scholarly journals Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain

1990 ◽  
Vol 270 (3) ◽  
pp. 715-719 ◽  
Author(s):  
A Höer ◽  
D Höer ◽  
E Oberdisse
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Kinetic Data ◽  
Gel Filtration ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Gel Filtration Chromatography ◽  
Pyrimidine Nucleotides ◽  
High Affinity ◽  
Marked Inhibition

We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.

scholarly journals Purification and some characteristics of the acetylxylan esterase from Schizophyllum commune

1994 ◽  
Vol 298 (3) ◽  
pp. 751-755 ◽  
Author(s):  
N Halgasová ◽  
E Kutejová ◽  
J Timko
Keyword(s):  
Enzyme Activity ◽  
Ion Exchange ◽  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Full Activity ◽  
Sds Page ◽  
Acetylxylan Esterase

Acetylxylan esterase from Schizophyllum commune was purified using ion-exchange and hydrophobic chromatography. The enzyme has a molecular mass of 31 kDa, as determined by SDS/PAGE, or 18 kDa, according to gel filtration. Glycosylation of the enzyme was not detected. Acetylxylan esterase is relatively stable under laboratory conditions; it retains full activity at pH 6.2-8.5 upon incubation at 25 degrees C for 7 h, but loses nearly the whole activity upon incubation at 60 degrees C for 30 min. The pH optimum of the enzyme activity is 7.7 and its temperature optimum lies between 30 and 45 degrees C. Ca2+ and Co2+ inhibit markedly the activity of acetylxylan esterase at a concentration of 10 mM, as do Mn2+, Zn2+, Fe2+ and Cu2+ at a concentration of 1 mM.

scholarly journals Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties

1990 ◽  
Vol 271 (1) ◽  
pp. 75-86 ◽  
Author(s):  
J Bielicki ◽  
C Freeman ◽  
P R Clements ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Human Kidney ◽  
Reduced Form ◽  
Dermatan Sulphate ◽  
Native Molecular Mass ◽  
Phosphate Ions

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

scholarly journals Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role

1995 ◽  
Vol 306 (1) ◽  
pp. 167-175 ◽  
Author(s):  
M A Gijón ◽  
C Pérez ◽  
E Méndez ◽  
M Sánchez Crespo
Keyword(s):  
Septic Shock ◽  
Enzyme Activity ◽  
Phospholipase A2 ◽  
Molecular Mass ◽  
Gel Filtration ◽  
High Density ◽  
Complement C3 ◽  
High Density Lipoproteins ◽  
Group Ii ◽  
Anaphylatoxin C3a

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)

1991 ◽  
Vol 274 (3) ◽  
pp. 657-662 ◽  
Author(s):  
R de Groot ◽  
D O Chaffin ◽  
M Kuehn ◽  
A L Smith
Keyword(s):  
Enzyme Activity ◽  
Haemophilus Influenzae ◽  
Molecular Mass ◽  
Isoelectric Focusing ◽  
Dihydrofolate Reductase ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Peptide Fragment ◽  
Total Enzyme Activity ◽  
Total Enzyme

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

scholarly journals Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties

1994 ◽  
Vol 304 (3) ◽  
pp. 987-992 ◽  
Author(s):  
A Amici ◽  
M Emanuelli ◽  
E Ferretti ◽  
N Raffaelli ◽  
S Ruggieri ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Point ◽  
Gel Filtration ◽  
Protein Band ◽  
Product Inhibition ◽  
Human Erythrocytes ◽  
Reaction Products ◽  
Methionine Residues ◽  
Formed Product ◽  
Unique Specificity

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3′-monophosphates. Km values for 3′dUMP, 3′UMP, 5′dUMP, 5′UMP, 5-fluoro-2′dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.

CLONING, EXPRESSION, BIOCHEMICAL PARTIAL CHARATERIZATION AND MODELING OF ESTERASE FROM ACINETOBACTER CALCOACETICUS SP DSM587

2013 ◽  
Vol 6 ◽  
Author(s):  
Inmaculada Navarro-González
Keyword(s):  
Molecular Mass ◽  
Acyl Chain ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Catalytic Triad ◽  
Maximum Activity ◽  
Structure Modeling ◽  
Gel Filtration Chromatography ◽  
Aminoacid Sequence ◽  
Chain Lengths

The aim of this paper has been to clone, express, purify and characterization the EstB from<br />Acinetobacter calcoaceticus encoded by the gen X8895. The esterase was cloned in Pet28a and<br />partially purified. The molecular mass of the purified enzyme was 36 kDa (by SDS) and 68.9<br />KDa (by gel filtration chromatography). The EstB showed a maximum activity at 35ºC and pH 8<br />and towards shorter acyl chain lengths (PNPA, PNPB, PNPC) and showed activity about Smethyltiobutanoate,<br />too. The catalytic triad has been predicted by aminoacid sequence<br />alignment and the structure modeling was performed used esterase 1QoR as template.

scholarly journals Human platelet heparanase: purification, characterization and catalytic activity

1998 ◽  
Vol 330 (3) ◽  
pp. 1341-1350 ◽  
Author(s):  
Craig FREEMAN ◽  
R. Christopher PARISH
Keyword(s):  
Molecular Mass ◽  
Tumour Cell ◽  
Human Platelet ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Aggregation ◽  
Capillary Endothelium ◽  
Gel Filtration Chromatography ◽  
Average Molecular Mass ◽  
Tumour Metastasis

Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C. R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.

scholarly journals Purification and heterogeneity of inorganic pyrophosphatase of pig scapula cartilage

1975 ◽  
Vol 147 (1) ◽  
pp. 103-109 ◽  
Author(s):  
R Felix ◽  
H Fleisch
Keyword(s):  
Enzyme Activity ◽  
Ion Exchange ◽  
High Purity ◽  
Cetylpyridinium Chloride ◽  
Gel Filtration ◽  
Inorganic Pyrophosphatase ◽  
Gel Filtration Chromatography ◽  
Enzyme Preparations ◽  
Specific Activities

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) was purified from pig scapula cartilage by fractionation with N-cetylpyridinium chloride and (NH4)2SO4, followed by ion-exchange and gel-filtration chromatography. Enzyme preparations of high purity were obtained, with specific activities (100-400 units/mg) higher than those previously described for mammalian pyrophosphatases. The enzyme activity could be separated into several subfractions on ion-exchange columns.

Characterization of dissolved material during the initial phase of softwood kraft pulping

TAPPI Journal ◽  
2013 ◽  
Vol 12 (1) ◽  
pp. 37-43 ◽  
Author(s):  
HANNU PAKKANEN ◽  
TEEMU PALOHEIMO ◽  
RAIMO ALÉN
Keyword(s):  
Mass Transfer ◽  
Molecular Mass ◽  
Initial Phase ◽  
Degradation Products ◽  
Kraft Pulping ◽  
Reaction Products ◽  
Cooking Temperature ◽  
Molecular Masses ◽  
Aliphatic Carboxylic Acids

The influence of various cooking parameters, such as effective alkali, cooking temperature, and cooking time on the formation of high molecular mass lignin-derived and low molecular mass carbohydrates-derived (aliphatic carboxylic acids) degradation products, mainly during the initial phase of softwood kraft pulping was studied. In addition, the mass transfer of all of these degradation products was clarified based on their concentrations in the cooking liquor inside and outside of the chips. The results indicated that the degradation of the major hemicellulose component, galactoglucomannan, typically was dependent on temperature, and the maximum degradation amount was about 60%. In addition, about 60 min at 284°F (140°C) was needed for leveling off the concentrations of the characteristic reaction products (3,4-dideoxy-pentonic and glucoisosaccharinic acids) between these cooking liquors. Compared with low molecular mass aliphatic acids, the mass transfer of soluble lignin fragments with much higher molecular masses was clearly slower.

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