Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

High glucose increases the expression of Gq/11alpha and PLC-beta proteins and associated signaling in vascular smooth muscle cells

The levels and activity of protein kinase C and diacylglycerol were shown to be upregulated in diabetes/hyperglycemia; however, studies on the expression of upstream signaling molecules of phosphatidylinositol turnover were lacking. The present study was therefore undertaken to examine whether hyperglycemia/diabetes could also modulate the expression of Gqalpha and phospholipase C-beta (PLC-beta) proteins and associated phosphatidylinositol turnover signaling in aortic vascular smooth muscle cells (VSMCs) and A10 VSMCs exposed to high glucose. Aortic VSMCs from streptozotocin-diabetic rats exhibited an increased expression of Gqalpha and PLC-beta1 proteins (60% and 30%, respectively) compared with control cells as determined by Western blot analysis. The pretreatment of A10 VSMCs with high glucose (26 mM) for 3 days also augmented the levels of Gqalpha, G11alpha, PLC-beta1 and -beta2 proteins by about 50, 35, 30, and 30%, respectively, compared with control cells that were restored to control levels by endothelin-1 (ET-1), ET types A and B (ET(A) and ET(B)) receptors, and angiotensin II type 1 (AT1) receptor antagonists. In addition, ET-1-stimulated inositol triphosphate formation was also significantly higher in VSMCs exposed to high glucose, whereas the basal levels of inositol triphosphate were not different between the two groups. Furthermore, the treatment of A10 VSMCs with angiotensin II and ET-1 also significantly increased the levels of Gq/11alpha and PLC-beta proteins that were restored toward control levels by ET(A)/ET(B) and AT1 receptor antagonists. These results suggest that high glucose augments the expression of Gq/11alpha, PLC-beta, and mediated signaling in VSMCs, which may be attributed to AT1, ET(A), and ET(B) receptors.     

Coated vesicles contain a phosphatidylinositol kinase

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.     

Enhanced phosphatidylinositol kinase activity is associated with early stages of hepatocarcinogenesis and hepatocellular carcinoma

Liver phosphatidylinositol (PI) kinase activity was determined in rats exposed to two different hepatocarcinogenic regimens. In contrast to partial treatment regimens the complete Solt and Farber hepatocarcinogenic regimen caused a significant increase in liver PI kinase activity at day 11 after partial hepatectomy. PI kinase activity in hepatocarcinomas removed 15 1/2 months after initiation of the complete Solt and Farber regimen was 2-fold higher than normal liver tissue surrounding the tumors. Compared to a choline supplemented diet a hepatocarcinogenic regimen consisting of a diet deficient in choline and methionine significantly increased liver PI kinase activity after 26 days. These data demonstrate that liver PI kinase activity is selectively elevated during hepatocarcinogenesis.     

Legionella micdadei protein kinase catalyzes phosphorylation of tubulin and phosphatidylinositol.

Legionella micdadei, a pathogen which enters into host phagocyte phagolysosomal structures, contains at least two protein kinases. We have purified to homogeneity the predominant, nucleotide-independent protein kinase and examined its ability to catalyze the transfer of phosphate from ATP to acceptors in human neutrophils. The L. micdadei protein kinase catalyzed the phosphorylation of proteins of 11.5, 14, 19, 23, 28, 34, and 38 kilodaltons (kDa) present in a Triton X-100 extract of neutrophil membranes and of 11.5, 13.5, 25, and 38 kDa in the neutrophil cytosol. Tubulin was a good substrate for the L. micdadei protein kinase in vitro. The bacterial kinase also catalyzed the phosphorylation of phosphatidylinositol (PI) at about half the rate at which histones were phosphorylated; phosphatidylinositol-4-phosphate (PIP) was not phosphorylated by the kinase. The PI kinase activity of the L. micdadei enzyme was optimum at pH 7.0, and the divalent cation requirement was satisfied best by Mg2+ and Ca2+. The maximum rate of PI phosphorylation was obtained with 0.6 mM PI; in the presence of MgCl2 (10 mM), the Km for PI was 0.9 mM and the Km for ATP was 1.5 mM. The detergents octyl-beta-D-glucoside (10 to 20 mM) and Triton X-100 (0.5%) stimulated kinase activity twofold when PI was the phosphate acceptor; however, only octyl glucoside stimulated histone kinase activity. Various membrane phospholipids inhibited PI kinase activity. The most potent phospholipid inhibitor was the product of the PI kinase reaction, PIP, which at a 0.6 mM concentration inhibited both PI and tubulin phosphorylation by 80%. The inhibition of kinase activity by PIP when histone served as the acceptor was noncompetitive in character. The L. micdadei kinase also phosphorylated PI in intact. (3H)inositol-labeled neutrophils. The PI kinase and histone kinase activities of teh L. micdadei kinase copurified and cofucused (pI, 5.8) when subjected to isoelectric focusing, suggesting that the two enzymatic activities reside in a single protein.     

Regulation of polyphosphoinositide synthesis in cardiac membranes

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Phospholipase C activation and diacylglycerol kinase inactivation lead to an increase in diacylglycerol content in spontaneously hypertensive rat

Activities of three kinases, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) kinases, and phospholipase C were measured in erythrocyte ghosts from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). PI kinase activity was significantly higher in SHR than WKY but there was no significant difference in PIP kinase activity between SHR and WKY. The activity of phospholipase C, which hydrolyzes PIP2, was also increased in SHR. However, DG kinase activity was, on the contrary, decreased in SHR. These results suggest that there is a tendency to accumulate DG in SHR. Indeed, DG content in erythrocytes of SHR increased 1.7-fold compared to that of WKY. Such DG accumulation may cause the sustained activation of protein kinase C in SHR, since DG is a physiological activator for protein kinase C.     

Effects of glucose infusion in exercising rats

To determine whether feedforward control of liver glycogenolysis during exercise is subject to negative feedback by elevated blood glucose, glucose was infused into exercising rats at a rate that elevated blood glucose greater than 10 mM. Liver glycogen content decreased 22.4 mg/g in saline-infused rats compared with 13.6 mg/g in glucose-infused rats during the first 40 min of treadmill running (21 m/min, 15% grade). Liver adenosine 3',5'-cyclic monophosphate (cAMP) concentration was significantly lower in the glucose-infused rats during the exercise bout. The concentration of hepatic fructose 2,6-bisphosphate remained elevated throughout the exercise bout in glucose-infused rats but decreased markedly in saline-infused rats. Plasma insulin concentration was higher and plasma glucagon concentration lower in glucose-infused rats than in saline-infused rats during exercise. Early in exercise, liver glycogenolysis proceeds in the glucose-infused rats despite the fact that glucose and insulin concentrations are markedly elevated and liver cAMP is unchanged from resting values. These observations suggest the existence of a cAMP-independent feedforward system for activation of liver glycogenolysis that can override classical negative feedback mechanisms during exercise.     

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.     

Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor

On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.     

Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.     

Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Effect of a prolonged protein-free diet on cell size, protein synthesis and degradation of rat liver

Rats were fed a protein-free diet. After 9 weeks the animals' weight decreased to about 50% of the original. The liver weight was also decreased to about half, and most interestingly the average size of the liver cells was reduced about 50%. Liver protein synthesis was approximately 75% of controls tested in an "in vitro" system. Polysomes were found disaggregated in livers of rats on protein-free diet. This was not due to a reduced content or translatability of mRNA. eIF-2 partially purified from livers of rats on protein-free diet had the same activity as that from controls. The decrease of ATP, ADP and AMP in livers of rats on protein-free diet (19%, 42% and 58% respectively) may be responsible for the decreased rate of initiation of protein synthesis. Proteolysis in liver cytosol from rats on protein-free diet was 50% higher than in controls mostly due to lysosomal proteolysis.     

Kinetics of diacylglycerol accumulation in response to vasopressin stimulation in hepatocytes of continuously endotoxaemic rats.

The content and composition of 1,2-diacyl-sn-glycerol (1,2-DAG) was determined in hepatocytes from saline (0.9% NaCl)- and Escherichia coli endotoxin (ET)-infused rats upon continuous vasopressin (VP) (10(-8) M) stimulation. In both experimental groups the accumulation of 1,2-DAG was detected after a lag period (2-5 min), was sustained up to the last time analysed (10 min), and C18:0- and C20:4-fatty-acid-containing-DAG accumulation preceded that of DAG containing other acyl groups. In hepatocytes from ET-infused rats the VP-induced accumulation of DAG was delayed and was decreased by 50%, showing a C18:0/C20:4 molar ratio of 1.6 as compared with 1.1 for cells from saline-infused rats. A similar lower cellular response to VP stimulation was observed in cells prelabelled with [14C]C20:4 fatty acid. The accumulation of [14C]C20:4-DAG (lower in ET than in saline-infused rats) was paralleled by a decrease in phosphatidylinositol (PI) labelling, whereas phosphatidic acid showed a transient increase by 5 min in saline- but not in ET-infused rats. The present results demonstrate that the previously reported impairment in the early degradation of poly-PI and later in the 'PI cycle' during VP stimulation [Rodriguez de Turco & Spitzer (1987) Metab. Clin. Exp. 36, 753-760] is also reflected at the level of their phosphodiesteratic product, DAG. Moreover, the kinetics of the accumulation of DAG acyl groups is consistent with the idea that the initial release of C18:0- and C20:4-DAG (possibly derived from inositol lipids) could regulate the subsequent enlargement of this pool by stimulating a phospholipase C-mediated degradation of other phospholipids (e.g. phosphatidylcholine).     

Pig liver phosphomevalone kinase. 1. Purification and properties

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.     

Phosphatidylinositol kinase of bovine adrenal chromaffin granules: kinetic properties and inhibition by low concentrations of Ca2+

The kinetics and regulatory properties of phosphatidylinositol (PI) kinase were studied in chromaffin granule ghosts isolated from the bovine adrenal medulla. Phosphatidylinositol 4-phosphate (PIP) was the major 32P-labelled phospholipid formed when the isolated membranes were phosphorylated by [gamma-32P]ATP. The PI kinase activity was rather independent of pH, but highly dependent on Mg2+ with a maximal stimulation at 60 mM Mg2+. By contrast, KCl and NaCl had a slight inhibitory effect. The Km value for MgATP was 44 and 62 microM in the presence of 1 and 20 mM MgCl2, respectively. The PI kinase was almost fully and reversibly inhibited by free Ca2+ (calmodulin-independent) in the nanomolar and low micromolar range, depending on the concentration of Mg2+. The inhibition was not dependent on Ca2+-stimulated protein phosphorylation, and it could not be explained by a dephosphorylation of PIP.     

The Inositol Lipids of Paramecium tetraurelia and Preliminary Characterizations of Phosphoinositide Kinase Activity in the Ciliary Membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphati-dylinositol-bis-phosphate (PI-P₂) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [γ-³²P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P₂. Kinase activity was activated by millimolar [Mg²⁺] and inhibited by millimolar [Ca²⁺]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated     

Activation of phosphatidylinositol-4-phosphate kinase in rat liver plasma membranes by polyamines

The formation of phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous substrate in rat liver plasma membranes was stimulated approximately 3-fold by 1 mM spermine, with half-maximal effect at 0.2 mM polyamine. This effect of spermine was due to enhancement of phosphatidylinositol-4-phosphate kinase activity rather than to a decrease in degradation of PIP2 formed or the substrate phosphatidylinositol 4-phosphate (PIP). The stimulation of phosphatidylinositol-4-phosphate kinase by spermine decreased to half at physiological ionic strength, and was not affected appreciably by variations in the concentration of ATP and MgCl2. Among several di- and polyamines only spermine and spermidine were effective. Although spermine may cause aggregation of membrane vesicles, thereby potentially increasing substrate availability for phosphatidylinositol-4-phosphate kinase, our results do not support such an explanation for the enhancement in enzyme activity. Phosphatidylinositol kinase activity, contrary to phosphatidylinositol-4-phosphate kinase, was not stimulated appreciably by spermine.