Activation of detergent-solubilized rat hepatic 5′-iodothyronine deiodinase by NADPH and nonglutathione cytosolic components

1988 ◽  
Vol 66 (1) ◽  
pp. 71-76
Author(s):  
Kenzo Sawada ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Specific Activity ◽  
Microsomal Membrane ◽  
Relative Mass ◽  
Fractional Precipitation ◽  
Iodothyronine Deiodinase ◽  
Low Concentrations ◽  
Similar Dependence ◽  
Maximal Activation ◽  
Cytosolic Factors

An investigation was made of the possible role of the hepatic microsomal membrane in the activation of 5′-iodothyronine deiodinase (5′-DI) by a cytosolic activating system consisting of fraction A (relative mass (Mr) > 60000), fraction B (Mr, approximately 13 000), and NADPH. Activation of 5′-DI in washed microsomes was compared with that of a microsome extract prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate and further purification by fractional precipitation with polyethylene glycol and by DEAE-Sephacel chromatography. All 5′-DI preparations exhibited qualitatively similar dependence upon NADPH and cytosolic factors in fractions A and B for 5′-DI activation and were relatively unresponsive to NADH. Activation of solubilized preparations, unlike that of intact microsomes, was more readily inhibited by low concentrations of detergent and not inhibited by NADPH concentrations above 0.25 mM. Attempted purification of 5′-DI failed to produce a substantial increase in specific activity of the enzyme. It is concluded that, while glutathione-independent cytosolic factors and NADPH can activate 5′-DI in the absence of an intact microsomal membrane, some membrane constituents removed during solubilization and purification of the enzyme are required for maximal activation.

ACC-N-Malonyltransferase activity during zygotic embryogenesis and germination of chick-pea seeds

2000 ◽  
Vol 10 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Clemente Martin-Remesal ◽  
Maria del Carmen Gomez-Jimenez ◽  
Angel J. Matilla
Keyword(s):  
Enzymatic Activity ◽  
Specific Activity ◽  
Embryonic Axis ◽  
Zygotic Embryogenesis ◽  
Chick Pea ◽  
Low Concentrations ◽  
Mtase Activity ◽  
Storage Organs ◽  
Radicle Emergence

AbstractSome physiological characteristics of ACC-Nmalonyltransferase (ACC-N-MTase) have been studied in the seeds of chick-pea (Cicer arietinum L.). This enzymatic activity was detectable during all periods of zygotic embryogenesis; however, the highest values were found in the dry seed. In dry seeds, the enzymatic activity was greater in the embryonic axis than in the cotyledons. During the onset of germination, activity increased more strongly in the cotyledons than in the embryonic axis, reaching its highest values in the storage organs coinciding with radicle emergence (18 to 24 h). Removal of the cotyledons strongly diminished enzymatic activity in the embryonic axis. Low concentrations of ethrel (50 µM) stimulated the axis ACC-N-MTase. The highest specific activity was found in the apical meristem of the embryonic axis, declining over the length of the organ. The role of ACC-N-MTase activity in the germinative process is discussed, together with the regulatory effect of ethylene on this ACCconjugating enzyme.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

scholarly journals Impaired platelet responses to thrombin and collagen in AKT-1–deficient mice

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1703-1710 ◽  
Author(s):  
Juhua Chen ◽  
Sarmishtha De ◽  
Derek S. Damron ◽  
William S. Chen ◽  
Nissim Hay ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granules ◽  
Fibrinogen Binding ◽  
Downstream Effectors ◽  
Low Concentrations ◽  
Phosphoinositide 3 Kinase ◽  
Deficient Mice ◽  
Granule Release

Abstract We investigated the role of Akt-1, one of the major downstream effectors of phosphoinositide 3-kinase (PI3K), in platelet function using mice in which the gene for Akt-1 had been inactivated. Using ex vivo techniques, we showed that Akt-1-deficient mice exhibited impaired platelet aggregation and spreading in response to various agonists. These differences were most apparent in platelets activated with low concentrations of thrombin. Although Akt-1 is not the predominant Akt isoform in mouse platelets, its absence diminished the amount of total phospho-Akt and inhibited increases in intracellular Ca2+ concentration in response to thrombin. Moreover, thrombin-induced platelet α-granule release as well as release of adenosine triphosphate from dense granules was also defective in Akt-1-null platelets. Although the absence of Akt-1 did not influence expression of the major platelet receptors for thrombin and collagen, fibrinogen binding in response to these agonists was significantly reduced. As a consequence of impaired αIIbβ3 activation and platelet aggregation, Akt-1 null mice showed significantly longer bleeding times than wild-type mice. (Blood. 2004;104:1703-1710)

Obesity and inflammatory bowel disease

2021 ◽  
Vol 75 (1) ◽  
pp. 20-28
Author(s):  
Vladimír Teplan ◽  
Milan Lukáš
Keyword(s):  
Inflammatory Bowel Disease ◽  
Adipose Tissue ◽  
Bowel Disease ◽  
Intestinal Inflammation ◽  
Perioperative Complications ◽  
Overweight And Obesity ◽  
Special Role ◽  
Low Concentrations ◽  
Inflammatory Bowel

The incidence and prevalence of overweight and obesity has dramatically increased in the last decades and is generally considered to be global pandemics. The incidence of inflammatory bowel disease (IBD) is rising parallel with overweight and obesity. Contrary to a conventional believe, about 15–40% patients with IBD are obese, which can contribute to the development and course of IBD, especially in Crohn’s disease. Although the findings of some cohort studies are still conflicting, recent results indicate a special role of visceral adipose tissue and particularly mesenteric adipose tissue known as creeping fat, leading to intestinal inflammation. The involvement of altered adipocyte function and deregulated production of adipokines such as leptin and adiponectin has been suggested in the pathogenesis of IBD. The emerging role of Western diet and microbiota can also open new possibilities in IBD management. The effect of obesity on the IBD-related therapy remains to be studied. The finding that obesity results in suboptimal response to the therapy, potentially promoting rapid clearance of biologic agents and thus leading to their low concentrations, has a great importance. Obesity also makes IBD colorectal surgery technically challenging and might increase a risk of perioperative complications.

Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport

1996 ◽  
Vol 271 (1) ◽  
pp. C255-C263 ◽  
Author(s):  
P. W. Flatman ◽  
N. C. Adragna ◽  
P. K. Lauf
Keyword(s):  
Protein Kinases ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Sheep Erythrocyte ◽  
Inhibition Constant ◽  
Calyculin A ◽  
Sheep Erythrocytes ◽  
A 23187 ◽  
Maximal Activation

K-Cl cotransport in sheep erythrocytes can be activated by treatment either with A-23187 and EDTA to reduce concentration of internal ionized Mg [Mg]i) to submicromolar levels, with staurosporine, a potent kinase inhibitor, or with N-ethylmaleimide (NEM). Activation by these maneuvers is prevented and reversed by genistein [inhibition constant (Ki) of 15 microM], which inhibits tyrosine kinases (TK). The related glycosidated compound genistin, which does not inhibit TK, does not inhibit transport, whereas another TK inhibitor, tyrphostin B46, inhibits both basal and stimulated transport (Ki of 28 microM). Cotransport activation by NEM is prevented and reversed by the phosphatase inhibitor, calyculin A, and activation by staurosporine occurs only if cells contain ATP. Increasing [Mg]i inhibits cotransport in the presence of calyculin A whether or not staurosporine is present as well. Our work suggests that genistein inhibits cotransport through a TK and that staurosporine and NEM activate cotransport, probably through inhibition of other kinases, causing stimulation through dephosphorylation of a protein (possibly the transporter itself) be a serine/threonine phosphatase. [Mg]i inhibits cotransport by activating a kinase (concentration for half-maximal activation of 10 microM) that phosphorylates this protein.

Effects of nonsulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism

1999 ◽  
Vol 277 (1) ◽  
pp. E144-E153 ◽  
Author(s):  
Deborah L. Bella ◽  
Christine Hahn ◽  
Martha H. Stipanuk
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Mrna Level ◽  
Basal Diet ◽  
Cysteine Dioxygenase ◽  
Sulfur Amino Acids ◽  
Subunit Mrna ◽  
Cysteine Metabolism ◽  
Glutamylcysteine Synthetase

To determine the role of nonsulfur vs. sulfur amino acids in regulation of cysteine metabolism, rats were fed a basal diet or diets supplemented with a mixture of nonsulfur amino acids (AA), sulfur amino acids (SAA), or both for 3 wk. Hepatic cysteine-sulfinate decarboxylase (CSDC), cysteine dioxygenase (CDO), and γ-glutamylcysteine synthetase (GCS) activity, concentration, and mRNA abundance were measured. Supplementation with AA alone had no effect on any of these measures. Supplementation of the basal diet with SAA, with or without AA, resulted in a higher CDO concentration (32–45 times basal), a lower CSDC mRNA level (49–64% of basal), and a lower GCS-heavy subunit mRNA level (70–76%). The presence of excess SAA and AA together resulted in an additional type of regulation: a lower specific activity of all three enzymes was observed in rats fed diets with an excess of AA and SAA. Both SAA and AA played a role in regulation of these three enzymes of cysteine metabolism, but SAA had the dominant effects, and effects of AA were not observed in the absence of SAA.

The Role of Factor XI in a Dilute Thromboplastin Assay of Extrinsic Coagulation Pathway

2001 ◽  
Vol 85 (06) ◽  
pp. 1055-1059 ◽  
Author(s):  
Shilong Xiong ◽  
Xiaofan He ◽  
Fayi Liu ◽  
Jianzhong Han ◽  
Juncheng Li ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Platelet Rich Plasma ◽  
Critical Role ◽  
Extrinsic Pathway ◽  
Thrombin Inhibition ◽  
Normal Plasma ◽  
Low Concentrations ◽  
Plasma Depletion ◽  
Coagulation Pathway

SummaryBlood coagulation has been thought to be composed of both intrinsic and extrinsic pathways. Recent evidence strongly supports the critical role of the extrinsic pathway in the initiation of blood coagulation. This investigation established an assay that examines the role of FXI in the thromboplastin-initiated (extrinsic) coagulation based on this new concept. Plasma clotting times were measured at different concentrations of thromboplastin with activated FXII inhibited (FXIIa-inhibited Diluted Thromboplastin Time, FXIIaiDTT). Only at low concentrations of thromboplastin was FXIIaiDTT of FXI-deficient plasma significantly prolonged than that of normal plasma. Depletion of FXI from normal plasma prolonged its FXIIaiDTT and replenishment of FXI shortened it. FXIIaiDTTs of both FVIII-deficient and FIX-deficient plasma were remarkably prolonged, and addition of normal plasma dose-dependently shortened it. Furthermore, earlier α-thrombin inhibition was directly correlated with decreasing FXa generation. The amount of FXa production was: platelet-rich plasma > platelet-poor plasma > FXI-deficient plasma. Therefore, our findings from the FXIIaiDTT assays not only support the critical role of extrinsic pathway in blood coagulation initiation, but also demonstrate the importance of FXI as an amplifier of thrombin generation in thromboplastin-initiated coagulation.

scholarly journals The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1970 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
P. S. G. Goldfarb ◽  
R. Rodnight
Keyword(s):  
Potassium Chloride ◽  
Magnesium Chloride ◽  
Adenosine Triphosphatase ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Protein Ratio ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

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