Liposome fusion catalytically induced by phospholipase C

Large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (50:25:25 mole ratio) were treated with phospholipase C. The early stages of phospholipid cleavage are accompanied by mixing of bilayer lipids (monitored by dequenching of octadecylrhodamine fluorescence) and leakage-free mixing of vesicle contents [measured by using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX)]. These results are interpreted in terms of vesicle fusion induced by the catalytic activity of phospholipase C. The use of sonicated unilamellar vesicles decreases the lag time, but does not modify the amplitude, of the fusion process. The presence of both phosphatidylethanolamine and cholesterol appears to be essential for measurable fusion effects to occur with low levels of phospholipid hydrolysis. Optimal fusion rates are observed with about 10-20 enzyme molecules per large unilamellar vesicle. This system of catalytically induced liposome fusion may be of relevance for the interpretation of physiological membrane fusion processes.     

Rapamycin partially prevents insulin resistance induced by chronic insulin treatment

Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes.     

Stimulation of phospholipase C activity by insulin is mediated by both isotypes of the human insulin receptor.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.     

Acid phosphatase from maize scutellum: Negative cooperativity suppression by glucose

Acid phosphatase purified from maize scutellum, upon acylation with succinic anhydride, still shows negative co-operativity for the hydrolysis of glucose-6-phosphate at pH 5.4. This phenomenon is abolished by glucose, for both native and succinylated enzymes, through stimulation of the initial velocities at sub-optimal substrate concentrations. However, negative co-operativity for the enzymatic hydrolysis of p-nitrophenylphosphate at pH 5.4 is suppressed only at high concentrations of glucose. Furthermore, the hydrolysis of p-nitrophenylphosphate is noncompetitively inhibited (low affinity form of the enzyme molecule) by glucose, which suggests the existence of different substrate binding sites.     

Intraperitoneal fluid accumulation induced by Clostridium perfringens alpha-toxin (phospholipase C)

We report that the intraperitoneal injection of Clostridium perfringens alpha-toxin into mice induces ascites. This phenomenon was monitored by measuring fluid volume and analyzing hematologic data. The mouse toxicity test provides a simple and useful model for examining C. perfringens alpha-toxin-induced vascular permeability.     

TPA inhibits insulin stimulated PIP hydrolysis in fat cell membranes: evidence for modulation of insulin dependent phospholipase C by proteinkinase C

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.     

The activation of phosphatidylinositol-specific phospholipase C by insulin in mammary epithelial cells of lactating mouse

We have previously demonstrated in vitro that, in the endoplasmic reticulum and Golgi apparatus of mammary epithelial cells of lactating and pregnant mice, inositol 1,4,5-trisphosphate releases Ca2+ that has been stored in these organelles. In this study, we examined whether insulin and prolactin, essential for the growth of mammary gland and for lactation, influenced the activity of phosphatidylinositol-specific phospholipase C in mammary cells. In the plasma membrane fraction of mammary epithelial cells of the DDY mouse strain 5 days after the start of lactation after the first pregnancy, and with phosphatidylinositol as substrate, it was shown that the activity of phospholipase C was enhanced by about four times in the presence of insulin compared with the control. Such enhancement was not found in the membrane fraction treated with prolactin.     

Negative cooperativity in Root-effect hemoglobins: role of heterogeneity

In some animals, the oxygen transport capacity of blood decreaseswhen pH is lowered, yielding oxygen binding curves with Hill-coefficientssmaller than unity. This so-called Root effect is observed inseveral fishes and is important for creating large oxygen partialpressures locally, for example in the swim bladder. While thereis general agreement on the physiological advantages of thiseffect, its molecular basis remains ambiguous. Various studiesshow that isoforms of hemoglobins usually are present in thehemolymph, when the Root effect is observed. Here, we show thatin such a case the mixture of these isoforms can exhibit apparentnegative cooperativity, although each component taken separatelycan be described by the MWC model. In other cases, isolatedisoforms exhibit true negative cooperativity. The well establishedMWC model describes many cooperative phenomena of enzymes andrespiratory proteins but is not capable of describing negativecooperativity. In order to model negative cooperativity withina single molecular species a decoupling model might be employed,as pointed out previously. However, simulations show that itis not mandatory to have species with negative cooperativity,in order to obtain the binding curves typically seen for wholeblood. These two aspects of the Root effect will be discussedon the basis of data from the literature.     

Catalytic cooperativity induced by SH1 labeling of myosin filaments

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of ornithine decarboxylase by treatment of guinea pig lymphocytes with phospholipase C

Treatment of guinea pig lymphocytes with Clostridium perfringens phospholipase C but not with Naja naja snake venom phospholipase A2 increased ornithine decarboxylase activity. The increase in ornithine decarboxylase activity was suppressed by actinomycin D or cycloheximide, suggesting that de novo syntheses of RNA and protein are necessary for the increase in the enzyme activity. These results suggest that the activation of phospholipase C rather than that of phospholipase A2 is responsible for induction of ornithine decarboxylase during lymphocyte transformation.     

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the G_q subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the G_q subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.     

The increase of phospholipase C activity induced by orotic acid is unrelated to lipid peroxidation.

Liver phospholipase-C (PL-C) activity proved to be promptly modified in rats fed with an orotic acid (OA) supplemented diet; an increased of PL-C basal activity was demonstrated after 2 days of diet. In the present work the possible involvement of lipid peroxidation was investigated, since 4-hydroxynonenal (HNE) and 4-hydroxyoctenal (HOE), two end-products of lipid peroxidation, have been shown to induce a strong stimulation of hepatic PL-C. Membrane-bound PL-C activity was evaluated together with the rate of TBArs production by liver homogenates obtained from rats fed with a diet containing 1% OA for 2 and 5 days. PL-C activity was measured by following the rate of formation of Ins-P3 from labelled PtdIns-P2 added to isolated liver membranes. TBArs production was unchanged in the livers of rats fed the OA diet, while basal and GTPgammaS-stimulated PL-C activity increased; furthermore PL-C stimulation by bombesin was deeply impaired by OA.