scholarly journals A monoclonal antibody distinguishes two types of phosphatidylinositol 4-kinase

1991 ◽  
Vol 273 (1) ◽  
pp. 63-66 ◽  
Author(s):  
G C Endemann ◽  
A Graziani ◽  
L C Cantley
Keyword(s):  
Monoclonal Antibody ◽  
Rat Brain ◽  
Rat Liver ◽  
Bovine Brain ◽  
Human Erythrocytes ◽  
Gene Products ◽  
Type Ii ◽  
Type Iii ◽  
Distinct Gene ◽  
Phosphatidylinositol 4

A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase.

scholarly journals Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes

1992 ◽  
Vol 284 (1) ◽  
pp. 39-45 ◽  
Author(s):  
A Graziani ◽  
L E Ling ◽  
G Endemann ◽  
C L Carpenter ◽  
L C Cantley
Keyword(s):  
Monoclonal Antibody ◽  
Human Erythrocyte ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Bovine Brain ◽  
Erythrocyte Membranes ◽  
Major Protein ◽  
Type Ii ◽  
Effective Inhibitor ◽  
Phosphatidylinositol 4

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

Nuclear localization of phosphatidylinositol 4-kinase β

2002 ◽  
Vol 115 (8) ◽  
pp. 1769-1775 ◽  
Author(s):  
Petra de Graaf ◽  
Elsa E. Klapisz ◽  
Thomas K. F. Schulz ◽  
Alfons F. M. Cremers ◽  
Arie J. Verkleij ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Kinase Activity ◽  
Insoluble Fraction ◽  
Type Ii ◽  
3T3 Cells ◽  
Type Iii ◽  
Pore Complexes ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase,whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,suggesting a specific function for this kinase in nuclear processes.

scholarly journals Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

2008 ◽  
Vol 19 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Andras Balla ◽  
Yeun Ju Kim ◽  
Peter Varnai ◽  
Zsofia Szentpetery ◽  
Zachary Knight ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Type Ii ◽  
Measurable Effect ◽  
Down Regulation ◽  
Type Iii ◽  
Hormone Sensitive ◽  
Phosphatidylinositol 4 ◽  
Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

scholarly journals Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases

1992 ◽  
Vol 288 (2) ◽  
pp. 637-642 ◽  
Author(s):  
N Divecha ◽  
C E L Brooksbank ◽  
R F Irvine
Keyword(s):  
Bovine Brain ◽  
Kinetic Properties ◽  
Type Ii ◽  
Purification And Characterization ◽  
Kinase C ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Single Polypeptide ◽  
Phosphatidylinositol 4

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

Labelling of non-A, non-B hepatitis infected chimpanzee hepatocytes with nuclease-gold conjugates

1984 ◽  
Vol 42 ◽  
pp. 250-251
Author(s):  
G. D. Gagne ◽  
M. F. Miller ◽  
D. A. Peterson
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Infection ◽  
Uranyl Acetate ◽  
Type I ◽  
Cellular Injury ◽  
Type Ii ◽  
Tubular Structures ◽  
Type Iii ◽  
Type Iv ◽  
Infected Chimpanzee

Experimental infection of chimpanzees with non-A, non-B hepatitis (NANB) or with delta agent hepatitis results in the appearance of characteristic cytoplasmic alterations in the hepatocytes. These alterations include spongelike inclusions (Type I), attached convoluted membranes (Type II), tubular structures (Type III), and microtubular aggregates (Type IV) (Fig. 1). Type I, II and III structures are, by association, believed to be derived from endoplasmic reticulum and may be morphogenetically related. Type IV structures are generally observed free in the cytoplasm but sometimes in the vicinity of type III structures. It is not known whether these structures are somehow involved in the replication and/or assembly of the putative NANB virus or whether they are simply nonspecific responses to cellular injury. When treated with uranyl acetate, type I, II and III structures stain intensely as if they might contain nucleic acids. If these structures do correspond to intermediates in the replication of a virus, one might expect them to contain DNA or RNA and the present study was undertaken to explore this possibility.

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