Soluble and membrane-bound polyphosphoinositide phosphohydrolases in mammalian erythrocytes

1988 ◽  
Vol 66 (3) ◽  
pp. 199-207 ◽  
Author(s):  
Suzanne E. Mack ◽  
Frederick B. St. C. Palmer
Keyword(s):  
Phosphatase Activity ◽  
Cetyltrimethylammonium Bromide ◽  
Cytosol Fraction ◽  
Sheep Erythrocytes ◽  
Phosphatidylinositol Bisphosphate ◽  
Membrane Activity ◽  
Phosphatidylinositol Phosphate ◽  
Membrane Bound ◽  
Triton X ◽  
Assay Conditions

Phosphatases and phosphodiesterases that hydrolyse polyphosphoinositides are described in both membrane and cytosol fractions of human, pig, rat, rabbit, and sheep erythrocytes using exogenous substrates. With suitably optimized assay conditions, Ca2+-dependent phosphatidylinositol bisphosphate (PIP2) phosphodiesterase activity was found in the hemoglobin-free cytosol fraction, as well as the membrane. Membrane activity is completely dependent upon Triton X-100 and salt and inhibited by cetyltrimethylammonium bromide (CTAB), while the soluble activity requires CTAB and is inhibited by Triton. A low Ca2+-dependent PIP2 phosphatase activity, not present in other tissues, was also detected. The cation-independent phosphatidylinositol phosphate (PIP) phosphatase is localized in the membrane in most species, while the diesterase and the PIP2 phosphatases (both Mg2+ and Ca2+ dependent) are localized in the cytosol. Rat and rabbit erythrocytes are atypical in having a substantial proportion of their Mg2+ -dependent PIP2 phosphatase activities in the membrane. All activities are lowest in sheep erythrocytes, except the PIP phosphatase, most of which is soluble in this species. Ca2+-dependent PIP2 phosphatase activity is not correlated with the activity or subcellular distribution of any of the other hydrolases and seems to be a separate enzyme. All the phosphoinositide hydrolase activities, particularly the diesterase, are orders of magnitude lower in erythrocytes than in other tissues. Both soluble and membrane diesterase activities are lost as erythrocytes age. Soluble polyphosphoinositide diesterase does not seem to be active with membrane-bound substrate, since pig and sheep erythrocytes that have negligible membrane activity do not respond to Ca2+ loading, yet have substantial diesterase activity in the cytosol. This supports the view that the diesterase is not physiologically functional in normal erythrocytes.

scholarly journals Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma

1976 ◽  
Vol 158 (3) ◽  
pp. 535-541 ◽  
Author(s):  
P J St Louis ◽  
P V Sulakhe
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Maximal Activity ◽  
Cardiac Sarcolemma ◽  
Guinea Pig Heart ◽  
Membrane Bound ◽  
Triton X ◽  
Assay Conditions

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an ‘activated’ form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.

Kinase A does not activate phosphatidylinositol phosphorylation in rat lymphocyte membranes

1986 ◽  
Vol 251 (6) ◽  
pp. C883-C886 ◽  
Author(s):  
A. Coco ◽  
I. G. Macara
Keyword(s):  
Protein Kinase ◽  
Initial Rate ◽  
Dependent Protein Kinase ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphatidylinositol Phosphate ◽  
Dependent Protein ◽  
Hydrolysis Of ◽  
Assay Conditions ◽  
Kinase A ◽  
Protein Kinase Kinase

The interaction of the dissociated catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (kinase A) and phosphatidylinositol metabolism has been studied in rat spleen lymphocyte membranes. As reported previously (Sarkadi et al., FEBS Lett. 152: 195-198, 1983) addition of kinase A increased by about twofold the phosphorylation of phosphatidylinositol in lymphocyte membranes at low ATP concentrations. However, we have found that this increase is an artifact of the assay conditions, and that the increase is a consequence of an inhibition of membrane adenosine triphosphatase activity by the protein kinase A. When lipid phosphorylation was measured under initial rate conditions, at high ATP concentrations, the increase was abolished. No effect of kinase A was observed on initial rates of the synthesis or hydrolysis of phosphatidylinositol phosphate. No phosphatidylinositol bisphosphate was produced in the membranes under any of the assay conditions used.

scholarly journals Corticotropin-(1–24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain

1981 ◽  
Vol 194 (1) ◽  
pp. 283-291 ◽  
Author(s):  
J Jolles ◽  
H Zwiers ◽  
A Dekker ◽  
K W A Wirtz ◽  
W H Gispen
Keyword(s):  
Phosphatidic Acid ◽  
Protein Phosphorylation ◽  
Subcellular Fraction ◽  
Membrane Phospholipids ◽  
Cytosol Fraction ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphorylation Of Proteins ◽  
Synaptosomal Fraction ◽  
Phosphatidylinositol Phosphate ◽  
Polyphosphoinositide Metabolism

1. Effects of corticotropin-(1–24)-tetracosapeptide on the endogenous phosphorylation of proteins and lipids were studied in a membrane/cytosol fraction prepared from a lysed crude mitochondrial/synaptosomal fraction. 2. The labelling of proteins and lipids was monitored by incubation of the subcellular fraction for 10s with [gamma-32P]ATP. 3. The phosphorylation of proteins was dose-dependently inhibited by the peptide (40% of control incubations at 100 microM-corticotropin). 4. Of the membrane phospholipids only phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidic acid became labelled. Corticotropin dose-dependently increased the formation of phosphatidylinositol bisphosphate and inhibited the production of phosphatidic acid (470% and 50% respectively of control incubations, at 100 microM of the peptide) and had no effect on phosphatidylinositol phosphate. 5. Phosphatase activity was observed to act on phosphatidylinositol bisphosphate, phosphatidylinositol phosphate and phosphoprotein but not on phosphatidic acid. 6. Corticotropin interacted with the kinases rather than with the phosphatases. 7. The formation of phosphatidylinositol bisphosphate and phosphatidic acid was maximal at 1–10mM-Mg2+ in the absence of Ca2+, and the production of phosphatidylinositol phosphate was maximal at 30mM-Mg2+. 8. The basal value of lipid phosphorylation decreased with increasing Ca2+ concentration. 9. Ca2+ abolished the effect of corticotropin on phosphatidylinositol bisphosphate formation (470%, 190% and 100% of control incubations at respectively 0, 0.1 and 1 mM-Ca2+). 10. The data provide evidence that the effects of corticotropin on protein phosphorylation and on polyphosphoinositide metabolism in brain membranes are related.

scholarly journals Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram

1993 ◽  
Vol 292 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Y Shakur ◽  
J G Pryde ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Thermal Inactivation ◽  
Cytosol Fraction ◽  
Terminal Domain ◽  
Low Concentrations ◽  
Membrane Bound ◽  
High Concentrations ◽  
Membrane Components ◽  
Triton X

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a ‘hypotonic shock’ treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

scholarly journals Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine β-hydroxylase

1979 ◽  
Vol 181 (1) ◽  
pp. 231-237 ◽  
Author(s):  
O J Bjerrum ◽  
K B Helle ◽  
E Bock
Keyword(s):  
Hydrophobic Interaction ◽  
Membrane Fraction ◽  
Cetyltrimethylammonium Bromide ◽  
Limited Proteolysis ◽  
Chromaffin Granules ◽  
Immunoelectrophoretic Analysis ◽  
Crossed Immunoelectrophoresis ◽  
Dopamine Beta Hydroxylase ◽  
Membrane Bound ◽  
Triton X

By means of a monospecific antibody, dopamine beta-hydroxylase was monitored immunoelectrophoretically in various extracts of chromaffin granules. Approximately one-third of the dopamine beta-hydroxylase present was located in the membrane fraction and could only be liberated with detergent. The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. Furthermore, immunoelectrophoretic analysis in the presence of Triton X-100 plus the cationic detergent cetyltrimethylammonium bromide indicates additional heterogeneity of the membrane-bound dopamine-beta-hydroxylase. By limited proteolysis with chymotrypsin and thermolysin the amphiphilic form could be convered into its hydrophilic counterpart.

Studies on the unmasking of membrane-bound alkaline phosphatase during the differentiation of Dictyostelium discoideum

1984 ◽  
Vol 62 (10) ◽  
pp. 970-974 ◽  
Author(s):  
D. V. Mohan Das ◽  
Gerald Weeks
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Dictyostelium Discoideum ◽  
Alkaline Phosphatase Activity ◽  
Developmental Regulation ◽  
Nucleotidase Activity ◽  
Nitrophenyl Phosphate ◽  
Single Protein ◽  
Membrane Bound ◽  
Triton X

Evidence has been presented recently to suggest that the 5′-nucleotidase and alkaline phosphatase activities of culmination phase cells of Dictyostelium discoideum are due to a single protein. However, we find that the membrane bound 5′-nucleotidase activity is only marginally activated by either 50 °C treatment or by dialysis, conditions that markedly activate alkaline phosphatase activity. In contrast, the 5′-nucleotidase activity of Triton X-100 extracts is activated by dialysis to the same extent as alkaline phosphatase activity. The available evidence suggests that, although a single protein is responsible for both alkaline phosphatase and 5′-nucleotidase activities, the characteristics of binding of the two substrates p-nitrophenyl phosphate and AMP to this enzyme are somewhat different. The alkaline phosphatase activity of dialyzed vegetative cell membranes is inhibited by the addition of concentrated dialyzate and this inhibition is reversed by further dialysis. The culmination phase enzyme is also inhibited by concentrated dialyzate from vegetative cells, suggesting that the removal of inhibitor from the enzyme can completely account for the developmental regulation. Dialyzates from culmination phase membranes do not inhibit enzyme activity, indicating the absence of inhibitor in these preparations. Alkaline phosphatase and 5′-nucleotidase activities of a partially purified enzyme preparation are equally inhibited by the addition of the concentrated dialyzate.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

1977 ◽  
Vol 55 (10) ◽  
pp. 1114-1117 ◽  
Author(s):  
Gerrit H. Bomhoff ◽  
Mary Spencer
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Ferrocytochrome C ◽  
Optimum Ph ◽  
Triton X ◽  
Assay Conditions ◽  
Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

Local ATP regeneration is important for sarcoplasmic reticulum Ca2+ pump function

1994 ◽  
Vol 267 (2) ◽  
pp. C357-C366 ◽  
Author(s):  
P. Korge ◽  
K. B. Campbell
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Muscle Relaxation ◽  
Creatine Phosphate ◽  
Coupling Ratio ◽  
Pump Function ◽  
Membrane Bound ◽  
Low Efficiency ◽  
Atp Regeneration ◽  
Assay Conditions

Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) vesicles was measured by monitoring Ca2+ uptake and efflux with a Ca(2+)-sensitive minielectrode and adenosinetriphosphatase (ATPase) activity of the same preparation under the same conditions. The efficiency of Ca2+ transport into SR vesicles, defined by the amount of Ca2+ transported per ATP hydrolyzed (coupling ratio), varied significantly depending on assay conditions. Coupling ratio increased in parallel with increase in precipitating anion concentration, which is supposed to decrease accumulation of free Ca2+ inside vesicles and its subsequent efflux. Membrane-bound creatine kinase-creatine phosphate (CK-CP) system, acting as a ADP sensor and local ATP regenerator, significantly improved Ca2+ pump function when the pump worked with low efficiency (coupling ratio < 1). The effect of CK-CP system on Ca2+ pump function was also dependent on extravesicular Ca2+ concentration ([Ca2+]o), the effect being most significant at high initial [Ca2+]o. Under conditions in which SR vesicles were allowed to decrease [Ca2+]o, as occurs also during muscle relaxation, plateau values of Ca(2+)-ATPase activity were reached at significantly higher [Ca2+]o (54 +/- 5.7, n = 6), compared with leaky vesicles or the condition in which [Ca2+]o was maintained. By preventing local accumulation of ADP, generated in ATPase reactions, CK-CP system also inhibited Ca2+ efflux under conditions in which this efflux was stimulated by the increase of free Ca2+ inside vesicles. This effect was at least partially responsible for the CK-CP-supported increase in Ca2+ uptake and coupling ratios that were more expressed at low precipitating anion concentration. We hypothesize that local ATP regeneration by CK-CP system is one mechanism the cell can use to improve Ca2+ uptake by SR in emergency conditions, where excessive increase in cytoplasmic [Ca2+] may have deleterious effects.

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