Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver

1987 ◽  
Vol 65 (10) ◽  
pp. 890-898 ◽  
Author(s):  
Frederick B. St. C. Palmer
Keyword(s):  
Rat Brain ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Liver Microsomes ◽  
Maximum Activity ◽  
Molar Ratio ◽  
Phosphatase Activities ◽  
Rat Brain And Liver ◽  
Ph Range

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg∙mL−1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0–8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl. Lack of inhibition by 2,3-bisphosphoglycerate and absence of stimulation by cysteine or dithioerythritol, as well as a different subcellular distribution in liver, excluded inositol-1,4,5-trisphosphate and inositol-1,4-bisphosphate phosphatases as sources of the lysoPtdIns(4,5)P2 and lysoPtdIns4P phosphatase activities.

scholarly journals Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes

1995 ◽  
Vol 310 (1) ◽  
pp. 221-224 ◽  
Author(s):  
J F St-Denis ◽  
B Annabi ◽  
H Khoury ◽  
G van de Werve
Keyword(s):  
Substrate Specificity ◽  
Membrane Permeability ◽  
Phosphatase Activity ◽  
Liver Microsomes ◽  
Microsomal Membrane ◽  
Phosphatase Activities ◽  
Phosphate Hydrolysis ◽  
Mannose 6 Phosphate ◽  
Stimulation Of

The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate.

scholarly journals Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C).

1988 ◽  
Vol 107 (3) ◽  
pp. 1001-1009 ◽  
Author(s):  
H S Shpetner ◽  
B M Paschal ◽  
R B Vallee
Keyword(s):  
Ionic Strength ◽  
Divalent Cations ◽  
High Sensitivity ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Maximum Activity ◽  
Scanning Transmission ◽  
Sulfhydryl Oxidation ◽  
The Brain

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.

scholarly journals Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

1989 ◽  
Vol 9 (10) ◽  
pp. 4187-4195 ◽  
Author(s):  
J C Vera ◽  
O M Rosen
Keyword(s):  
Glucose Uptake ◽  
Rat Brain ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Rat Brain And Liver ◽  
Insulin Responsiveness

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.

scholarly journals DNA interference by a mesophilic Argonaute protein, CbcAgo

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 321
Author(s):  
Nieves García-Quintans ◽  
Laurie Bowden ◽  
José Berenguer ◽  
Mario Mencía
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Pyrococcus Furiosus ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximum Activity ◽  
Argonaute Protein ◽  
Starting Point ◽  
Mesophilic Bacterium

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools  for mammalian cells.

1,4 Butanediol and ethanol compete for degradation in rat brain and liver in vitro

Alcohol ◽  
1986 ◽  
Vol 3 (6) ◽  
pp. 367-370 ◽  
Author(s):  
Flavio Poldrugo ◽  
O.Carter Snead
Keyword(s):  
Rat Brain ◽  
Rat Brain And Liver

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

1987 ◽  
Vol 65 (11) ◽  
pp. 2338-2345 ◽  
Author(s):  
Gordon T. Bolger ◽  
Kendall A. Marcus ◽  
Ronald Thibou ◽  
Phil Skolnick ◽  
Ben Avi Weissman
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Divalent Cations ◽  
Opiate Receptors ◽  
Opiate Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Regulatory Effects ◽  
In Vitro Effects

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5′-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity μ-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris–HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+,2.9 mM and Li, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the μ-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris–HCl. In 50 mM Tris–HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris–HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited, [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris–HCl, respectively. These findings indicate that the in vitro effects of Na+ at μ- and possibly other opiate receptors in rat brain are dependent on the concentration of Tris–HCl used in the assay buffer, lower concentrations of Tris-HCl revealing novel regulatory effects for Na+ at μ-opiate receptors.

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity

1989 ◽  
Vol 9 (10) ◽  
pp. 4187-4195
Author(s):  
J C Vera ◽  
O M Rosen
Keyword(s):  
Glucose Uptake ◽  
Rat Brain ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Rat Brain And Liver ◽  
Insulin Responsiveness

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.

scholarly journals rnrGene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

2011 ◽  
Vol 77 (22) ◽  
pp. 7896-7904 ◽  
Author(s):  
Shaheen Sulthana ◽  
Purusharth I. Rajyaguru ◽  
Pragya Mittal ◽  
Malay K. Ray
Keyword(s):  
Pseudomonas Syringae ◽  
Divalent Cations ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Rnase R ◽  
Content Type ◽  
Antarctic Bacterium ◽  
The Antarctic

ABSTRACTRNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacteriumPseudomonas syringaeLz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His6-tagged form of theP. syringaeRNase R (RNase RPs), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg2+and Mn2+) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase RPsexhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structuredmalE-malFRNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5′-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain byEscherichia coliRNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase RPsin vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase RPsactivity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.

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