scholarly journals Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium

1996 ◽  
Vol 314 (2) ◽  
pp. 491-495 ◽  
Author(s):  
Peter VAN DIJKEN ◽  
Jan C. T. BERGSMA ◽  
Hoebert S. HIEMSTRA ◽  
Berber DE VRIES ◽  
Jeroen VAN DER KAAY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Inositol Monophosphatase

The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a non-competitive manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity.

scholarly journals Dephosphorylation of 1d-myo-inositol 1,4-bisphosphate in rat liver

1988 ◽  
Vol 254 (3) ◽  
pp. 655-660 ◽  
Author(s):  
A J Morris ◽  
D J Storey ◽  
C P Downes ◽  
R H Michell
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Substrate Concentration ◽  
Gel Filtration ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Inositol Monophosphatase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Purification and characterization of a Ca2+-dependent novel lectin from Nymphaea nouchali tuber with antiproliferative activities

2011 ◽  
Vol 31 (6) ◽  
pp. 465-475 ◽  
Author(s):  
Syed Rashel Kabir ◽  
Md. Abu Zubair ◽  
Md. Nurujjaman ◽  
Md. Azizul Haque ◽  
Imtiaj Hasan ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Deae Cellulose ◽  
Ehrlich Ascites Carcinoma ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Neutral Sugar ◽  
The Stability

A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5–9 and temperatures of 30–60°C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC50 value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl2 indicated the requirement of Ca2+ for the stability of NNTL.

Abstract 97: Increased Plasma Level of ApoCIII-rich Electronegative HDL May Contribute to Cognitive Impairment in Alzheimer’s Disease

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Hua-Chen Chan ◽  
Hsiao-Ting Lu ◽  
Mei-Chuan Chou ◽  
Ming-Hsien Tsai ◽  
Wei-Hsiang Chen ◽  
...  
Keyword(s):  
Cognitive Impairment ◽  
Cholesterol Efflux ◽  
Chemical Properties ◽  
Density Lipoprotein ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Paraoxonase 1 ◽  
Cholesterol Efflux Capacity ◽  
Tnf Α

Background: High-density lipoprotein (HDL), the only lipoprotein class that can cross the blood brain barrier bidirectionally, is positively associated with cognitive functions. To delineate HDL’s role in Alzhenimer’s disease (AD), we analyzed the chemical properties of plasma HDL from AD and healthy normal adult (control) subjects. Methods and results: By using anion-exchange chromatography, we divided HDL into 5 increasingly electronegative subfractions, H1-H5. Compared to the control cohort (4.24±3.22%; n=20), HDL from AD patients (23.48±17.83%; n=30) had a 5.5-fold increase of H5 ( P <0.001; Figure ), accompanied by a decreased protein/lipid ratio attributed to a significant reduction of albumin essential for prevention of amyloid beta (Aβ) aggregation. As determined by LC/MS E and ProteinLynx Global SERVER (PLGS), AD-HDL was had a rich content of apolipoprotein (apo)CIII, but diminished amounts of sphingosine-1-phosphate (S1P)-associated apoM and antioxidative paraoxonase 1 (PON1). Exposure of murine RAW 264.7 macrophages to H5 induced vibrant expression of ganglioside GM1 in colocalization with apoCIII on lipid rafts, alongside a concomitant increase of TNF-α detectable in the cultured medium ( Figure ). LC/MS E examination localized posttranslational oxidation exclusively in ApoA1 residues of H5 in AD-HDL, which exhibited a compromised cholesterol efflux capacity. Conclusions: Plasma HDL from AD patients has a high proportion of H5, an apoCIII-rich electronegative HDL subfraction. The associated reduction in functional (albumin, S1P, apoM) and increase in proinflammatory (apoCIII, PON1, TNF-α) components may favor Aβ assembly and neuroinflammation. Additionally, a compromised cholesterol-efflux capacity of AD-HDL may also contribute to vascular cognitive impairment.

scholarly journals Interferon alpha associated with systemic lupus erythematosus is not intrinsically acid labile.

1989 ◽  
Vol 169 (3) ◽  
pp. 987-993 ◽  
Author(s):  
A M Yee ◽  
J P Buyon ◽  
Y K Yip
Keyword(s):  
Anion Exchange ◽  
Lupus Erythematosus ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Ifn Alpha ◽  
Plasma Factor ◽  
Acid Stable ◽  
Acid Labile

The physicochemical properties of apparently acid-labile IFN-alpha from patients with SLE have been studied. The antigenicity, apparent molecular size, and isoelectric point of SLE IFN-alpha are indistinguishable from those of conventional, previously characterized, acid-stable subspecies of IFN-alpha. However, after partial purification by anion-exchange chromatography, SLE IFN-alpha no longer exhibits acid lability, suggesting that other plasma factor(s) are responsible for the acid lability of SLE IFN-alpha. Addition of SLE plasma, but not normal plasma, to conventional acid-stable IFN-alpha renders the exogenous IFN-alpha acid labile. Preliminary results demonstrate that an acid-dependent IFN-inactivating activity can be partially purified from SLE plasma by anion-exchange chromatography.

Pectinesterases From Mature, Unripe Peach Fruit Bind Strongly to Pectic Polysaccharides

1995 ◽  
Vol 22 (6) ◽  
pp. 977 ◽  
Author(s):  
H Glover ◽  
CJ Brady
Keyword(s):  
Enzyme Activity ◽  
Strong Association ◽  
Exchange Chromatography ◽  
Tissue Type ◽  
Ripe Fruit ◽  
Peach Fruit ◽  
Pectic Polysaccharides ◽  
Green Fruit ◽  
Large Molecular Weight

Contrary to previous findings, the level of the pectin de-esterifying enzyme, pectinesterase (PE; EC 3.1 .1. 11), is shown to be much higher in mature, green peach fruit than in ripe fruit. Aqueous buffers readily extracted three pectinesterase isoforms from ripe fruit but only a portion of the activity from mature, green fruit. In mature, green fruit extracts the enzyme precipitated when the ionic strength was lowered; consequently isoforms could not be recovered by ion exchange chromatography. In extraction residues from mature, green fruit, residual PE could be measured as active enzyme and, when denatured, could be detected by immunological techniques. Extraction of the enzyme was enhanced after digestion of the tissue with pectin lyase. The extracted enzyme fractionated as a large molecular weight complex rich in uronic acid, rhamnose, galactose and arabinose. After further digestion with endo-β-1,4- galactanase, the enzyme was in two fractions of smaller size but with residual carbohydrate. When mature, green and ripe fruit tissue were co-extracted, the recovered activity was as predicted from independently extracted tissues demonstrating that enzyme activity was not influenced by inhibitors contributed by either tissue type. Isoforms known to be present in the ripe fruit were recovered from extracts of the mixed tissues. It is concluded that PE in extracts of mature, green fruit has a strong association with pectic polymers and this has lead to its underestimation in previous studies. It is suggested that such an association with pectin polymers in vivo may regulate enzyme activity and enzyme turnover.

scholarly journals Sesquiterpene α-Farnesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Development in Apples

2000 ◽  
Vol 125 (1) ◽  
pp. 111-119 ◽  
Author(s):  
H.P. Vasantha Rupasinghe ◽  
Gopinadhan Paliyath ◽  
Dennis P. Murr
Keyword(s):  
Metal Ion ◽  
Maximal Activity ◽  
Exchange Chromatography ◽  
Cytosolic Fraction ◽  
Skin Tissue ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Farnesyl Pyrophosphate ◽  
Superficial Scald

To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg2+ or Mn2+ for activity. It exhibited allosteric kinetics, S(0.5) for farnesyl pyrophosphate being 84±18 μmol·L-1, and a Hill coefficient (nH) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.

scholarly journals Purification and Properties of a Glucuronan Lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472)

2001 ◽  
Vol 67 (11) ◽  
pp. 5197-5203 ◽  
Author(s):  
Alexandre Da Costa ◽  
Philippe Michaud ◽  
Emmanuel Petit ◽  
Alain Heyraud ◽  
Philippe Colin-Morel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Sinorhizobium Meliloti ◽  
Specific Activity ◽  
Protein Sequences ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn2+, Cu2+, and Hg2+ (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified fromS. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.

scholarly journals Evidence for N-linked glycosylation in Toxoplasma gondii

1993 ◽  
Vol 291 (3) ◽  
pp. 713-721 ◽  
Author(s):  
M Odenthal-Schnittler ◽  
S Tomavo ◽  
D Becker ◽  
J F Dubremetz ◽  
R T Schwarz
Keyword(s):  
Toxoplasma Gondii ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Common Precursor ◽  
The Common ◽  
Oligosaccharide Structures

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

scholarly journals Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

2015 ◽  
Vol 112 (5) ◽  
pp. 1356-1361 ◽  
Author(s):  
Tao Liu ◽  
Yong Zhang ◽  
Yan Liu ◽  
Ying Wang ◽  
Haiqun Jia ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
3D Structure ◽  
Human Growth ◽  
Fold Increase ◽  
Obese Mouse ◽  
Human Antibody ◽  
Humanized Antibody ◽  
Long Acting

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

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