Diphosphoinositide metabolism in bovine adrenal medulla

1976 ◽  
Vol 54 (8) ◽  
pp. 746-753 ◽  
Author(s):  
Y. A. Lefebvre ◽  
D. A. White ◽  
J. N. Hawthorne
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Microsomal Fraction ◽  
Kinase Activity ◽  
Hydrolytic Activity ◽  
Vesicle Membrane ◽  
Bovine Adrenal Medulla ◽  
Phosphatidylinositol Kinase ◽  
Membrane Bound ◽  
Phosphatidylinositol 4

(1) A phosphatidylinositol kinase (EC 2.7.1.67) ofa chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue.(2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane.(3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.

scholarly journals Identification and characterization of differentially active pools of type IIα phosphatidylinositol 4-kinase activity in unstimulated A431 cells

2003 ◽  
Vol 376 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Mark G. WAUGH ◽  
Shane MINOGUE ◽  
Deena BLUMENKRANTZ ◽  
J. Simon ANDERSON ◽  
J. Justin HSUAN
Keyword(s):  
Kinase Activity ◽  
Cell Signalling ◽  
Cell Types ◽  
Intrinsic Activity ◽  
A431 Cells ◽  
Cell Functions ◽  
Subcellular Membrane ◽  
Phosphatidylinositol Kinase ◽  
Wide Range ◽  
Phosphatidylinositol 4

The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIα (type IIα phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIα possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in Km for phosphatidylinositol and a 3.5-fold difference in Vmax, thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIα at the level of individual organelles rather than whole-cell lysates.

scholarly journals Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat β-cell tumour

1984 ◽  
Vol 219 (2) ◽  
pp. 471-480 ◽  
Author(s):  
N E Tooke ◽  
C N Hales ◽  
J C Hutton
Keyword(s):  
Plasma Membrane ◽  
Beta Cell ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Secretory Granules ◽  
Cell Tumour ◽  
Density Gradients ◽  
Phosphatidylinositol Kinase ◽  
Phosphatidylinositol 4

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

PROPERTIES OF MEMBRANE-BOUND DOPAMINES-HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA

1977 ◽  
Vol 29 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Dominique Aunis ◽  
Martine Bouclier ◽  
M. Pescheloche ◽  
P. Mandel
Keyword(s):  
Adrenal Medulla ◽  
Chromaffin Granules ◽  
Bovine Adrenal Medulla ◽  
Membrane Bound
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