scholarly journals Biochemical and cytochemical studies on enzymes that dephosphorylate inositol (1,4,5)-trisphosphate in neutrophils.

1991 ◽  
Vol 39 (3) ◽  
pp. 321-329 ◽  
Author(s):  
J A Badwey ◽  
J M Robinson
Keyword(s):  
Soluble Enzyme ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Cell Stimulation ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Biochemical Studies ◽  
Cytochemical Reaction ◽  
Membrane Bound ◽  
C 50

Guinea pig neutrophils contain membrane-bound and soluble phosphatases that catalyze the dephosphorylation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. The activities were 5.1 +/- 0.2 and 1.3 +/- 0.2 (SD; n = 5) nmoles phosphate (Pi) released/min/10(7) cell equivalents, respectively. The membrane-bound enzyme dephosphorylated many substrates (e.g., beta-glycerophosphate), exhibited alkaline pH optima, and was inhibited by levamisole. In contrast, the soluble phosphatase was specific for Ins(1,4,5)P3, exhibited a neutral pH optimum, and was insensitive to levamisole. A cerium-based ultrastructural cytochemical procedure was employed to identify the subcellular sites of the membrane-bound activity. Staining was observed on the exterior of the plasmalemma and in a population of granules. Staining in the granules was observed only in permeabilized cells. Treatment of neutrophils with p-diazobenzenesulfonate (DBSA) (4.0 mM) for 20 min at 37 degrees C blocked the cytochemical reaction on the cell surface using beta-glycerophosphate as the substrate, but did not affect the staining of the granules on subsequent permeabilization. In biochemical studies, this treatment with DBSA inhibited the membrane-bound activity by c. 50% but did not affect the soluble phosphatase. Therefore, the membrane-bound phosphatase is, in fact, an alkaline phosphatase that resides in locales not accessible to Ins(1,4,5)P3 generated during cell stimulation. Breakdown of Ins(1,4,5)P3 generated during cell stimulation, therefore, would be catalyzed by the soluble enzyme.

Genetic and Biochemical Studies on the Conversion of Dihydroflavonols to Flavonols in Flowers of Petunia hybrida

1986 ◽  
Vol 41 (1-2) ◽  
pp. 179-186 ◽  
Author(s):  
G. Forkmann ◽  
P. de Vlaming ◽  
R. Spribille ◽  
H. Wiering ◽  
A. W. Schram
Keyword(s):  
Enzyme Activity ◽  
Petunia Hybrida ◽  
Soluble Enzyme ◽  
Flower Buds ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Biochemical Studies ◽  
High Enzyme ◽  
Substantial Correlation ◽  
High Enzyme Activity

Abstract Soluble enzyme preparations from flower buds of Petunia hybrida catalyzed the conversion of dihydroflavonols to flavonols. Dihydrokaempferol and dihydroquercetin were readily converted to the respective flavonols, whereas dihydromyricetin was a poor substrate. The reaction required 2-oxoglutarate, ascorbate and Fe2+ as cofactors and had a pH optimum at about 6.5. In the presence of the dominant allele Fl, high enzyme activity for flavonol formation was found, whereas in enzyme preparations from flower buds of recessive genotypes (fl/fl) only low enzyme activity could be observed. A substantial correlation was found between enzyme activity for flavonol formation and the flavonol content of buds and flowers during development.

scholarly journals sn-Glycero(3)phosphoinositol glycerophosphohydrolase. A new phosphodiesterase in rat tissues

1979 ◽  
Vol 182 (1) ◽  
pp. 39-45 ◽  
Author(s):  
R M C Dawson ◽  
N Hemington ◽  
D E Richards ◽  
R F Irvine
Keyword(s):  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Temperature Stability ◽  
Rat Tissues ◽  
Alkaline Ph ◽  
Stability Studies ◽  
Ph Optimum ◽  
Excess Substrate ◽  
Membrane Bound ◽  
Cyclic Phosphate

1. A phosphodiesterase that cleaves glycerophosphoinositol into glycerophosphate and inositol has been detected in rat tissues. 2. The enzyme requires Mg2+ (Mn2+) and has a pH optimum of 7.7. 3. The richest sources of the enzyme are kidney and intestinal mucosa. In pancreas subcellular fractions it occurs largely in the microsomal fraction. 4. The enzyme is inhibited by excess substrate and by the reaction product glycerophosphate. 5. Temperature-stability studies and other observations distinguish the enzyme from other membrane-bound phosphodiesterases active at an alkaline pH e.g. glycerophosphoinositol inositophosphohydrolase, glycerophosphocholine diesterase, inositol cyclic phosphate phosphodiesterase and phosphodiesterase I.

PHOSPHODIESTERASE AND ACID PHOSPHATASE ACTIVITIES OF AVIAN BONE MARROW CELLS

1965 ◽  
Vol 43 (8) ◽  
pp. 1319-1328 ◽  
Author(s):  
Gerald Kingsley Bristow ◽  
Esther W. Yamada
Keyword(s):  
Bone Marrow ◽  
Acid Phosphatase ◽  
Bone Marrow Cells ◽  
Intracellular Distribution ◽  
Specific Substrate ◽  
Magnesium Ions ◽  
Differential Centrifugation ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Rna And Dna

Avian bone marrow has been found to contain a phosphodiesterase as well as an acid phosphatase. Some properties of these enzymes have been described. Because the phosphodiesterase of this tissue has an alkaline pH optimum, is activated by magnesium ions, and acts on the specific substrate p-nitrophenyl thymidine 5′-phosphate, it is probably a phosphodiesterase I such as is present in snake venom and other tissues.The intracellular distribution of these two enzymes in normal and regenerating bone marrow was studied. Subcellular fractions were prepared by differential centrifugation or by centrifugation through sucrose density gradients. The RNA and DNA content of each fraction was determined. By the methods used no differences in the properties or intracellular distribution of the two enzymes in normal and regenerating bone marrow were found.

Assessment of transport capacity of plasmalemmal Ca2+ pump in smooth muscle

1988 ◽  
Vol 255 (2) ◽  
pp. C226-C236 ◽  
Author(s):  
P. A. Lucchesi ◽  
R. A. Cooney ◽  
C. Mangsen-Baker ◽  
T. W. Honeyman ◽  
C. R. Scheid
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Reliable Estimate ◽  
Flux Rate ◽  
Transport Capacity ◽  
High Affinity ◽  
Transmembrane Flux ◽  
Inositol 1,4,5 Trisphosphate ◽  
Biochemical Studies

In resting smooth muscle, a variety of Ca2+ extrusion processes offset the inward Ca2+ leak. Biochemical studies suggest that the plasmalemmal Ca2+ pump dominates this process; however, this contention could not be proven without a reliable estimate of the inward Ca2+ leak that must be opposed by active transport. Recent studies using dispersed cells from the toad stomach provided such an estimate; thus we examined the capacity of the plasmalemmal Ca2+ pump in this tissue. Membranes were prepared using nitrogen cavitation, high-salt extraction, and flotation on discontinuous sucrose gradients. These membrane vesicles were enriched 16- to 24-fold for plasma membrane markers and exhibited an ATP-dependent uptake of 45Ca that was insensitive to azide or oxalate but sensitive to orthovanadate inhibition and calmodulin stimulation. 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by caffeine, inositol 1,4,5-trisphosphate, or GTP. Uptake exhibited a high affinity for Ca2+ (Km 0.2 microM) and a high-transport capacity, producing greater than 12,000-fold gradient for Ca2+ and a transmembrane flux rate greater than that observed in resting smooth muscle cells. Thus this enzyme is capable of maintaining steady-state Ca2+ levels in smooth muscle.

Soluble neutral maltase–glucoamylase from the small intestine: separation and characterization of components with differing affinity for concanavalin A

1982 ◽  
Vol 60 (11) ◽  
pp. 1007-1013 ◽  
Author(s):  
G. Forstner ◽  
A. Salvatore ◽  
L. Lee ◽  
J. Forstner
Keyword(s):  
Concanavalin A ◽  
Gradient Elution ◽  
Soluble Form ◽  
Rat Intestine ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Membrane Bound ◽  
Sepharose 4B

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M α-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M α-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M α-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.

scholarly journals Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

1974 ◽  
Vol 144 (2) ◽  
pp. 281-292 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Heat Sensitivity ◽  
Adult Rats ◽  
Ph Optimum ◽  
Maltase Activity ◽  
Acid Maltase ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
The Relationship

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

scholarly journals Effect of sphingosine derivatives on calcium fluxes in thyroid FRTL-5 cells

1994 ◽  
Vol 299 (1) ◽  
pp. 213-218 ◽  
Author(s):  
K Törnquist ◽  
E Ekokoski
Keyword(s):  
Elevated Level ◽  
Channel Blocker ◽  
Thyroid Stimulating Hormone ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Myristate Acetate ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dose Dependent ◽  
In Cells

The effects of sphingosine derivatives on Ca2+ fluxes were investigated in thyroid FRTL-5 cells labelled with Fura 2. Addition of sphingosylphosphocholine (SPC) or sphingosine (SP) increased intracellular free Ca2+ ([Ca2+]i) in a dose-dependent manner. At the highest dose tested (30 microM), the response was biphasic: a rapid transient increase in [Ca2+]i, followed by a new, elevated, level of [Ca2+]i. Both phases of the SPC-evoked increase in [Ca2+]i were dependent on extracellular Ca2+, whereas only the SP-evoked elevated level of [Ca2+]i was dependent on the influx of Ca2+. Both compounds released sequestered Ca2+ from thapsigargin- and inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools. In addition, the increase in [Ca2+]i in response to SPC, but not to SP, was attenuated in cells treated with phorbol myristate acetate or with the putative Ca(2+)-channel blocker SKF 96365, and in cells pretreated with pertussis toxin for 24 h. SPC did not activate the production of IP3. Furthermore, both SPC and SP released sequestered Ca2+ from permeabilized cells. We observed that SPC, but not SP, stimulated release of [3H]arachidonate from cells prelabelled with [3H]arachidonate for 24 h. Both SPC and SP stimulated the incorporation of [3H]thymidine into DNA in cells grown in the absence of thyroid-stimulating hormone (TSH). The results suggest that sphingosine derivatives are putative regulators of Ca2+ fluxes in FRTL-5 cells, and that SP and SPC may act on [Ca2+]i via different mechanisms. Furthermore, both SP and SPC may be of importance in modulating thyroid-cell proliferation.

Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells

1999 ◽  
Vol 276 (1) ◽  
pp. L81-L89 ◽  
Author(s):  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Keith A. Jones ◽  
David O. Warner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptor ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Tracheal Smooth Muscle ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra

1995 ◽  
Vol 269 (3) ◽  
pp. C698-C705 ◽  
Author(s):  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Dissociation Constants ◽  
Time Dependent ◽  
Isosbestic Point ◽  
Half Time ◽  
Permeabilized Cells ◽  
Aortic Smooth Muscle ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.

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