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.

scholarly journals Salivary apyrase of Rhodnius prolixus. Kinetics and purification

1986 ◽  
Vol 233 (3) ◽  
pp. 885-891 ◽  
Author(s):  
J J F Sarkis ◽  
J A Guimarães ◽  
J M C Ribeiro
Keyword(s):  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Rhodnius Prolixus ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Inorganic Pyrophosphatase ◽  
Blood Sucking ◽  
Specific Activities

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5′-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.

scholarly journals 554. Efficient Scalable Purification of rAAV1 Using Ion-Exchange and Gel-Filtration Chromatography To Avoid Ultracentrifugation Procedure

2015 ◽  
Vol 23 ◽  
pp. S222
Author(s):  
Taro Tomono ◽  
Yukihiko Hirai ◽  
Hironori Okada ◽  
Tomoko Chiyo ◽  
Takashi Shimada ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Gel Filtration Chromatography

scholarly journals Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

1969 ◽  
Vol 111 (2) ◽  
pp. 139-146 ◽  
Author(s):  
A. Dahlqvist ◽  
U. Telenius
Keyword(s):  
Ion Exchange ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Heat Inactivation ◽  
Enzyme Preparations ◽  
Small Intestinal ◽  
Two Peaks ◽  
Second Peak ◽  
Intestinal Disaccharidases

1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the ‘void volume’. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based on heat inactivation [maltase Ia (=isomaltase), maltase Ib (=invertase or sucrase), maltase II and maltase III] until increased knowledge about the specificity and structure of these enzymes makes possible a more rational nomenclature.

scholarly journals Location of an epitopic site on epiglycanin by molecular immunoelectron microscopy

1985 ◽  
Vol 227 (1) ◽  
pp. 231-237 ◽  
Author(s):  
J K Wold ◽  
H S Slayter ◽  
J F Codington ◽  
R W Jeanloz
Keyword(s):  
Electron Microscopy ◽  
Ion Exchange ◽  
Immunoelectron Microscopy ◽  
Gel Filtration ◽  
Strong Tendency ◽  
Gel Filtration Chromatography ◽  
Shadow Casting ◽  
Probable Presence ◽  
Antigen Antibody

Antibodies of the IgM type present in rabbit anti-epiglycanin antiserum were purified by (NH4)2SO4 precipitation and by ion-exchange, affinity and gel-filtration chromatography. After papain treatment of this fraction, followed by gel filtration, the fraction with highest apparent Mr was incubated with epiglycanin, and the antigen-antibody complexes separated by gel filtration. These were examined by electron microscopy, using rotational shadow casting, and the photographs of the complexes were mapped for the locations of the antibody molecules on the extended epiglycanin molecules. Distribution of the frequency of attachment of immunoglobulin showed a strong tendency toward binding at one end of epiglycanin, suggesting the probable presence of only one epitope, probably a glycopeptide structure containing a beta-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-D-galactose chain.

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.

Specificity of human beta-choriogonadotropin assays for the hormone and for an immunoreactive fragment present in urine during normal pregnancy.

1983 ◽  
Vol 29 (4) ◽  
pp. 667-671 ◽  
Author(s):  
H R Schroeder ◽  
C M Halter
Keyword(s):  
Concanavalin A ◽  
High Purity ◽  
Gel Filtration ◽  
Normal Pregnancy ◽  
Beta Subunit ◽  
Relative Molecular Mass ◽  
Human Choriogonadotropin ◽  
Conformational Determinant ◽  
Reference Preparation ◽  
Specific Activities

Abstract We evaluated four high-purity commercial preparations of human choriogonadotropin (hCG; Boehringer-Mannheim, Calbiochem, Cambridge, and Radioassay Systems) by two radioimmunoassays and one radioreceptor procedure. Their specific activities were less than half that of the first International Reference Preparation of hCG for immunoassay. In addition, a fragment that was immunoreactive in radioimmunoassays for hCG with antisera to the beta-subunit conformational determinant was isolated from crude hCG (Roussel Corp.). The fragment adsorbed to concanavalin A and exhibited a relative molecular mass of 12 000 by gel filtration. In a study with 94 urines from women three to 24 weeks pregnant, the fragment contributed more than 70% of the immunoreactivity detected by the above radioimmunoassays for hCG in 70% of the samples. The fragment was present in urine throughout pregnancy, but was not detected in the serum of any of seven pregnant women tested.

Some properties of a highly thermostable α-amylase from a Thermoactinomyces sp.

1984 ◽  
Vol 30 (6) ◽  
pp. 780-785 ◽  
Author(s):  
S. K. C. Obi ◽  
F. J. C. Odibo
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzyme Activation ◽  
Cow Dung ◽  
C 30 ◽  
Hydrolysis Of

Thermostable α-amylase from Thermoactinomyces sp. No. 15, isolated from cow dung, was partially purified and characterized. The enzyme was purified (318-fold) by acetone precipitation, ion-exchange chromatography, and gel filtration techniques. The molecular weight was estimated to be 47 800. Optimum enzyme activity was recorded at pH 7 and at 80 °C. The enzyme was stable at pH 5.0–10.0 and retained 74% activity at 100 °C (30 min). Enzyme activation was observed in the presence of Mn2+, Ag+, and Fe2+, but Hg2+ and Zn2+ were inhibitory. Products of hydrolysis of native starches were mainly glucose and maltose.

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