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At least 19 recordsLinked to original sources
PubMed1999

A test of the cytosolic apolipoprotein E hypothesis fails to detect the escape of apolipoprotein E from the endocytic pathway into the cytosol and shows that direct expression of apolipoprotein E in the cytosol is cytotoxic.

Genetic evidence indicates that apolipoprotein E4 (apoE4) is a risk factor for the development of Alzheimer's disease. A controversial hypothesis proposes that apoE, a typical secretory protein, accesses the neuronal cytosol in which apoE3, but not apoE4, protects tau from hyperphosphorylation. However, no conclusive evidence for the presence of apoE in the cytosolic compartment has been presented. We designed a novel assay to test whether apoE can access the cytosol via escape from the endocytic pathway by incorporating a nuclear localization signal (NLS) into apoE. Control experiments demonstrated that apoE plus NLS (apoE+NLS) is chaperoned to the nucleus if it reaches the cytosolic compartment. When exogenous apoE+NLS was endocytosed by neuronal cells, no nuclear apoE was detected, indicating that apoE remains within the endocytic pathway and does not escape into the cytosol. Furthermore, we show that direct cytosolic expression of apoE is cytotoxic. These data argue that effects of apoE on the neuronal cytoskeleton and on neurite outgrowth are not mediated via cytosolic interactions but rather by actions originating at the cell surface.

Apolipoproteins E↗
PubMed1986

Acceptors for cyclic AMP-dependent and calcium ion-dependent protein kinases in rat brain cytosol fractions: a comparison of occluded (synaptosomal) cytosol with non-occluded cytosol.

Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinase activities of Mr 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.

Animals↗
PubMed1994

Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein, Gi alpha.

In a previous report we showed that glucocorticoid inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Gi alpha levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Gi alpha. In order to establish the nature of the coupling between cytosolic Gi alpha and cytosolic PLC we examined the effects of G-protein activators, and inhibitors on cytosolic PLC activity from rat splenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. 1) Neither GTP nor its nonhydrolyzable analogue, GTP gamma S, at 100 microM had any effect on the calcium stimulated as well as the basal PLC activity. 2) However, affinity purified antibodies to Gi alpha 1 and Gi alpha 2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50-70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. 3) Administration of Gi alpha 1 antisense oligonucleotides to splenocytes for 48 h produced 25-40% decrease in cytosolic Gi alpha 1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25-50% compared to control conditions. This suggest that cytosolic Gi alpha is associated with the activation of splenocyte soluble PLC. 4) Pertussis toxin administered in vivo significantly reduced cytosolic Gi alpha immunoreactivity and soluble PLC activity when PI was used as substrate, providing additional evidence that cytosolic Gi alpha is associated with the activation of soluble PLC. 5) Another agent that has been used extensively to define G-protein coupled processes is NaF/AlCl3. NaF (5 mM; with or without AlCl3) inhibited soluble PLC activity with PIP2 as substrate, in contrast to the stimulatory effect that has been reported in the activation of membrane PLC. 6) Because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperizine (50 microM, TFP), an inhibitor of protein phosphatase 2B; TFP (50 microM) significantly inhibited soluble PLC activity when PI was used as substrate. These results suggest a direct involvement of cytosolic Gi alpha in the activation of soluble PLC from splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Gi alpha coupled PLC is addressed in the second paper.

Animals↗
PubMed1985

Stereospecific transport of triiodothyronine from plasma to cytosol and from cytosol to nucleus in rat liver, kidney, brain, and heart.

We have investigated the transport of L- and D-triiodothyronine (T3) from plasma to cellular cytoplasm and from cytoplasm to nucleus by estimating the concentration of free hormone in these compartments in rat liver, kidney, brain, and heart. We assessed the distribution of T3 in various tissues and its metabolism by standard isotopic techniques and measured plasma and cytosolic tissue T3 by radioimmunoassay. In addition, we determined the fraction of radiosensitive T3 associated with the cytosol in individual tissues and estimated the cytosolic volume per gram of tissue. Equilibrium dialysis allowed us to determine the binding power of cytosols and plasma, and in vitro saturation techniques provided values for the affinity (ka) for L- and D-T3 of isolated nuclei in aqueous solution at 37 degrees C. We calculated the free cytosolic hormone from the product of cytosolic T3 and the binding power of cytosol for T3, and the free intranuclear T3 from the ka and previously determined ratio of occupied-to-unoccupied binding sites under steady state conditions in euthyroid animals. Our results showed that the free cytosolic/free plasma concentrations for L-T3 and D-T3, respectively, were: liver 2.8, 21.6; kidney 1.17, 63.3; heart 1.31, 1.58; brain 0.86, 0.24. The free nuclear/free cytosolic ratios for L-T3 and D-T3, respectively, were: liver 58.2, 3.70; kidney 55.9, 1.54; heart 80.6, 24.9; and brain 251, 108.6. Our findings suggest that stereospecific transport occurs both from plasma to cytosol and from cytosol to nucleus. The high gradients from cytosol to nucleus imply that there is an energy-dependent process and appear to account for the differences in the nuclear association constant determined in vivo and in vitro.

Animals↗
PubMed1990

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.

Alkalies↗
PubMed1990

Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors.

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.

Cell-Free System↗
PubMed1986

Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses.

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea-pig hearts. Whole-tissue adenylate data from freeze-clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non-aqueous media. Adenosine and inosine in venous cardiac effluents were measured by high-performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non-aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near-quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were predominantly located in the cytosol of the well-oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP [( CrP]/[Cr] X [Pi]) and hence that of ATP [( ATP]/[ADP] X [Pi]) assuming the creatine kinase at near-equilibrium at a near-constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearly up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass-action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher-power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearly correlated (r = 0.920) with the calculated free cytosolic AMP concentration. Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1990

Agonist-evoked alkaline shift in the cytosolic pH set point for activation of Na+/H+ antiport in human platelets. The role of cytosolic Ca2+ and protein kinase C.

This work examines the roles of elevated cytosolic Ca2+ and stimulation of protein kinase C in agonist and non-agonist-mediated alkaline shift of the cytosolic pH set point for activation of Na+/H+ antiport in human platelets. Ca2(+)-depleted and control platelets were subjected to phorbol 12-myristate 13-acetate, thrombin, vasopressin, and ionomycin in 1 mM or Ca2+ free, nominally bicarbonate free media. To measure the cytosolic pH set point for Na+/H+ antiport activation, cells were acidified to different levels using the sodium propionate method. In some experiments protein kinase C was inhibited by staurosporine. Both protein kinase C stimulation and elevation of cytosolic Ca2+ can produce an alkaline shift in the pH set point for activation of Na+/H+ antiport in human platelets. However, the effect of Ca2+i on the pH set point predominates that of protein kinase C stimulation. Cytosolic Ca2+ is a prerequisite for agonist-evoked alkaline shift in the cytosolic pH set point for activation of Na+/H+ antiport. The cytosolic Ca2+ level is also essential for maintaining the basal cytosolic pH. These findings underscore the central role of cytosolic Ca2+ under basal and stimulated states in regulating cytosolic pH of human platelets.

Alkaloids↗
PubMed2000

The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species.

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.

Adenosine Triphosphatases↗
PubMed1998

Folding in vivo of a newly translated yeast cytosolic enzyme is mediated by the SSA class of cytosolic yeast Hsp70 proteins.

The nature of chaperone action in the eukaryotic cytosol that assists newly translated cytosolic proteins to reach the native state has remained poorly defined. Actin, tubulin, and Galpha transducin are assisted by the cytosolic chaperonin, CCT, but many other proteins, for example, ornithine transcarbamoylase (OTC), a cytosolic homotrimeric enzyme of yeast, do not require CCT action. Here, we observe that yeast cytosolic OTC is assisted to its native state by the SSA class of yeast cytosolic Hsp70 proteins. In vitro, refolding of OTC diluted from denaturant was assisted by crude yeast cytosol and ATP and found to be directed by SSA1/2. In vivo, when OTC was induced in a temperature-sensitive SSA-deficient strain, it exhibited reduced specific activity, and nonnative subunits were detected in the soluble fraction. These findings indicate that, in vivo, the Hsp70 system assists in folding at least some newly translated cytosolic enzymes, most likely functioning in a posttranslational manner.

Adenosine Triphosphate↗
PubMed2004

Calpain and other cytosolic proteases can contribute to the degradation of retro-translocated prion protein in the cytosol.

PrP, a cell surface-localized N-linked glycoprotein, is required for the pathogenesis of prion diseases. Recent studies have revealed that prion protein (PrP) becomes neurotoxic and prone to aggregation when it is in the cytosol, suggesting that cytosolic PrP may play a role in the pathogenesis of prion disease. Retro-translocation of PrP from the endoplasmic reticulum to the cytosol for proteasome degradation offers a natural route for PrP to enter the cytosol, but whether PrP is subject to retrotranslocation is controversial. In this study, we investigated the metabolism of endogenous wild-type PrP in several cell lines and in primary mouse cortical neurons. Our results suggest that a portion of the endogenous wild-type PrP is retro-translocated to the cytosol and degraded by the proteasome. Moreover, we also found that calpain and other cytosolic proteases could degrade PrP in the cytosol when the proteasome activity is compromised. These results provide the foundation for the hypothesis that cytosolic PrP may be involved in the pathogenesis of prion disease.

Animals↗
PubMed1991

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats.

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.

Animals↗
PubMed2005

Inositol 1,4,5-triphosphate-mediated shuttling between intracellular stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils.

The current study was designed to probe Ca2+ shuttling between intracellular stores and the cytosol as a potential mechanism contributing to the prolongation of elevated Ca2+ transients in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation were measured using spectrofluorimetric and radiometric procedures, respectively, while inositol 1,4,5-triphosphate (IP3) was measured using a radioreceptor assay. The Ca2+-chelating agent, ethylene glycol-bis (beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA; 10mM), was used to exclude store-operated influx of Ca2+ into neutrophils, while the IP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 microM), added to the cells 10s after FMLP (0.01 and 1 microM), at which time the increases in IP3 and cytosolic Ca2+ were maximal, was used to eliminate both sustained release from stores and influx of Ca2+. Addition of FMLP at 0.01 or 1 microM resulted in equivalent peak increases in cytosolic Ca2+, while the increase in IP3 was greater and the rate of clearance of Ca2+ from the cytosol slower, in cells activated with 1 microM FMLP. Treatment of the cells with either EGTA or 2-APB following addition of 1 microM FMLP, completely (EGTA) or almost completely (2-APB) abolished the influx of Ca2+ and accelerated the rate of clearance of the cation from the cytosol. Post-peak cytosolic Ca2+ concentrations were lower, and the Ca2+ content of the stores higher, in cells treated with 2-APB. The involvement of IP3 was confirmed by similar findings in cells treated with U-73122 (1 microM), a selective inhibitor of phospholipase C. Taken together, these observations are compatible with IP3-mediated Ca2+ shuttling in neutrophils activated with FMLP.

Boron Compounds↗
PubMed1981

Metabolism in the cytosol of intact isolated cattle rod outer segments as indicator for cytosolic calcium and magnesium ions.

The metabolism of the chromophore of rhodopsin in the cytosol compartment of isolated intact cattle rod outer segments was used as an indicator for changes of the cytosolic Mg2+ and Ca2+ concentration upon changes of the external Mg2+ and Ca2+ concentration. The reduction of retinal to retinol upon photolysis of rhodopsin in situ in intact rod outer segments was critically dependent on the availability of cytosolic Mg2+. The latter is necessary as chelator of endogenous adenosine 5'-triphosphate (ATP). Lowering the cytosolic Ca2+ concentration beneath 10(-7) M resulted in an inhibition of the rate of retinol formation. This is presumably due to a light-activated process, which competes with retinol formation for the supply of high-energy phosphate from a common pool. These results led to the following conclusions. Changes of the external Mg2+ concentration are only followed by substantial changes of the cytosolic Mg2+ concentration when the ionophore A23187 is present. Changes of the external Ca2+ concentration are followed by parallel changes of the cytosolic Ca2+ concentration either when external Na+ is present or in the presence of A23187. Li+ and K+ could not substitute for Na+ in the former case, but K+ diminished the effectivity of Na+ at low Na+ concentrations and enhanced it at high Na+ concentrations. It is concluded that the control of cytosolic Ca2+ concentration in isolated intact rod outer segments is predominantly provided for by Na-Ca exchange, i.e., by coupled fluxes.

Adenosine Triphosphate↗
PubMed1998

Practical assay method of cytosolic acetoacetyl-CoA thiolase by rapid release of cytosolic enzymes from cultured lymphocytes using digitonin.

We designed a simple approach to determine cytosolic acetoacetyl-CoA thiolase (CT) activity for differential diagnosis of ketone body catabolic defects, using rapid cell-subfractionation of cultured lymphocytes with digitonin. Efficiency of cell subfractionation was determined by measurement of lactate dehydrogenase and citrate synthetase as marker enzymes for cytosol and organelle fractions, respectively, and confirmed by immunotitration and immunoblotting using antibodies against cytosolic and mitochondrial thiolases, respectively. In the condition of best separation taken in the presence of 1 mg/ml digitonin, acetoacetyl-CoA thiolase activities in the presence of K+ ion in the cytosol and organelle fractions were 138.3+/-39.2 and 84.0+/-16.2 nmol/min/ml, respectively. The thiolase activity in the organelle fraction was doubled by the presence of K+ ion, whereas that in the cytosol fraction was not affected. The thiolase activity in the organelle fraction was reduced by the treatment of anti-mitochondrial acetoacetyl-CoA thiolase (T2) antibody but not by anti-CT antibody. On the other hand, that in the cytosol fraction was significantly decreased by anti-CT antibody but not by anti-T2 antibody. These data suggested that T2 was collected in the organelle fraction, and that CT activity could be assessed by measurement of the thiolase activity in the cytosolic fraction. Succinyl-CoA: 3-ketoacid CoA transferase (SCOT), whose defect is the third inherited disorder of ketone body catabolism, was collected in the organelle fraction. Hence, this method will prove to be useful for accurate assessment of defects of CT as well as T2 or SCOT, all involved in ketone body catabolism.

Acetyl-CoA C-Acetyltransferase↗
PubMed2001

Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.

Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.

Adenosine Diphosphate↗
PubMed1997

Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase.

Cytosolic and lysosomal acid phosphatases have the ability to hydrolyze orthophosphoric monoesters below pH 5-6. However, it is thought they may have different intracellular roles. To clarify their properties, substrate specificity, inhibitor sensitivity and the modulation of enzyme by redox conditions were determined using bovine liver enzymes. DEAE-cellulose chromatography following (NH4)2SO4 fractionation revealed three forms of cytosolic acid phosphatases as in the KCl gradient (0-500 mM). After Sephadex G-75 gel filtration, the enzymes appeared as single bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their activities for D-erythrose 4-phosphate co-purified with p-nitrophenylphosphatase activities in all steps. In contrast the lysosomal enzyme was purified by Octyl-Sepharose column chromatography after n-butanol treatment, (NH4)2SO4 fractionation, Bio gel P-200 gel filtration and DE-52 chromatography. The relative molecular masses (M(r)) determined by SDS-PAGE indicated that M(r) of the cytosolic enzymes (16,000) was less that of lysosomal enzyme (160,000). The cytosolic enzymes were active against sugar phosphates and were inhibited by 1 mM Cu2+. In addition, the cytosolic enzymes were inactivated by 5 mM oxidized glutathione and protected by 10 mM reduced glutathione (in the presence or absence of thioltransferase), suggesting that sensitive cysteinyl residue(s) existed. The lysosomal enzyme was active against various substrates and was strongly inhibited by 1 mM Cu2+ and 2 mM fluoride. The results presented here suggest that cytosolic enzymes have different properties from those of lysosomal enzyme with respect to substrates, inhibitors and regulation of activity.

Acid Phosphatase↗
PubMed2004

Dual sensitivity of sarcoplasmic/endoplasmic Ca2+-ATPase to cytosolic and endoplasmic reticulum Ca2+ as a mechanism of modulating cytosolic Ca2+ oscillations.

The effects of ER (endoplasmic reticulum) Ca2+ on cytosolic Ca2+ oscillations in pancreatic acinar cells were investigated using mathematical models of the Ca2+ oscillations. We first examined the mathematical model of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) to reproduce the highly co-operative inhibitory effect of Ca2+ in the ER lumen on ER Ca2+ uptake in the acinar cells. The model predicts that luminal Ca2+ would most probably inhibit the conversion of the conformation state with luminal Ca2+-binding sites (E2) into the conformation state with cytoplasmic Ca2+-binding sites (E1). The SERCA model derived from this prediction showed dose-response relationships to cytosolic and luminal Ca2+ concentrations that were consistent with the experimental data from the acinar cells. According to a mathematical model of cytosolic Ca2+ oscillations based on the modified SERCA model, a small decrease in the concentration of endoplasmic reticulum Ca2+ (approx. 20% of the total) was sufficient to abolish the oscillations. When a single type of IP3R (IP3 receptor) was included in the model, store depletion decreased the spike frequency. However, the frequency became less sensitive to store depletion when we added another type of IP3R with higher sensitivity to the concentration of free Ca2+ in the cytosol. Bifurcation analysis of the mathematical model showed that the loss of Ca2+ from the ER lumen decreased the sensitivity of cytosolic Ca2+ oscillations to IP3 [Ins(1,4,5)P3]. The addition of a high-affinity IP3R did not alter this property, but significantly decreased the sensitivity of the spike frequency to IP3. Our mathematical model demonstrates how luminal Ca2+, through its effect on Ca2+ uptake, can control cytosolic Ca2+ oscillations.

Binding Sites↗