scholarly journals A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-d-myo-inositol 3,4,5-trisphosphate

1991 ◽  
Vol 280 (2) ◽  
pp. 533-539 ◽  
Author(s):  
G Reiser ◽  
R Schäfer ◽  
F Donié ◽  
E Hülser ◽  
M Nehls-Sahabandu ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Anion Exchange ◽  
Protein Band ◽  
Anion Exchange Chromatography ◽  
Bovine Brain ◽  
Exchange Chromatography ◽  
Receptor Protein ◽  
Radioactive Label ◽  
Binding Reaction ◽  
High Affinity

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.

scholarly journals A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

1995 ◽  
Vol 309 (2) ◽  
pp. 557-567 ◽  
Author(s):  
T Treptau ◽  
R Kissmehl ◽  
J D Wissmann ◽  
H Plattner
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Molecular Mass ◽  
Protein Kinases ◽  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Paramecium Tetraurelia ◽  
Release Channel

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 (‘parafusin’) would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

scholarly journals Exo-Arabinanase of Penicillium chrysogenum Able To Release Arabinobiose from α-1,5-l-Arabinan

2001 ◽  
Vol 67 (7) ◽  
pp. 3319-3321 ◽  
Author(s):  
Tatsuji Sakamoto ◽  
Jean-Fran�ois Thibault
Keyword(s):  
Molecular Mass ◽  
Ammonium Sulfate ◽  
Anion Exchange ◽  
Culture Filtrate ◽  
Mode Of Action ◽  
Penicillium Chrysogenum ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Apparent Molecular Mass ◽  
Ammonium Sulfate Precipitation

ABSTRACT An exo-arabinanase, designated Abnx, was purified from a culture filtrate of Penicillium chrysogenum 31B by ammonium sulfate precipitation, anion-exchange chromatography, and hydrophobic chromatography. Abnx had an apparent molecular mass of 47 kDa. The enzyme released only arabinobiose from the nonreducing terminus of α-1,5-l-arabinan and showed no activity towardsp-nitrophenyl-α-l-arabinofuranoside and α-1,5-l-arabinofuranobiose. Abnx is the first enzyme with this mode of action.

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography

2021 ◽  
pp. 111853
Author(s):  
Joao Carlos Simoes-Cardoso ◽  
Nanako Hoshino ◽  
Yusuke Yoshimura ◽  
Chyi-Shin Chen ◽  
Cristina Dias-Cabral ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enthalpy Change ◽  
Adsorption Enthalpy
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