase (6%) and (Na+ + K+)ATPase (28%). The losses in band 3 protein, glycophorin A (calculated from the decrease of sialic acid) and in the number of the intramembranous particles correspond to one another. The calmodulin concentration of the vesicles was estimated to be 35% that of the erythrocytes in maximum. The findings indicate a binding of these components to the membrane skeleton. The nature of the binding of the transmembrane proteins as well as the consequences of these results regarding the composition of the intramembranous particles are discussed.

Calmodulin↗
PubMed1984

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes.

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.

Adenosine Triphosphate↗
PubMed1984

Ca/EGTA solutions: comparison between measured and calculated free calcium ion concentrations in the micromolar range.

We prepared a series of Ca/EGTA solutions and compared the pCa2+free values obtained by calculation and measurement with an ion sensitive electrode. Two possible reasons for the discrepancies between these values were studied: (1) the effect of various association constants used for the calculation of pCa2+free, and (2) the influence of deviations of the calcium total concentration in the Ca/EGTA solutions from the values used for the calculation. The results obtained showed that the main reason for differences between calculated and measured pCa2+free values was the nonequimolarity of both stock solutions CaCl2 and EGTA. A linear dependence of measured electrode potentials with Nernstian behaviour on the calculated pCa2+free in the Ca/EGTA solutions has been obtained with carefully equalized stock solutions.

Buffers↗