scholarly journals Rat brain inositol 1,4,5-trisphosphate 3-kinase. Ca2+-sensitivity, purification and antibody production

1990 ◽  
Vol 268 (1) ◽  
pp. 213-217 ◽  
Author(s):  
K Takazawa ◽  
M Lemos ◽  
A Delvaux ◽  
C Lejeune ◽  
J E Dumont ◽  
...  
Keyword(s):  
Western Blot ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Rat Brain ◽  
Antibody Production ◽  
Kinase Activity ◽  
Bovine Brain ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Blue Sepharose

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

1990 ◽  
Vol 272 (1) ◽  
pp. 107-112 ◽  
Author(s):  
K Takazawa ◽  
J Vandekerckhove ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rat Brain ◽  
Kinase Activity ◽  
Limited Proteolysis ◽  
Cloning And Expression ◽  
Terminal Part ◽  
Dependent Protein Kinase ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5′ end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

scholarly journals Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme

1991 ◽  
Vol 278 (3) ◽  
pp. 883-886 ◽  
Author(s):  
K Takazawa ◽  
J Perret ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Amino Acid ◽  
Kinase Activity ◽  
Cloning And Expression ◽  
Fusion Product ◽  
Molecular Evidence ◽  
Reading Frame ◽  
Acid Protein ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3′ end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.

scholarly journals Syringe Purification and HRP-Conjugation of Goat IgGs Used in Quality Control of Erythropoietin

2018 ◽  
Vol 6 (2) ◽  
pp. 11
Author(s):  
Marcel Bassil ◽  
Nataliia Pavliuchenko ◽  
Elia Raya
Keyword(s):  
Quality Control ◽  
Horseradish Peroxidase ◽  
Western Blot ◽  
Affinity Chromatography ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Protein G ◽  
Sds Page

Goat anti-rabbit IgGs were purified from serum using Protein G affinity chromatography. The purity of eluted fractions was tested using SDS-PAGE, and the functionality of IgGs was tested in Western Blot analysis of Erythropoietin, after conjugation with Horseradish Peroxidase.

scholarly journals Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line

1991 ◽  
Vol 280 (2) ◽  
pp. 427-430 ◽  
Author(s):  
H Schohn ◽  
J L Guéant ◽  
M Girr ◽  
E Nexø ◽  
L Baricault ◽  
...  
Keyword(s):  
Cell Culture ◽  
Western Blot ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Cell Line ◽  
Binding Protein ◽  
Gel Filtration ◽  
Secreted Protein ◽  
Sds Page ◽  
Ht 29

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

scholarly journals The dephosphorylation pathway of d-myo-inositol 1,3,4,5-tetrakisphosphate in rat brain

1987 ◽  
Vol 247 (3) ◽  
pp. 635-639 ◽  
Author(s):  
C Erneux ◽  
A Delvaux ◽  
C Moreau ◽  
J E Dumont
Keyword(s):  
Rat Brain ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Phosphatase Activities ◽  
Inositol 1 ◽  
The Third ◽  
Inositol 1,4,5 Trisphosphate ◽  
Hydrolysis Of ◽  
Blue Sepharose

Dephosphorylation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was measured in both the soluble and the particulate fractions of rat brain homogenates. Analysis of the hydrolysis of [4,5-32P]Ins(1,3,4,5)P4 showed that for both fractions the 5-phosphate of Ins(1,3,4,5)P4 was removed and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] was specifically produced. In the soluble fraction, Ins(1,3,4)P3 was further hydrolysed at the 1-phosphate position to inositol 3,4-bisphosphate[Ins(3,4)P2]. DEAE-cellulose chromatography of the soluble fraction separated the phosphatase activities into three peaks. The first hydrolysed both Ins(1,3,4,5)P4 and inositol 1,4,5-trisphosphate, the second inositol 1-phosphate and the third Ins(1,3,4)P3 and inositol 1,4-bisphosphate, [Ins(1,4)P2]. Further purification of the third peak on either Sephacryl S-200 or Blue Sepharose could not dissociate these two activities [i.e. with Ins(1,4)P2 and Ins(1,3,4)P3 as substrates]. The dephosphorylation of Ins(1,3,4)P3 could be inhibited by the addition of Li+.

scholarly journals Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.

1993 ◽  
Vol 40 (2) ◽  
pp. 261-267 ◽  
Author(s):  
J Ignacak ◽  
M Gumińska
Keyword(s):  
Affinity Chromatography ◽  
Pyruvate Kinase ◽  
Rat Liver ◽  
Kinase Activity ◽  
Signal Molecules ◽  
Pyruvate Kinase Activity ◽  
Normal Rat ◽  
Morris Hepatoma ◽  
Blue Sepharose ◽  
Main Substrate

Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 382-388 ◽  
Author(s):  
Dulce Veloso ◽  
Robert W Colman
Keyword(s):  
Complex Formation ◽  
Western Blot ◽  
Human Plasma ◽  
Contact System ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Sds Page ◽  
Activation Products ◽  
Major Antigen ◽  
Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

Ethanol and inositol 1,4,5-trisphosphate mobilize calcium from rat brain microsomes

Alcohol ◽  
1989 ◽  
Vol 6 (6) ◽  
pp. 431-436 ◽  
Author(s):  
Tina Machu ◽  
John J. Woodward ◽  
Steven W. Leslie
Keyword(s):  
Rat Brain ◽  
Inositol 1,4,5 Trisphosphate ◽  
Brain Microsomes
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