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K H Krause

Publications and source records attributed to K H Krause.

At least 73 records · Page 4Linked to original sources
PubMed1993

The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes.

Cytolytic T lymphocytes (CTL), natural killer cells, and lymphokine-activated killer (LAK) cells are cytolytic cells known to release the cytolytic protein perforin and a family of proteases, named granzymes, from cytoplasmic stores upon interaction with target cells. We now report the purification of an additional major 60-kD granule-associated protein (grp 60) from human LAK cells and from mouse cytolytic T cells. The NH2-terminal amino acid sequence of the polypeptide was found to be identical to calreticulin. Calreticulin is a calcium storage protein and carries a COOH-terminal KDEL sequence, known to act as a retention signal for proteins destined to the lumen of the endoplasmic reticulum. In CTLs, however, calreticulin colocalizes with the lytic perforin to the lysosome-like secretory granules, as confirmed by double label immunofluorescence confocal microscopy. Moreover, when the release of granule-associated proteins was triggered by stimulation of the T cell receptor complex, calreticulin was released along with granzymes A and D. Since perforin is activated and becomes lytic in the presence of calcium, we propose that the role of calreticulin is to prevent organelle autolysis due to the protein's calcium chelator capacity.

Amino Acid Sequence↗
PubMed1993

Inositol 1,4,5-trisphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver.

Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.

Animals↗
PubMed1993

Proton channels, plasma membrane potential, and respiratory burst in human neutrophils.

When confronted with invading microorganisms, neutrophils undergo a number of nearly synchronous reactions including the generation of microbicidal reactive oxygen intermediates by the NADPH oxidase. These reactions are accompanied by a slow depolarization, from resting values of-60 mV to levels probably exceeding 0 mV. The depolarization is transient, indicating that a compensatory charge transport mechanism is activated. Activation of the oxidase system causes a massive burst of metabolic acid generation that would, if uncompensated, lower the intracellular pH of neutrophils by over 5 units, to lethal levels (pH = 2). Neutrophils must therefore possess particularly effective regulatory systems to avoid excessive cytosolic acidification. The recently described H+ conductance of neutrophils may counteract both the acidification and the depolarization. Activation of the H+ conductance occurs at depolarizing voltages and is promoted by cytosolic acidification, a combination that takes place during the respiratory burst. The NADPH oxidase of neutrophils is thus associated to an unusual, particularly efficient mechanism of H+ export and charge compensation. The sequential activation of these two systems causes neutrophils to depolarize through the activation of an electron transport chain, and to repolarize through the activation of a H+ conductance.

Cell Membrane↗
PubMed1993

Highly co-operative Ca2+ activation of intermediate-conductance K+ channels in granulocytes from a human cell line.

1. To study Ca(2+)-activated K+ currents in dimethyl sulphoxide (DMSO)-differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-L-phenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)-activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nM [Ca2+]i, and a highly co-operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin-sensitive, intermediate-conductance Ca(2+)-activated K+ channel in HL-60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co-operativity and voltage independence) are similar to the Ca2+ activation of the apamin-sensitive small-conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL-60 granulocytes.

Apamin↗
PubMed1993

Ion channels and receptor-mediated Ca2+ influx in neutrophil granulocytes.

Although generally classified as non-excitable cells, human neutrophils possess a variety of ion channels that play a crucial role in the regulation of cellular activity. The mechanism of receptor-mediated Ca2+ influx in neutrophils is complex. Receptor agonists empty intracellular Ca2+ stores via generation of Ins(1,4,5)P3. The emptying of intracellular Ca2+ stores leads by an hitherto not understood mechanism to the activation of Ca2+ influx across the plasma membrane. Neutrophils possess at least 2 types of K+ channels. Voltage-activated K+ channels, important for the maintenance of the resting potential, and Ca2+ activated K+ channels, important for the repolarization after cellular activation. Neutrophils also possess voltage- and pH activated H+ channels that serve to extrude protons, generated by the neutrophil respiratory burst. Neutrophils depolarize in response to activation by agonists. The mechanism of neutrophil depolarization involves electron transport by the respiratory burst oxidase. Neutrophil depolarization serves as a negative feed-back mechanism, it also activates the H+ channels and thereby stimulates extrusion of protons.

Calcium↗
PubMed1993

Proton currents in human granulocytes: regulation by membrane potential and intracellular pH.

1. To determine whether conductive pathways contribute to the H+ efflux from granulocytes, we used the whole-cell patch-clamp technique combined with microfluorimetric determinations of cytosolic pH (pHi) in single, dimethylsulphoxide-differentiated HL-60 cells. 2. In voltage-clamp mode, depolarization of the cell from the resting potential (around -60 mV) to +60 mV caused an increase in pHi that was accompanied by a sizeable outward current. 3. Ion substitution experiments and analysis of the reversal potential of tail currents indicated that the outward current is carried largely by H+ ions. 4. Full activation of the H+ current occurred within 1-2s after depolarization and deactivation within 100-200 ms upon repolarization. 5. This H+ conductance was strongly dependent on pHi, being larger at acidic pH. In addition, at low pHi the threshold for voltage activation of the H+ conductance was shifted to more negative values. 6. Addition of millimolar concentrations of Cd2+ and Zn2+ to the bath solution reduced the maximum H+ conductance and shifted the voltage dependence of the H+ conductance to more positive potentials. The effects were reversible. 7. In conclusion, our results demonstrate that granulocytic HL-60 cells possess a voltage-gated and pHi-sensitive H+ conductance. Because both a depolarization and a cytosolic acidification occur during the activation of granulocytes, this conductance may play a role in pHi homeostasis of granulocytes during microbial killing.

Cations, Divalent↗
PubMed1992

Differential effects on neutrophil activation of staurosporin and its protein kinase C-selective derivative cgp 41231.

The relative insensitivity of the chemoattractant-induced respiratory burst to non-specific kinase inhibitors, such as staurosporin, is widely considered as evidence against the involvement of protein kinase C in signal transduction by chemoattractants. In this study we compared the effect on neutrophil activation of the non-specific kinase inhibitor staurosporin with the effect of its protein kinase C-selective derivative cgp 41251. Staurosporin activates secondary granule release by itself and enhances chemoattractant-induced primary granule release; it inhibits superoxide production in response to phorbol esters at low concentrations, but superoxide production in response to chemoattractants only at considerably higher concentrations. In contrast, cgp 41251 did not interfere with granule release, but inhibited phorbol ester- and chemoattractant-induced superoxide production with similar potency. These results suggest that many of the staurosporin effects, including its low potency to inhibit chemoattractant-induced superoxide production, are due to protein kinase C-independent effects. The results obtained with cgp 41251 are compatible with a role of protein kinase C in the mediation of the chemoattractant-induced respiratory burst of human neutrophils.

Alkaloids↗
PubMed1992

Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells.

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.

Biological Transport↗
PubMed1992

Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells.

To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.

Calcium-Binding Proteins↗
PubMed1992

s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin.

Cyclophilins (cyclosporin A-binding proteins) are conserved, ubiquitous, and abundant proteins that accelerate the isomerization of XaaPro peptide bonds and the refolding of proteins in vitro. s-Cyclophilin is a member of the cyclophilin family with unique NH2- and COOH-terminal extensions, and with a signal sequence. We now report that s-cyclophilin is retained in the cell, and that the conserved s-cyclophilin-specific COOH-terminal extension VEKPFAIAKE is sufficient to direct a secretory protein to s-cyclophilin containing structures. Antibodies to s-cyclophilin-specific peptides were produced and the location of the protein was determined by an immunocytochemical study at the light microscopic level. s-Cyclophilin colocalized with the Ca(2+)-binding protein calreticulin and, to a lesser extent, with the microsomal Ca(2+)-ATPase in the myogenic cell line L6, and with the Ca(2+)-binding protein calsequestrin in skeletal muscle. In activated platelets, s-cyclophilin immunoreactivity was detected in a ring-like structure that might correspond to the Ca(2+)-storing and -releasing dense tubular network. In spreading cells, s-cyclophilin containing vesicular structures accumulated at actin-rich protrusion sites. While s-cyclophilin consistently codistributed with Ca2+ storage site markers, the distribution of s-cyclophilin immunoreactivity was not identical to that of ER markers. To determine whether the COOH-terminal extension of s-cyclophilin was involved in its intracellular transport we added this sequence to the COOH-terminus of the secretory protein glia-derived nexin. Appropriate constructs were expressed transiently in cultured cells and proteins were detected with specific antibodies. We found that glia-derived nexin with the COOH-terminal sequence VEKPFAIAKE (but not with the control sequence GLVVMNIT) colocalized with endogenous s-cyclophilin, indicating that the sequence contained retention information. These results indicate that s-cyclophilin is a retained component of an intracellular organelle and that it may accumulate in specialized portions of the ER, and possibly in calciosomes. Because of its conserved structure, widespread distribution, and abundance s-cyclophilin may be a useful marker to study the biogenesis and distribution of ER subcompartments.

Amino Acid Isomerases↗
PubMed1992

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Calcium↗
PubMed1991

Ca(2+)-storage organelles.

Intracellular Ca(2+)-storage organelles are found in virtually all eukaryotic cells. They play an important role in the regulation of the cytosolic free Ca2+ concentration and, thereby, in the regulation of cellular activity. Ca(2+)-storage organelles consist, in the simplest model of a Ca2+ pump, of a Ca(2+)-storage protein and a Ca(2+)-release channel. The primary structure of these functionally important proteins of Ca(2+)-storage organelles is similar in different cell types and conserved through evolution. In contrast, their spatial arrangement and, thus, the architecture of Ca(2+)-storage organelles may vary dramatically from one cell type to another.

Biological Transport, Active↗
PubMed1991

Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding sites.

Ca2+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Ca(2+)-binding sites with a mean dissociation constant (KD) of 0.79 mM were titrated in the absence, and 23 sites with a KD of 0.88 mM in the presence of 3 mM Mg2+. No cooperativity was observed. For Mg2+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg2+/protein with a KD of 2mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca(2+)- and Mg(2+)-binding sites. After correction for proton release, the enthalpy change for Ca2+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg2+) was therefore virtually the sole driving force for Ca2+ binding. Mg2+ binding is slightly more exothermic (-12.6 kJ/protein), but as for Ca2+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg(2+)-saturated calsequestrin was clearly different from that in the Ca(2+)-saturated protein, but that the (Ca2+ + Mg2+)-saturated protein was not distinct from the Ca(2+)-saturated protein. Thus, in addition to the thermodynamic characterization of the Ca2+/calsequestrin interaction, our data indicate that Ca2+ and Mg2+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca(2+)-Mg2+ exchange in vivo.

Animals↗
PubMed1990

Sequence similarity of calreticulin with a Ca2(+)-binding protein that co-purifies with an Ins(1,4,5)P3-sensitive Ca2+ store in HL-60 cells.

HL-60 cells possess a 60 kDa Ca2(+)-binding protein that is contained in a discrete subcellular compartment, referred to as calciosomes. Subcellular fractionation studies have suggested that, in HL-60 cells, this intracellular compartment is an Ins(1,4,5)P3-sensitive Ca2+ store. In order to investigate the structural relationship of the 60 kDa Ca2(+)-binding protein of HL-60 cells to other Ca2(+)-binding proteins, we have purified the protein by ammonium sulphate extraction, acid precipitation, and DEAE-cellulose and phenyl-Sepharose column chromatography. The N-terminal sequence of the protein shows 93% identity with rabbit muscle calreticulin, a recently cloned sarcoplasmic reticulum Ca2(+)-binding protein. No amino acid sequence similarity with calsequestrin was found, although the purified protein cross-reacted with anti-calsequestrin antibodies. The calreticulin-related protein of HL-60 cells might play a role as an intravesicular Ca2(+)-binding protein of an Ins(1,4,5)P3-sensitive Ca2+ store.

Amino Acid Sequence↗
PubMed1990

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes.

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.

Acetylcholine↗