A small G-protein involved in phosphatidylinositol-4-phosphate kinase activation.

A guanine nucleotide binding protein tentatively designated Gs(PIPK) which activates purified phosphatidylinositol-4-phosphate kinase in vitro, has been partially purified from rat liver membranes and identified as a small G-protein with a molecular mass between 20 and 25 kDa.

Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein.

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.

Evidence for a cholera-toxin-sensitive G-protein involved in the regulation of phosphatidylinositol 4-phosphate kinase of rat liver membranes.

Studies on the phosphorylation of inositol phospholipids of rat liver membranes have shown that [gamma S]pppG stimulates 32P incorporation from [gamma-32P]ATP into PI and PIP. This effect appeared specific for stable GTP analogues and could not be reproduced by other compounds. ADP-ribosylation of the membranes with cholera toxin resulted in a large decrease of PIP2 without changes in the level of PIP. Since an activation of phospholipase C can be ruled out, the lowering of PIP2 is explained on the basis of an inhibition of PIP kinase (EC 2.7.1.68). From these results it appears that a novel cholera-toxin-sensitive G-protein is involved in the regulation of PIP kinase.

Interaction of insulin receptors with lipid bilayers and specific and nonspecific binding of insulin to supported membranes.

In the first part, we study the interaction of the insulin receptor with model membranes of dimyristoylphosphatidylcholine (DMPC) by various techniques, including calorimetry, densitometry, static light scattering, and electron microscopy. By analyzing the pronounced depression of the lipid chain melting transition in terms of the Van Laar-Hildebrand theory of regular dilute solutions, an (exothermic) interaction energy of Wp = 2000 kJ.mol-1 is found for the receptor and of WL = 0.6 kJ.mol-1 for the lipid. This is interpreted in terms of an adsorption of the 2 hydrophilic head groups of the receptor to the membrane surface so that 1 protein interacts with about 2000 lipids. This number is verified by freeze-fracture electron microscopy. Binding of insulin induces a remarkable decoupling of the receptor head group from the membrane, pointing to a pronounced conformational change. In the second part, we introduce a simple fluorescence technique by which adsorption isotherms of water-soluble and fluorescent-labeled substrates, such as insulin, to membranes may be determined. It is based on the selective evanescent field excitation of ligands adsorbed to supported planar bilayers on argon-sputtered glass plates. These are deposited by the monolayer transfer technique or by vesicle condensation. The reconstituted receptor exhibits a weak (binding constant Kw = 3 X 10(9) L.M-1) and a strong (binding constant Ks greater than 10(10) L.M-1) binding site. Insulin exhibits a weak but remarkable nonspecific binding to bilayers of pure DMPC and DMPC containing 20% positively charged lipid and a strong binding to DMPC containing negatively charged lipids such as phosphatidylserine or ganglioside (GT1b).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of phosphatidylinositol 4-phosphate phosphorylation in human placenta membranes by GTP gamma S.

In human placenta membranes the rate limiting enzyme for PIP2 formation from PI is PIP kinase. GTP gamma S is shown to activate PIP kinase by increasing Vmax of the enzyme. It is suggested that a guanine nucleotide regulatory protein is involved in the activation of PIP kinase although coupling with a specific receptor is not yet known. Since PIP2 is the preferred substrate of phospholipase C, the possibility exists that an increase of PIP2 due to activation of PIP kinase leads to an enhancement of phospholipase C activity and hence to an increased production of IP3 and DAG.

On the nature of the glucose tolerance factor from yeast.

The aim of this study was to purify the glucose tolerance factor (GFT) from yeast, and to gain further knowledge on its chemical structure. Activity of GTF was determined by its potency to stimulate 1-14C glucose oxidation of rat adipose tissue, in vitro, in the presence of maximally effective insulin concentrations. While described procedures Toepfer, Merth, Polansky, Roginski and Wolf (1977) resulted in some 3-fold purification, specific activity was no more increased by further fractionation on HPLC and/or TLC. Instead separation on TLC yielded several fractions with GTF activity, two of them enriched in aspartate and cystine/cystein, respectively, being the most important. L-aspartate, when added at comparable concentrations in vitro displayed similar GTF-activity as did adenosine, another component present in yeast extracts. It is concluded that GTF-activity of yeasts is not attributable to a single molecular species but rather to the combined action of amino acids and nucleosides especially aspartate, and adenosine.

Evidence for phosphorylation of actin by the insulin receptor-associated protein kinase from human placenta.

A purified preparation of rabbit muscle actin (43-kDa protein) is phosphorylated at tyrosine residues when incubated with solubilized insulin receptor from human placenta. Phosphorylation of the 95-kDa receptor subunit and of 43-kDa protein is stimulated by insulin and vanadate, respectively; however, the mode of action of the two agents is distinguishable.

Phosphorylation--dephosphorylation of purified insulin receptor from human placenta. Effect of insulin.

The insulin receptor of human placenta even after extensive purification is phosphorylated in the presence of [gamma-32P]ATP and NaF, and is dephosphorylated again on incubation in NaF-free medium. Insulin stimulates phosphate incorporation into the Mr 95 000 subunit probably by activation of the phosphorylation step. Our data suggest that the insulin receptor contains both kinase and phosphatase activities that may control the phosphorylation state of the receptor.