scholarly journals Zn2+ depletion blocks endosome fusion

1995 ◽  
Vol 312 (3) ◽  
pp. 919-923 ◽  
Author(s):  
A Aballay ◽  
M N Sarrouf ◽  
M I Colombo ◽  
P D Stahl ◽  
L S Mayorga
Keyword(s):  
Acetic Acid ◽  
Metal Ion ◽  
Endocytic Pathway ◽  
Intact Cells ◽  
Free System ◽  
Present Evidence ◽  
Gtp Binding ◽  
Low Concentrations ◽  
A Cell ◽  
Control Fusion

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM. Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 microM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn(2+)-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.

Bioenergetics Studies of the Cyanobacterium Anabaena variabilis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1280-1284 ◽  
Author(s):  
Siegfried Scherer ◽  
Heike Sadowski ◽  
Peter Böger
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Cytochrome B ◽  
Anabaena Variabilis ◽  
Electron Donors ◽  
Free System ◽  
Low Concentrations ◽  
Cyanobacterium Anabaena ◽  
A Cell ◽  
C Complex

A cell-free system exhibiting both photophosphorylation (P/2e= 1) and oxidative phosphoryltion (P/O up to 0.8) is described for the cyanobacterium Anabaena variabilis. NADH ant NADPH were found to be equally effective as electron donors for oxidative phosphorylation. Low concentrations of UHDBT, an inhibitor of the cytochrome b/c complex of mitochondria ant loroplasts, were found to inhibit photosystem-II electron transport reactions, but did not affet the cytochrome b6/f-complex of Anabaena. The inhibition by myxothiazol, antimycin and heptyihydroxyquinoline corroborates the hypothesis that both respiration and photosynthesis share the cytochrome b6/f-complex.

scholarly journals Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

1987 ◽  
Vol 104 (4) ◽  
pp. 1105-1115 ◽  
Author(s):  
K Matuoka ◽  
M Namba ◽  
Y Mitsui
Keyword(s):  
Cell Proliferation ◽  
Cell Density ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Synthetase Activity ◽  
Intact Cells ◽  
Free System ◽  
Glycosaminoglycan Synthesis ◽  
Cell Free System ◽  
A Cell

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity

1981 ◽  
Vol 200 (3) ◽  
pp. 685-690 ◽  
Author(s):  
K Grankvist
Keyword(s):  
Superoxide Dismutase ◽  
Hydroxyl Radicals ◽  
Metal Ion ◽  
Reduced Glutathione ◽  
Chemical Reactivity ◽  
Free System ◽  
A Cell ◽  
Radical Generation ◽  
Metal Ion Chelators

Chemiluminescence of luminol in a cell-free system was used to investigate the mechanism of alloxan-dependent free-radical generation. In the presence of alloxan and reduced glutathione (GSH), luminescence was greatly stimulated by FeSO4. Replacing GSH by oxidized glutathione or NAD(P)(H), or replacing FeSO4 by CuSO4, ZNSO4 or FeCl3, did not yield chemiluminescence. The chemiluminescence of a mixture of alloxan. GSH, FeSO4 and luminol was inhibited by catalase, superoxide dismutase, scavengers of hydroxyl radicals (sodium benzoate, n-butanol, D-mannitol, dimethyl sulphoxide) or metal-ion chelators (EDTA, diethylenetriaminepenta-acetic acid, diethyldithiocarbamate. desferroxamine), D-glucose, L-glucose, D-mannose, D-fructose, 3-O-methyl-D-glucose, NAD+, NADH, NADP+ or NADPH, but not by urea or enzymically inactive superoxide dismutase. The results support the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals generated in an iron-catalysed reaction. Protection against alloxan in vivo depends both on the chemical reactivity of protector with radicals or radical-generating systems and on the stereospecific requirement of some strategic site in the B-cell.

scholarly journals Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells

1998 ◽  
Vol 336 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Fumikazu OKAJIMA ◽  
Koichi SATO ◽  
Hideaki TOMURA ◽  
Atsushi KUWABARA ◽  
Hiromi NOCHI ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Inhibitory Effect ◽  
Fatty Acid Residue ◽  
Stimulatory Action ◽  
Intact Cells ◽  
Free System ◽  
A Cell ◽  
G Protein Coupled

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.

Synthesis of β-glucan by cell-free extracts of Aureobasidium pullulans

1987 ◽  
Vol 33 (2) ◽  
pp. 123-127 ◽  
Author(s):  
Malcolm A. J. Finkelman ◽  
Alex Vardanis
Keyword(s):  
Enzyme Activity ◽  
Acetic Acid ◽  
Bovine Serum Albumin ◽  
Aureobasidium Pullulans ◽  
Enzyme Preparation ◽  
Glucanase Activity ◽  
Free System ◽  
Enzyme Preparations ◽  
Partial Activation ◽  
A Cell

A cell-free system catalysing the synthesis of β-glucan from uridine-diphosphoglucose was prepared from Aureobasidium pullulans. The activity was stable in the presence of 1 M sucrose and 10 mM MgSO4. The polymer produced was insoluble in H2O or acetic acid (0.5 M) and soluble in NaOH (0.5 M). Several enzyme preparations containing β-glucanase activity degraded the polymer to various extents. Synthesis of the polymer was enhanced by the presence of cellobiose and bovine serum albumin, but not by NaF or ATP. A Lineweaver–Burke plot of enzyme activity versus substrate concentration revealed biphasic kinetics. The enzyme preparation was subject to partial activation by trypsin and chymotrypsin.

scholarly journals Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the α-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the β-subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 659-665 ◽  
Author(s):  
David C. A. JOHN ◽  
Neil J. BULLEID
Keyword(s):  
Heavy Chain ◽  
Binding Protein ◽  
Immunoglobulin Heavy Chain ◽  
Β Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Free System ◽  
A Cell ◽  
Α Subunits

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

scholarly journals Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

1995 ◽  
Vol 311 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P G Heyworth ◽  
R W Erickson ◽  
J Ding ◽  
J T Curnutte ◽  
J A Badwey
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.

scholarly journals The Role of Intraorganellar Ca2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

2000 ◽  
Vol 149 (5) ◽  
pp. 1053-1062 ◽  
Author(s):  
Paul R. Pryor ◽  
Barbara M. Mullock ◽  
Nicholas A. Bright ◽  
Sally R. Gray ◽  
J. Paul Luzio
Keyword(s):  
Dynamic Equilibrium ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Free System ◽  
Rab Protein ◽  
Late Endosomes ◽  
The Reformation ◽  
A Cell ◽  
Gdp Dissociation Inhibitor

We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Mechanisms of caspase-1 activation by P2X7receptor-mediated K+release

2004 ◽  
Vol 286 (5) ◽  
pp. C1100-C1108 ◽  
Author(s):  
J. Michelle Kahlenberg ◽  
George R. Dubyak
Keyword(s):  
Proteolytic Processing ◽  
Intact Cells ◽  
Free System ◽  
Murine Macrophages ◽  
Caspase 1 ◽  
In Vitro Processing ◽  
Monocytes And Macrophages ◽  
Bromoenol Lactone ◽  
A Cell

The mechanisms underlying caspase-1 activation and IL-1β processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1β secretion. Analysis of in vitro IL-1β and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1β. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X7receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1β. This acceleration of in vitro processing was strictly dependent on loss of intracellular K+from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K+loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca2+-independent phospholipase A2. Caspase-1 activation and IL-1β processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K+efflux via P2X7nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.

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