Breakdown of phosphatidylinositol in soybean callus

We have investigated the breakdown of membrane-bound phosphatidylinositol (PI) in homogenates of soybean (Glycine max) callus. The breakdown of PI was stimulated by the detergent deoxycholate. At pH 7.0 and 1·gl^-1 of deoxycholate the loss of PI was rapid and extensive: more than 80% was broken down within 10 min. The breakdown of PI was also stimulated by millimolar concentrations of Ca²⁺. The products of breakdown of added PI (purified from soybean callus) in this system were identified from their chromatographic mobilities as 1,2-diacylglycerol, myo-inositol 1-phosphate and myo-inositol 1:2-cyclic monophosphate.Abbreviations DOC deoxycholate - EDTA ethylenedi-aminetetraacetic acid,-acetate - Pi Inorganic phosphate - PI phosphatidylinositol - PS phosphatidylserine - TLC thinlayer chromatography     

d-myoInositol 1:2-cyclic phosphate 2-phosphohydrolase

1. An enzyme in extracts of mammalian tissues catalyses the hydrolysis of d-myoinositol 1:2-cyclic phosphate (an intermediary in the enzymic degradation of phosphatidylinositol) to produce d-myoinositol 1-phosphate. 2. The enantiomorph of the substrate is not attacked. 3. The pH optimum is about 8.1-8.3 and the reaction is stimulated by Mg(2+) ions. 4. Extracts from rat kidney cortex and medulla are very rich sources of the enzyme; brain, testis and small intestine contain intermediary activities, and other tissues contain very small amounts.     

Phosphatidylinositol-cleaving activity in smooth muscle from rat vas deferens

• 1.1. Phosphatidylinositol-cleaving activity was studied in subcellular fractions from smooth muscle of rat vas deferens.
• 2.2. In the presence of calcium ions and deoxycholate most of the endogenous phosphatidylinositol was broken-down in 60 min, whilst the other phospholipids were stable.
• 3.3. The enzymatic activity responsible for this breakdown catalyses a phospholipase C-type cleavage of the glycerol-phosphate bond, the water soluble products from exogenous [³²P]-labelled phosphatidylinositol being d-myoinositol 1:2-cyclic phosphate (702-80%) and d-myoinositol 1-phosphate (202-30%).
• 4.4. Activity was abolished by 1 mM ethanedioxybis(ethylamine)tetra-acetate (EGTA) and in the presence of deoxycholate both the soluble and total particulate fractions showed maximum activity at pH 6.52-6.8. The soluble fraction showed a second peak of activity at pH 5.52-5.8 that was independent of deoxycholate; this was not observed in the particulate fraction.
• 5.5. About two-thirds of the activity was soluble. The remaining activity was particulate, with a preferential concentration in the microsomal fraction.

     

Hormonal regulation of phosphatidylinositol breakdown

Cyclic AMP and Ca2+ are intracellular mediators of hormone action. Catecholamines interact with beta adrenoceptors to activate adenylate cyclase or with alpha 2 adrenoceptors to inhibit adenylate cyclase. Alpha 1 adrenoceptor activation results in elevation of cytosol Ca2+ and an increased breakdown of phosphatidylinositol. In blowfly salivary glands, 5-hydroxytryptamine (5-HT) interacts with beta type receptors resulting in adenylate cyclase activation while alpha type receptors are involved in phosphatidylinositol breakdown and elevation of cytosol Ca2+. The link between Ca2+ mobilization and phosphatidylinositol breakdown remains to be established but breakdown of the receptor-regulated pool of phosphatidylinositol is not secondary to the rise in Ca2+. Direct addition of 5-HT to cell-free homogenates of blowfly salivary glands results in activation of phosphatidylinositol breakdown in the absence of Ca2+. In rat liver plasma membrane preparations, vasopressin increases phosphatidylinositol breakdown in the absence of Ca2+ or cytosol if deoxycholate is present. The data do not indicate whether hormone activation increases the availability of substrate to enzymatic hydrolysis or activates phospholipase C. However, they demonstrate that hormones directly accelerate phosphatidylinositol breakdown.     

THE DISTRIBUTION OF CALCIUM-DEPENDENT PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The properties of Ca2⁺-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in ₃₂P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

A plausible role for a membrane-bound cyclic AMP phosphodiesterase in cellular slime mold chemotaxis.

1. Kinetics of membrane-bound cyclic AMP phosphodiesterase of the cellular slime mold, Dictyostelium discoideum, were studied under two conditions: in the 27 000 times g sediment of cell homogenates (particle-bound phosphodiesterase) and in cell suspensions using external cyclic AMP as a substrate (cell-bound phosphodiesterase). Both methods revealed non-Michaelian kinetics with interaction coefficients less than 1. 2. The membrane-bound phosphodiesterase has a specificity different from that of the cyclic AMP receptor, also present at the cell surface. 3. The membrane-bound enzyme was solubilized by lithium 3, 5-diiodosalicylate and partially purified. In this state the non-linear kinetics were still retained; however, the enzyme was not inhibited by the D. discoideum inhibitor, unlike the cell-bound phosphodiesterase in vivo. This indicates that both enzymes share an inhibitor binding site and that this site is cryptic in the cell-bound state. 4. Production of periodic cyclic AMP pulses by centers, and their relay by other cells, is believed to occur during aggregation. It is suggested that the cell-bound enzyme determines a "time window" significantly smaller than the period of pulsing, and optimizes stimulation of the cyclic AMP receptors in chemotaxis and signal relaying.     

Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of d-myoinositol 1:2-cyclic phosphate 2-phosphohydrolase in rat kidney

1. On subcellular fractionation of rat kidney homogenates by differential and density-gradient centrifugation, the bulk of the inositol 1:2-cyclic phosphate 2-phosphohydrolase activity remains with the alkaline phosphatase activity, suggesting localization in the brush borders of the proximal tubules. 2. Histochemical studies with a medium containing inositol 1:2-cyclic phosphate and Escherichia coli phosphomonoesterase show Gomori staining around the brush borders of the proximal tubules in the outer cortex only. 3. Serial sections across the kidney from cortex perimeter to papilla suggest that the inositol 1:2-cyclic phosphate 2-phosphohydrolase has a limited distribution along the proximal tubule of the nephron, probably being limited to the pars convoluta, whereas the alkaline phosphatase extends along the pars recta.     

Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation.

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.     

Effects of the wasp venom peptide, mastoparan, on a phosphoinositide-specific phospholipase C purified from rabbit brain membranes

The wasp venom peptide, mastoparan (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2), activated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis as catalyzed by a phosphoinositide-specific phospholipase C (PLC-Im) purified from rabbit brain membranes. This activation was found when the molar ratio of mastoparan to PIP2 was less than 1 and when the concentration of PIP2 exceeded 10 microM. PIP2 breakdown was inhibited at both high and low substrate concentrations if the molar ratio of mastoparan to PIP2 was greater than 1. The stimulatory effect of mastoparan correlated with its ability to restrict aggregation of PIP2 into higher order structures (liposomes or mixed deoxycholate/phospholipid micelles) as the concentration of PIP2 was increased to 10 microM or greater. Mastoparan stimulation of PIP2 breakdown required the presence of a higher calcium concentration than was necessary for detection of enzyme activity. Both the stimulatory and inhibitory effects of mastoparan on PIP2 hydrolysis were lost if 2.5 mM deoxycholate was present in the assays. Hydrolysis of phosphatidylinositol (PI) by PLC-Im was inhibited at all concentrations of mastoparan tested. These results show that both PIP2 and PI are suitable substrates for PLC-Im, depending on the physical characteristics of their aggregates in aqueous suspension. An amphiphilic alpha-helix-forming peptide such as mastoparan may modulate phospholipase C activity due to the peptide's interaction with phospholipid substrates.     

Phosphatidylinositol-specific phospholipase C of murine lymphocytes

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.     

An enzymatically coupled assay for rat brain polyphosphoinositide phosphodiesterase in an optimized reaction mixture

Bovine intestinal alkaline phosphatase was found to hydrolyze inositol phosphates many times faster than the monoester phosphate groups of the polyphosphoinositides. A convenient and sensitive in vitro assay for the Ca2+-dependent polyphosphoinositide phosphodiesterase was devised in which inositol trisphosphate released from exogenous phosphatidylinositol 4,5-bisphosphate was hydrolyzed by alkaline phosphatase. The resulting inorganic phosphate was measured by an automated method after solubilization of the reaction mixture with sodium dodecyl sulfate. The phosphodiesterase was maximally stimulated by combining the known positive effects of cetyltrimethylammonium bromide (at the optimum detergent-to-substrate ratio of 2.3), monovalent cations (0.1 M KCl), and Ca2+ (0.5 mM) with the additional enhancement by Triton X-100 (0.2% w/v). Activities obtained for rat brain homogenates and microsomal and cytosol fractions were 126 +/- 3.8 (17), 110 +/- 5.7 (10), and 252 +/- 15.5 (8) nmol X min-1 X mg protein-1 (mean +/- SE for n determinations), respectively.     

Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems

The plasma membrane ATP-phosphohydrolase (ATPase) from red beet (Beta vulgaris L.) storage tissue was solubilized with the zwitterionic detergent Zwittergent 3-14 from a plasma membrane-enriched fraction which was extracted with the anionic detergent, sodium deoxycholate. For both the extraction of extraneous proteins by deoxycholate and the solubilization of active plasma membrane ATPase by Zwittergent 3-14, the optimal concentration of detergent was 0.1% (weight per volume) with a detergent to protein ratio of 1.0 (milligram per milligram). The properties of the solubilized ATPase were found to be similar to the membrane-bound enzyme with respect to pH optimum, substrate specificity, inhibitor sensitivity, and kinetics of K⁺ stimulation. The solubilized ATPase preparation formed a rapidly turning over phosphoenzyme, the breakdown velocity of which was increased in the presence of 50 millimolar KCl. Solubilization with 0.1% Zwittergent 3-14 following extraction with 0.1% deoxycholate resulted in an increase in both ATPase activity and steady state phosphoenzyme level; however, a direct correspondence between the increase in ATPase activity and phosphorylation level did not exist. It is proposed that this discrepancy may be the result of a detergent-mediated modification of kinetic rate constants in the mechanism of the enzyme.     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

Dibucaine,chlorpromazine, and detergents mediate membrane breakdown in potato tuber homogenates

Various strategies were evaluated for their ability to minimize the rate of breakdown of endogenous membrane lipids during cell fractionation studies with potato tubers. Buffering the homogenates at pH 7.5 to 8.0 resulted in significantly lower rates of phosphatidylcholine (PC) hydrolysis than were observed at lower pH values. Several potential inhibitors were added to homogenates to evaluate their ability to inhibit membrane lipid hydrolysis. The addition of bovine serum albumin (1% w/v) inhibited the rate of PC hydrolysis by 50%. The addition of low concentrations (25–100 μM) dibucaine (nupercaine) inhibited PC hydrolysis, but at higher concentrations (1-2 mM) it caused a 5- to 6-fold stimulation. Because dibucaine is a calmodulin antagonist, two other calmodulin antagonists (trifluoperazine and chlorpromazine) were tested and found to exhibit similar patterns of inhibition and stimulation. Similarly, the addition of low concentrations of deoxycholate also inhibited PC hydrolysis and high concentrations stimulated it. These results indicate that the hydrophobic properties of deoxycholate, dibucaine, and other calmodülin antagonists may explain their unusual effects on the rates of PC hydrolysis in potato tuber homogenates. Although the addition of exogenous calcium increased the rate of PC hydrolysis, the addition of calmodulin (bovine brain) had no effect. Other experiments revealed that the addition of 1% bovine serum albumin improved the yield and stability of mitochondrial and microsomal fractions from potato tubers. In constrast, the addition of 100 μM dibucaine caused deleterious effects.     

Characterization of nucleoside diphosphatase in the onion root tip. III. Solubilization and activation of membrane-bound enzyme(s)

The latency of nucleoside diphosphatase (NDPase) in onions root homogenates has been examined by comparing the activation of NDPase activity resulting from detergent treatment with that due to storage of homogenates for several days in the cold. Both detergent treatment and cold storage activated NDPase approximately two-fold. In both cases this activation was paralleled by the loss of enzyme activity from the membrane fractions and its appearance in the supernatants. Electrophoresis of these supernatants revealed an identifical isoenzyme pattern of 5 NDPase bands for both preparations. Enzyme kinetic studies demonstrated that NDPase from the detergent-treated homogenate and the homogenate stored in the cold as well as NDPase from the membrane and supernatant fractions from each of the homogenates all had the same Km value. These data suggest that latency of NDPase is the result of a breakdown of cellular membranes and subsequent release of NDPase. Abbreviations: DOC, deoxycholate; NDPase, nucleoside diphosphatase; IDP, inosine 5'-diphosphate; UDP, uridine 5'-diphosphate; GDP, guanosine 5'-diphosphate.     

Norepinephrine causes alpha 1-adrenergic receptor-mediated decrease of phosphatidylinositol in isolated rat liver plasma membranes supplemented with cytosol

When purified rat liver plasma membranes were incubated with norepinephrine, rat liver cytosol, and Ca2+, the amount of membrane-bound phosphatidylinositol was reduced by up to 50%. The levels of other major membrane phospholipids underwent negligible change. The decrease in phosphatidylinositol levels was not observed if norepinephrine was omitted or cytosol was absent. The disappearance of phospholipid persisted when soluble Ca2+ was depleted by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The decrease was prevented by phentolamine, benextramine, and prazosin, but not by sotalol. The results show that norepinephrine elicits a specific alpha 1-adrenergic receptor-mediated breakdown of phosphatidylinositol in isolated plasma membranes which is dependent on the presence of cytosol.     

Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.