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PubMed1989

The diacylglycerol analogue, 1,2-sn-dioctanoylglycerol, induces an increase in cytosolic free Ca2+ and cytosolic acidification of T lymphocytes through a protein kinase C-independent process.

In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.

Animals↗
PubMed1999

The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase.

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.

Amino Acid Sequence↗
PubMed2003

A low-starch barley mutant, risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit.

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.

Adenosine Diphosphate Glucose↗
PubMed1987

Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism.

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.

Alkaloids↗
PubMed1998

Activators of epithelial Na+ channels inhibit cytosolic feedback control. Evidence for the existence of a G protein-coupled receptor for cytosolic Na+.

We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl-, acting, respectively, via Go and Gi proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+]o), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na+]o. Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl- on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl- unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated alpha-subunit of Go in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.

4-Chloromercuribenzenesulfonate↗
PubMed2003

Sodium chloride reduces growth and cytosolic calcium, but does not affect cytosolic pH, in root hairs of Arabidopsis thaliana L.

The effects of salinity (NaCl) stress on growth, cytosolic Ca(2+) gradients and cytosolic pH homeostasis of root hairs of Arabidopsis thaliana are assessed here. Neither cytosolic Ca(2+) nor pH at the hair apex were significantly affected by 20 min exposure of up to 90 mM NaCl or of up to 5 mM extracellular Ca(2+). Exposure to increasing NaCl concentrations, up to 90 mM, for 2 d or 6 d reduced hair extension, and this inhibition was relieved by supplemental extracellular Ca(2+). Such extended salinity stress reduced the magnitude of the Ca(2+) gradient in the apical 12 microm of hairs at all NaCl concentrations tested (up to 90 mM), including NaCl concentrations that did not reduce hair extension. The magnitude of the tip-focused gradient was also reduced in root hairs of plants grown with low (0.5 mM) extracellular Ca(2+) when compared to those in 5 mM extracellular Ca(2+), regardless of the presence of NaCl. Up to 90 mM NaCl did not affect cytosolic pH of root hairs in any of the treatments. It is concluded that NaCl inhibition of root hair extension in the long term may operate via alterations in the tip-focused Ca(2+) gradient that regulates root hair growth. However, NaCl-induced alterations in this gradient do not always lead to detectably altered growth kinetics. Short-term signalling events in response to NaCl may operate by a means other than altering Ca(2+) at the root hair apex. Salinity stress in root hairs does not appear to be mediated by effects on cytosolic pH.

Adaptation, Physiological↗
PubMed1999

[Cooperation of membrane proteins and cytosolic proteins in metabolic regulation--involvement of binding of hexokinase to mitochondria in regulation of glucose metabolism and association and complex formation between membrane proteins and cytosolic proteins in regulation of active oxygen production].

Changes in amount and activity of enzyme protein are critical factors in regulating intracellular metabolisms. However, since the metabolisms are proceeding in environment with complex architecture consisted of various membranes, spatial factors should be taken into consideration for the regulation. In this review, involvement of interaction between cytosolic and membrane proteins in metabolic regulation are discussed. It had been reported that hexokinase activity was found in mitochondrial fraction in spite of almost exclusive distribution of other glycolytic enzymes to soluble fraction, the tendency being marked in the brain and many types of tumor cells whereas mitochondrial hexokinase activity was quite low in the liver. Interested in such enzyme and tissue specificities, we investigated the significance and mechanism of the unique intracellular distribution of hexokinase. We found that mitochondria-bound hexokinase was more active than the cytosolic type in producing glucose 6-phosphate (G6P), probably due to the advantage in utilizing ATP produced in mitochondria. In addition, we also found that the binding stabilized hexokinase against G6P inhibition. As to the binding, it was reported that G6P released hexokinase from mitochondria while Mg2+ promoted the binding. In this respect, we found that polyamines promoted the binding at much lower concentration than that of Mg2+, and mitochondria-bound form had small hydrophobic domain at terminal region for the binding to porin on the outer membrane. Then, we found a protease which specifically cleaved the domain with little effect on catalytic activity and molecular size of the bindable form. Such a modifying protease was purified and identified as lysosomal cathepsin L. The protease activity was high in the liver and low in the brain, suggesting that the difference in the activity was responsible for the afore-mentioned tissue specificity. On the other hand, we examined regulatory mechanism for active oxygen production in neutrophils, since the production of superoxide anion (O2-) by NADPH oxidase was very low at the resting state while markedly increased on phagocytosis and chemical stimulation. Since the stimulants for the activation were so various in chemical nature, we postulated mechanism to converge the stimulation to the activation. Incidentally, we found increase in phosphorylation of 46-47 K protein, irrespective of the type of stimulation. Use of inhibitors and examination on the phosphorylation condition indicated protein kinase C (PKC) as the phosphorylating enzyme. In addition, we observed the 46-47 K protein existed in cytosol at resting state, while it was translocated to cell membranes in concurrence with the phosphorylation. Similar findings were obtained in many laboratories and those proteins were named cytosolic activating factors (and then p47-phox, etc.). These proteins associate with membrane proteins to constitutes the active from of NADPH oxidase. Next, we examined mechanism to shut off the O2- production, and found that the inactivation through disassembly of the constituents was attained by dephosphorylation of phosphorylated p47-phox by cytosolic protein phosphatase. Then we have also found that protein kinases other than PKC were involved in regulation of NADPH oxidase activity. Though phosphorylation of p47-phox etc. is deeply involved in the activation of NADPH oxidase, membrane perturbation, so-called priming, is required for the activation. We also reported some possible indications for the priming, and possible involvement of cytoskeletons in O2- production. Apart from protein phosphorylation, it has been reported that amphiphilic acidic compounds are potent activator for NADPH oxidase. We also have examined their effects to find that these compounds also caused the assembly of the NADPH oxidase constituents. Reversely, amphiphilic basic compounds suppressed suggesting significance of introduction of negative charge in NADPH oxidase activat

Animals↗
PubMed1999

Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2.

Proapoptotic members of the Bcl-2 family, including Bax, Bak, and Bid, directly trigger the mitochondrial release of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. One of the crucial steps before Bax can exert its proapoptotic activity is translocation from the cytoplasm to the mitochondria, but the molecular mechanism of this translocation is not understood. To investigate the mechanism of apoptosis-associated Bax translocation, we used an in vitro system comprising isolated mitochondria and cytosol. We found that both endogenous and exogenous added recombinant Bax translocated to the mitochondria more efficiently in the presence of cytosol from cells with VP16-induced apoptosis than with cytoplasm from normal cells. This apoptosis-dependent promotion of Bax translocation was not seen with cytosol that was prepared from VP16-treated cells expressing Bcl-2. Cytosol from cells with VP16-induced apoptosis, but not that from normal cells or Bcl-2-expressing cells, induced cytochrome c release from isolated mitochondria, which, as assessed by immunodepletion experiments, was mainly mediated by Bax. These results suggest that Bcl-2 exerts its antiapoptotic activity partly by inhibiting the translocation of Bax through the modification of cytosolic factors that are involved in such translocation during apoptosis.

Apoptosis↗
PubMed1980

Calcium uptake and release by rat liver mitochondria in the presence of rat liver cytosol or the components of cytosol.

A study has been made of factors present in rat liver cytosol that might regulate the calcium content of mitochondria. A cytosol preparation containing all the components of molecular weight greater than 10,000 prevented uptake and caused early release of accumulated calcium. These effects were due to free long-chain fatty acids and their coenzyme A derivatives present in the cytosol, and these inhibitory effects were controlled by inclusion of Mg2+, carnitine, and adenosine triphosphate at physiological levels in the incubation medium. Palmitoyl carnitine was a good substrate for calcium uptake and did not cause release of calcium from mitochondria. A specific fatty acid-binding protein was found in cytosol which may be the intracellular transport protein for fatty acids.

Adenosine Triphosphate↗
PubMed1996

Cisplatin-induced nephrotoxicity in vitro: increases in cytosolic calcium concentration and the inhibition of cytosolic and mitochondrial protein kinase C.

Exposure of rat kidney cortical slices to cisplatin (2 mM) significantly increased the activity of cytosolic phosphorylase a, indicating that the concentration of cytosolic Ca2+ was increased. Dithiothreitol (DTT), a sulphydryl reducing agent, markedly reversed this effect but N,N'-diphenylphenylenediamine (DPPD), an antioxidant, did not. Cisplatin inhibited protein kinase C (PKC) activity in both mitochondrial and cytosolic fractions after the slices were exposed to cisplatin. Both DTT and DPPD prevented these inhibitory effects of cisplatin on PKC but diethylmaleate, a glutathione depletor, potentiated this inhibitory effect. These results suggest that the increase in cytosolic Ca2+ is related to depletion of SH-groups, but independent of lipid peroxidation, whereas inhibition of PKC may be associated with cisplatin-induced depletion of thiols and with lipid peroxidation.

Animals↗
PubMed1996

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium.

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.

Amino Acid Sequence↗
PubMed2006

FGF-1 and FGF-2 require the cytosolic chaperone Hsp90 for translocation into the cytosol and the cell nucleus.

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane.

Animals↗
PubMed1998

Cytosolic beta-cyanoalanine synthase activity attributed to cysteine synthases in cocklebur seeds. Purification and characterization of cytosolic cysteine synthases.

The activity of beta-cyanoalanine synthase (CAS, EC 4.4.1.9) in cotyledons of cocklebur seeds (Xanthium pennsylvanicum Wallr.) was detected both in the soluble and particulate fractions. The CAS activity of the soluble fraction (cytosolic CAS activity) was 10 times higher than that of the particulate fraction. The CAS activity of the particulate fraction was confirmed to be localized in the mitochondria. Both enzymatic activities were clearly separated by non-denaturing PAGE. The enzyme with cytosolic CAS activity has been extensively purified and separated into three different forms designated as cyt-1, cyt-2, and cyt-3. According to the SDS-PAGE analysis, the three enzymes are estimated to be a homodimer composed of 35-kDa subunits. The purified enzymes showed CS activity. Partial amino acid sequences of cyt-1 were determined and had a high homology with cysteine synthases (CS, EC 4.2.99.8) from other plant sources. The catalytic action of the purified CSs in converting cyanide and cysteine into H2S and beta-cyanoalanine was confirmed by the detection of significant 14CN incorporation into beta-cyanoalanine. These results indicated that cytosolic CAS activity is due to cytosolic CS and suggested that the CAS activity of CS is likely to be involved in cyanide metabolism in plant tissues.

Alanine↗
PubMed1994

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets.

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

Blood Platelets↗
PubMed1987

[Studies on cytosol thyroid hormone binding proteins in the rat liver: Part III. Partial purification and binding characteristics of thyroxine-binding protein in hypothyroid rat liver cytosol and serum].

Cytosolic thyroxine-binding protein (CT4BP) was partially purified from rat liver cytosol obtained 10 days after thyroidectomy using Sephadex G-200 gel filtration, and its binding characteristics were analyzed in displacement experiments using a charcoal binding method to separate bound and free hormones. Serum T4-binding proteins were also partially purified, and their binding characteristics were similarly determined. Sephadex G-200 gel filtration of liver cytosol from thyroidectomized rats revealed that CT4BP had an apparent molecular weight of 100 X 10(3) daltons. CT4BP had a very high affinity constant (Ka) of 2.2 +/- 10(10) M-1 and a small maximum binding capacity (MBC) of 5.1 X 10(-9) g/mg. protein for T4. Relative affinities of T4 analogues for CT4BP (if the affinity of L-T4 was assigned a value of 100, then D-T4 would have a value of 25.3; L-T3, 16.6; D-T3, 2.3; reverse T3, 1.4 and both Tetrac and Triac less than 1) showed that CT4BP had a rigid specificity for alanine-side chain of T4-molecule. This CT4BP was not demonstrated when cytosol from normal rat liver was used. Sephadex G-200 gel filtration of rat serum obtained 10 days after thyroidectomy revealed two T4-binding proteins. The faster peak (Peak I; MW about 100 X 10(3) daltons) was eluted before the albumin peak, and the slower peak (Peak II; MW about 56 X 10(3) daltons) appeared after the albumin peak. Peak I was barely detectable when normal rat serum was used. Peak I had a higher Ka of 2.0 X 10(10) M-1 and a smaller MBC of 3.9 X 10(-9) g/mg. protein than Peak II (Ka; 8.9 X 10(8) M-1, MBC; 3.7 +/- 10(-7) g/mg. protein). Relative affinities of T4 analogues for Peak I (L-T4 100, D-T4, 34.9, L-T3 11.1, D-T3 1.8, reverse T3 6.8, Tetrac 0.25 and Triac 0.1) showed that Peak I had a rigid specificity to alanine-side chain of T4 molecule, but Peak II had little specificity to this side chain (L-T4 100, D-T4 9.2, L-T3 2.1, D-T3 1.0, reverse T3 14.3, Tetrac 69 and Triac 26.3). Thus, Peak I had a similar binding characteristics to those of human thyroxine-binding globulin (TBG), and Peak II was comparable to human thyroxine-binding prealbumin (TBPA). The results that both molecular weight and binding characteristics were similar between CT4BP and Peak I suggest that both proteins are identical, being comparable to human TBG. This must be clarified in future.

Animals↗
PubMed1986

Binding of cytosol 3H-17, beta-estradiol to cytosol receptors in the liver of female rats.

The article is a study of the specific binding of 3H-17, beta-estradiol to cytosol receptors in the liver of female, sexually mature albino rats: sham-operated, ovariectomized and treated after the ovariectomy with 17-beta-estradiol (in doses of 20 micrograms/100 g body weight for 11 days). The binding of the labelled hormone in the liver cytosol isolated from ovariectomized rats is considerably reduced compared with the sham-operated animals. In the cytosol from the liver of the estradiol-treated rats the formation of a hormone-receptor complex is substantially increased compared with the ovariectomized animals. It is assumed that the formation of estradiol-receptor complexes in the liver depends on the concentration of the cytosol receptors, which is determined by the concentration of oestrogens in the blood.

Animals↗
PubMed1980

Cytosolic modulators of activities of microsomal enzyme of cholesterol biosynthesis. Role of a cytosolic protein with properties similar to Z-protein (fatty acid-binding protein).

Rat liver cytosol contains proteins and smaller molecules that affect activities of the microsomal enzymes of cholesterol biosynthesis. One protein (mr = 12,000) has been purified by chromatography, gel filtration, and preparative isoelectric focusing. Properties of the protein on sodium dodecyl sulfate-, and anodic-, and cathodic-disc gel electrophoresis are reported in addition to gel filtration and electrofocusing that were used for purification. By comparison of these properties, amino acid compositions, ligand binding, and the abundance in various tissues the protein appears to be very similar to Z-protein that has been shown by others to be identical with hepatic fatty-acid binding protein. The same properties also appear to be very similar to sterol carrier protein. A cytosolic metabolite, heme, which stimulates 4-methyl sterol oxidase, is bound to the protein during purification. When endogeneous heme is removed, both protein and heme are required for maximal stimulation of microsomal 4-methyl sterol oxidase activity. Hemoglobin produces an equal extent of stimulation presumably by heme group exchange, but the nonexchangeable heme group of cytochrome c is ineffective. Thus, the cytosolic protein may promote uptake and retention of relatively more water-soluble substances into the hydrophobic environment of the membrane-bound enzymes of cholesterol biosynthesis. Z-protein exhibits affinity for an exceptionally wide variety of ligands (e.g. fatty acids, azodyes, organic anions, bile pigments, etc) that may either stimulate or inhibit the microsomal enzymes. Accordingly, with the suggestion that the cytosolic protein is Z-protein, the wide variety of potential interactions of both endogenous metabolites and exogenous xenobiotics may account, in part, for facile modulations of activities of the rate-limiting microsomal synthetic enzymes in various physiological and nutritional states.

Amino Acids↗
PubMed1998

Control of Cl- efflux in chara corallina by cytosolic pH, free ca2+, and phosphorylation indicates a role of plasma membrane anion channels in cytosolic pH regulation

Enhanced Cl- efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl- efflux via two mechanisms. The first is a direct effect of pHc on Cl- efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl- efflux. Cl- efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl- efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl- efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.

Journal Article↗