Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum

1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.

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Purification of an alkaline phosphatase-lipoprotein-X complex.

Clinical Chemistry ◽

10.1093/clinchem/26.5.604 ◽

1980 ◽

Vol 26 (5) ◽

pp. 604-608 ◽

Cited By ~ 9

Author(s):

R N Weijers ◽

H Kruijswijk ◽

J M Baak

Keyword(s):

Alkaline Phosphatase ◽

Affinity Chromatography ◽

Molecular Mass ◽

Gel Filtration ◽

Relative Molecular Mass ◽

Agarose Gel ◽

Extrahepatic Cholestasis ◽

Abnormal Position ◽

Alkaline Phosphatase Isoenzyme ◽

Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

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Determination of the molecular weights of alginates by agarose gel filtration

Journal of Chromatography A ◽

10.1016/s0021-9673(01)92676-7 ◽

1989 ◽

Vol 465 (3) ◽

pp. 386-389 ◽

Cited By ~ 6

Author(s):

Michio Fujihara ◽

Terukazu Nagumo

Keyword(s):

Gel Filtration ◽

Agarose Gel ◽

Molecular Weights

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One-step simultaneous purification of three water-soluble constituents in crude extracts from Danshen by adsorption chromatography on oligo-β-cyclodextrin substituted agarose gel media

Process Biochemistry ◽

10.1016/j.procbio.2006.09.015 ◽

2007 ◽

Vol 42 (3) ◽

pp. 480-485 ◽

Cited By ~ 12

Author(s):

Jun Xu ◽

Tianwei Tan ◽

Jan-Christer Janson

Keyword(s):

Water Soluble ◽

Adsorption Chromatography ◽

Agarose Gel ◽

Crude Extracts ◽

One Step

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Soluble Fibrin Monomer Complexes Demonstrated by Agarose Gel Filtration and by Adsorption on Insolubilized Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651369 ◽

1975 ◽

Vol 34 (01) ◽

pp. 216-222 ◽

Cited By ~ 3

Author(s):

R von Hugo ◽

R Hafter ◽

A Stemberger ◽

H Graeff

Keyword(s):

Molecular Weight ◽

Electrophoretic Mobility ◽

Gel Filtration ◽

Adsorption Chromatography ◽

Agarose Gel ◽

Soluble Fibrin ◽

Relative Electrophoretic Mobility ◽

Fibrin Monomer

SummaryIncubation of fibrinogen with small amounts of thrombin resulted in the occurrence of soluble fibrin monomer complexes. These complexes consisted predominantly of a derivative with a higher molecular weight than that of fibrinogen. It was characterized by its relative electrophoretic mobility in 5% PAA gel (0.28 × 10-5 cm2/V × sec) and its elution position prior to the fibrinogen peak following gel filtration. Using adsorption chromatography on insolubilized fibrinogen the derivative dissociated at a ratio of almost 1 : 1 into one part which was adsorbed and into fibrinogen which was not adsorbed. The part which was adsorbed seemed to be the thrombin mediated fibrin monomer. This study confirms the concept that dissociable dimeric fibrinogen-fibrin monomer complexes occur after limited action of thrombin on fibrinogen.

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In Vitro Formation of Fibrin-Monomer Complexes in the Presence of Isotope Labelled Fibrinogen-Fibrin Degradation Products. An Agarose Gel Filtration Study

10.1055/s-0039-1689556 ◽

1975 ◽

Author(s):

F. Asbeck ◽

van de J. Loo

Keyword(s):

Molecular Weight ◽

Complex Formation ◽

Degradation Products ◽

Gel Filtration ◽

Agarose Gel ◽

Fibrinogen Degradation Products ◽

Molecular Weights ◽

Fibrin Degradation Products ◽

Fibrin Monomer

Human citrated plasmas were mixed with purified 131I-fibrinogen and 131I-fibrinogen degradation products (FDP) or 125I-fibrin degradation products (fdp). After incubation with small amounts of thrombin (0.01–0.02 imits/ml Pl.), these mixtures were gel filtrated on Biogel A5m columns and the elution patterns of the 131I- and -labelled materials were determined.In control experiments without thrombin incubation, no complex formation between fibrinogen, FDP or fdp in citrated plasmas could be detected. This was even true for fdp with a higher molecular weight than fibrinogen.After thrombin incubation, up to 11% fibrin-monomer complexes were formed. Irrespective of their molecular weights, labelled fdp were not incorporated into these complexes.Only large FDP – presumably derivative X – did partially copolymerize with fibrin-monomer complexes in citrated plasmas.

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One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel

Journal of Separation Science ◽

10.1002/jssc.201100006 ◽

2011 ◽

Vol 34 (9) ◽

pp. 995-998 ◽

Cited By ~ 6

Author(s):

Dan Liu ◽

Yan Ma ◽

Ye Wang ◽

Zhiguo Su ◽

Ming Gu ◽

...

Keyword(s):

Adsorption Chromatography ◽

Agarose Gel ◽

Separation And Purification ◽

Hydrolysable Tannins ◽

One Step

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Soluble Fibrin Monomer Complexes Demonstrated by Agarose Gel Filtration and by Adsorption on Insolubilized Fibrinogen. A Comparative Study

10.1055/s-0039-1689554 ◽

1975 ◽

Author(s):

von R. Hugo ◽

R. Hafter ◽

A. Stemberger ◽

H. Graeft

Keyword(s):

Molecular Weight ◽

Comparative Study ◽

Electrophoretic Mobility ◽

Gel Filtration ◽

Adsorption Chromatography ◽

Agarose Gel ◽

Soluble Fibrin ◽

Relative Electrophoretic Mobility ◽

Fibrin Monomer

Incubation of fibrinogen with small amounts of thrombin resulted in the occurrence of soluble fibrin monomer complexes. These complexes consisted predominantly of a derivative with a higher molecular weight than that of fibrinogen. It was characterized by its relative electrophoretic mobility in 5% PAA gel (0.28 × 10-5 cm2/V×sec) and its elution position prior to the fibrinogen peak following gel filtration. Using adsorption chromatography on insolubilized fibrinogen the derivative dissociated at a ratio of almost 1 : 1 into one part which was adsorbed and into fibrinogen which was not adsorbed. The part which was adsorbed seemed to be the thrombin mediated fibrin monomer. This study confirms the concept that dissociable dimeric fibrinogonfibrin monomer complexes occur after limited action of thrombin on fibrinogen.(Supported by DFG ; SFB 51, grant no. 2/Gra – B/6.)

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Design and Applications of Sensitive Enzyme Immunoassays Specific for Clostridial Enoate Reductases

Zeitschrift für Naturforschung C ◽

10.1515/znc-1989-5-602 ◽

1989 ◽

Vol 44 (5-6) ◽

pp. 345-352 ◽

Cited By ~ 4

Author(s):

Günter Krause ◽

Helmut Simon

Keyword(s):

Protein A ◽

Gel Filtration ◽

Staphylococcal Protein A ◽

Horse Radish Peroxidase ◽

Western Blots ◽

Membrane Filters ◽

Enzyme Immunoassays ◽

Molecular Weights ◽

Crude Extracts ◽

Enoate Reductase

Abstract Rabbit antisera raised against purified enoate reductase from Clostridium tyrobutyricum (DSM 1460) and horse-radish peroxidase-conjugated staphylococcal protein A or anti-rabbit immuno globulin G, respectively, were used to develop enzyme immunoassays. Sensitivity limits of the assay are about 250 pg antigen if the enzyme immunoassays are performed on membrane filters and examined visually, and 20 pg for tests in aqueous solution with spectrophotometrical evaluation. The procedures were applied for dot and Western blots as well as colony lifts. Immunologi cal distances between enoate reductases from different Clostridia were determined and the amounts of antigen present in bacterial crude extracts were estimated. In crude extracts of C. thermoaceticum a protein of approximately the size of the enoate reductase and its subunits from C. tyrobutyricum was immunologically detected. Gel filtration chromatography of identically pretreated crude extracts from C. thermoaceticum and C. tyrobutyricum produced immunological signals at similar molecular weights and revealed a lesser tendency of the presumed thermophilic enoatereductase from C. thermoaceticum to disintegrate into its subunits and fragments as compared to its mesophilic counterpart.

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