scholarly journals Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase

1988 ◽  
Vol 251 (1) ◽  
pp. 157-163 ◽  
Author(s):  
A J Morris ◽  
K J Murray ◽  
P J England ◽  
C P Downes ◽  
R H Michell
Keyword(s):  
Rat Brain ◽  
Inositol Phosphates ◽  
Gel Filtration ◽  
Partial Purification ◽  
First Order Kinetics ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate ◽  
Dependent Protein ◽  
Metabolic Properties ◽  
Sepharose 4B

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 × 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5′-[gamma−thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.

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 Purification and characterization of myosin light-chain kinase from the rat pancreas

1989 ◽  
Vol 258 (3) ◽  
pp. 739-747 ◽  
Author(s):  
M Bissonnette ◽  
D Kuhn ◽  
P de Lanerolle
Keyword(s):  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Gel Filtration ◽  
Type I ◽  
Rat Pancreas ◽  
Absolute Dependence ◽  
Dependent Protein ◽  
Sepharose 4B

We have partially purified a protein kinase from rat pancreas that phosphorylates two light-chain subunits of pancreatic myosin, a doublet with components of 18 and 20 kDa. This protein kinase was purified approx. 1000-fold by sequential (NH4)2SO4 fractionation, gel filtration, ion-exchange and affinity chromatography on calmodulin-Sepharose 4B. The resultant enzyme preparation is free of cyclic AMP-dependent protein kinase, protein kinase C and calmodulin-dependent type I or II kinase activities. The purified protein kinase is completely dependent on Ca2+ and calmodulin, and phosphorylates a 20 kDa light-chain subunit of intact gizzard myosin, suggesting that it belongs to a class of enzymes known as myosin light-chain kinase (MLCK). The apparent Km values of the putative pancreatic MLCK for ATP (73 microM), gizzard myosin light chains (18 microM) and calmodulin (2 nM) are similar to those reported for MLCKs isolated from smooth muscle, platelet and other sources. The enzyme is half-maximally activated at a free Ca2+ concentration of 2.5 microM. A single component of the affinity-purified kinase reacts with antibodies to turkey gizzard MLCK. The apparent molecular mass of this component is 138 kDa. Immunoprecipitation of a pancreatic homogenate with these antibodies decreases calmodulin-dependent kinase activity for pancreatic myosin by over 85%. The immunoprecipitate contains a single electrophoretic band of 138 kDa. Tryptic phosphopeptide analyses of pancreatic myosin, phosphorylated by either gizzard or pancreatic MLCK, are identical. Thus the enzyme that we have purified from rat pancreas is a MLCK, as judged by (1) absolute dependence on Ca2+ and calmodulin, (2) high affinity for calmodulin, (3) narrow substrate specificity for the light-chain subunit of myosin, and (4) reactivity with antibodies to turkey gizzard MLCK. These studies establish the existence of a pancreatic MLCK which may be responsible for regulating myosin phosphorylation and enzyme secretion in situ.

scholarly journals Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: implications for membrane traffic and Ca2+ homoeostasis

1997 ◽  
Vol 324 (2) ◽  
pp. 579-589 ◽  
Author(s):  
Salvador SORIANO ◽  
Stephen THOMAS ◽  
Stephen HIGH ◽  
Gareth GRIFFITHS ◽  
Clive D'SANTOS ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Direct Evidence ◽  
Brefeldin A ◽  
Membrane Association ◽  
Protein Protein Interactions ◽  
And Topology ◽  
Inositol 1,4,5 Trisphosphate ◽  
Peripheral Membrane Protein ◽  
Inositol Tetrakisphosphate ◽  
Dependent Protein

We previously reported the isolation of a rat cDNA clone encoding a protein with significant sequence homology to the B isoform of human myo-inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase B); this protein was thus designated rat IP3 3-kinase B [Thomas, Brake, Luzio, Stanley and Banting (1994) Biochim. Biophys. Acta 1220, 219–222]. However, no IP3 kinase isoform had been shown to generate the physiologically important isoform of inositol tetrakisphosphate, i.e. inositol 1,3,4,5-tetrakisphosphate. We now present direct evidence that the putative rat IP3 3-kinase B is genuinely an IP3 3-kinase. We also show that the enzyme exists both as a peripheral membrane protein tightly associated with the cytosolic face of the extended endoplasmic reticulum network, and as a cytosolic protein. Association of the IP3 3-kinase with membranes is not affected by treatment with brefeldin A, Na2CO3 (pH 11.5), 2 M NaCl, or alteration of [Ca2+]. However, treatment of isolated membranes with 4 M urea leads to dissociation of the kinase from the membrane, implying that membrane association involves specific, conformation-dependent protein–protein interactions. The fact that IP3 3-kinase B is localized exclusively to membranes of Ca2+ stores, is consistent with a model where this kinase plays a role in IP3-dependent Ca2+ release.

scholarly journals Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation

1988 ◽  
Vol 253 (3) ◽  
pp. 721-733 ◽  
Author(s):  
L R Stephens ◽  
P T Hawkins ◽  
C J Barker ◽  
C P Downes
Keyword(s):  
Rat Brain ◽  
Inositol Phosphates ◽  
Platelet Activating Factor ◽  
Chemical Properties ◽  
Brain Homogenate ◽  
Receptor Activation ◽  
Murine Bone Marrow ◽  
Astrocytoma Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Stimulation Of

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.

scholarly journals Binding of inositol phosphates and induction of Ca2+ release from pituitary microsomal fractions

1987 ◽  
Vol 244 (2) ◽  
pp. 493-496 ◽  
Author(s):  
A Spät ◽  
G L Lukács ◽  
I Eberhardt ◽  
L Kiesel ◽  
B Runnebaum
Keyword(s):  
Anterior Pituitary ◽  
Inositol Phosphates ◽  
Maximal Effect ◽  
Reversible Binding ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate

Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible binding of inositol 1,4,5-[32P]trisphosphate; 50% of the labelled ligand is displaced by 3.5 nM-inositol 1,4,5-trisphosphate. 0.5 microM-inositol 1,4-bisphosphate and 10 microM-ATP. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from the microsomal vesicles (half-maximal effect at 290 nM), and its action is potentiated by inositol tetrakisphosphate (half-maximal effect at 4 microM).

Regulation of plasma membrane-bound Ca(2+)-ATPase pump by inositol phosphates in rat brain

1992 ◽  
Vol 262 (3) ◽  
pp. F411-F416
Author(s):  
C. L. Fraser ◽  
P. Sarnacki
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Rat Brain ◽  
Inositol Phosphates ◽  
Stimulatory Action ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Bound

We have previously shown that inositol 1,4,5-trisphosphate (1,4,5-IP3) may participate in signal transduction in brain by inhibiting plasma membrane-bound Na(+)-Ca2+ exchanger. This study was therefore designed to determine whether 1,4,5-IP3 and/or inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) might also affect Ca2+ transport by the plasma membrane Ca(2+)-ATPase pump. Our data show that 1,4,5-IP3 significantly (P less than 0.005) stimulates Ca2+ transport, whereas 1,3,4,5-IP4 significantly (P less than 0.006) inhibits transport by the pump. However, in the presence of both 1,4,5-IP3 and 1,3,4,5-IP4, the stimulatory effect of 1,4,5-IP3 is dominant. Thus Ca2+ transport was significantly stimulated as though 1,4,5-IP3 alone was present. We also observed that 1,3,4-IP3, which had no effect on Ca2+ transport by itself, antagonizes the stimulatory action of 1,4,5-IP3 and potentiates the inhibition of Ca2+ transport by 1,3,4,5-IP4. Half-maximal activities were observed at 10(-8) M. Our data suggest that 1,3,4,5-IP4, 1,4,5-IP3, and 1,3,4-IP3 may participate in signal transduction in brain by regulating the plasma membrane-bound Ca(2+)-ATPase pump.

scholarly journals Extraction and partial Purification of ipopolysaccaride (LPS) from E. coli O157:H7 ioslate

2009 ◽  
Vol 3 (1) ◽  
pp. 10-14
Author(s):  
Ashwak B. J. Al-Hashimi ◽  
Essam F. A. Al-Jumaily ◽  
Amina N. Al-Thiwini ◽  
Hitham A. Al-Omari
Keyword(s):  
Molecular Weight ◽  
Nucleic Acids ◽  
Petroleum Ether ◽  
Wave Length ◽  
Gel Filtration ◽  
Partial Purification ◽  
E Coli ◽  
Ether Mixture ◽  
Two Peaks ◽  
Sepharose 4B

Lipopolysaccharide (LPS) was partialy purified from E. coli O157:H7 local isolate by Sepharose – 4B gel filtration chromatography, after extraction by phenol-chloroform –petroleum ether mixture. The results show the presence of two peaks of proteins; while one peak of carbohydrates when tested at wave length 490nm. Nucleic acids were not found in the sample after partial purification. Electrophoresis pattern shows one large band with a molecular weight of 69000 daltons.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

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