Properties and distribution of phosphatidylinositol-specific phospholipase C in human and horse platelets.
Abstract: Phospholipase C has been studied in homogenates, total particulate and soluble fractions of horse and human platelets. This enzyme, assayed with exogenous L-3-phosphatidyl[14C]inositol, is predominantly localized in the soluble fraction and its distribution parallels that of lactate dehydrogenase. A small percentage of activity present in the particulate fraction seems to be due to contamination with soluble enzyme. Enzyme from horse and human platelets appears identical, having a Km of 0.10-0.15 mM, acid pH optimum (pH 5.5) and showing Ca2+-dependency and weak inhibition by deoxycholate. Analysis of the reaction products shows the formation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate in almost equal amounts. Platelet stimulation with thrombin does not seem to induce association of the cytosolic activity to the membranes. The cytosolic activity is not affected by pretreatment of the intact platelets with prostacyclin or thrombin. Degradation of phosphatidylinositol present in a membrane fraction isolated from platelets by cytosolic phospholipase C requires addition of deoxycholate. Our information suggests that the degradation of phosphatidylinositol in stimulated platelets is mainly achieved by exposure of the substrate to the cytosolic enzyme and by an increase of the free Ca2+ concentration needed for optimal phospholipase C activity.
Publication Date: 1983-07-12 PubMed ID: 6860706DOI: 10.1016/0005-2760(83)90131-5Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The paper deals with the investigation of an enzyme known as Phospholipase C in horse and human blood platelets, focusing on its properties, distribution, and activities.
Overview of Research
In this research, scientists studied Phospholipase C in horse and human platelets. The enzyme was tested with exogenous L-3-phosphatidyl[14C]inositol and was mostly found in the soluble fraction, much like lactate dehydrogenase. Most notably, they found that this enzyme operation is not influenced by prostacyclin or thrombin pre-exposure.
- The study covers the properties and distribution of Phospholipase C in different components, such as homogenates, total particulate, and soluble parts of the platelets in both humans and horses.
- For the study, the enzyme was assessed using exogenous L-3-phosphatidyl[14C]inositol. It was primarily located in the soluble part, and its distribution was parallel to that of enzyme lactate dehydrogenase (LDH).
- A small amount of enzymatic activity was noted in the particulate fraction, which researchers suggested could likely be due to contamination from the soluble enzyme component.
Phospholipase C from Human and Horse Platelets
The enzyme phospholipase C from horse and human platelets seem to share identical properties.
- Both enzymes have the same Km value of 0.10-0.15 mM, indicating the same binding affinity for their molecules.
- Both have an optimal activity at an acid pH of 5.5 and are calcium-dependent. This means they function best under slightly acidic conditions and require calcium to be present for optimal enzymatic activity.
- They both showed weak inhibition to deoxycholate, which inhibits the overall enzyme functionality.
- When analyzed, the products of the reaction were mainly myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, roughly in equal proportions.
Effects from Stimuli on Phospholipase C
In this study, the researchers observed how the enzyme’s functionality changed when influenced by stimuli like thrombin and prostacyclin.
- Stimulating platelets with thrombin didn’t seem to increase the association of the enzyme’s activity from the cytosol (liquid part inside cells) to the membranes.
- This cytosolic activity of the phospholipase C also didn’t change when platelets were pretreated with prostacyclin or thrombin.
- The enzyme’s ability to degrade phosphatidylinositol (a type of fat molecule in cell membranes) in membrane fractions required the addition of deoxycholate.
- The degradation of phosphatidylinositol in activated platelets is thought to be mainly due to the exposure of the substrate to the cytosolic enzyme, along with an increase in free calcium concentration – vital for optimal phospholipase C activity.
Cite This Article
APA
Siess W, Lapetina EG.
(1983).
Properties and distribution of phosphatidylinositol-specific phospholipase C in human and horse platelets.
Biochim Biophys Acta, 752(2), 329-338.
https://doi.org/10.1016/0005-2760(83)90131-5 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Blood Platelets / enzymology
- Cell Membrane / enzymology
- Chemical Phenomena
- Chemistry
- Cytosol / enzymology
- Horses
- Humans
- L-Lactate Dehydrogenase / blood
- Phosphatidylinositols / blood
- Phospholipases / blood
- Solubility
- Species Specificity
- Subcellular Fractions / enzymology
- Type C Phospholipases / blood
Citations
This article has been cited 15 times.- Tate BF, Schaller GE, Sussman MR, Crain RC. Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa.. Plant Physiol 1989 Dec;91(4):1275-9.
- Ponzoni M, Cornaglia-Ferraris P. Interferon-gamma-stimulated and GTP-binding-proteins-mediated phospholipase A2 activation in human neuroblasts.. Biochem J 1993 Sep 15;294 ( Pt 3)(Pt 3):893-8.
- König B, König W. The role of the phosphatidylinositol turnover in 12-hydroxyeicosatetraenoic acid generation from human platelets by Escherichia coli alpha-haemolysin, thrombin and fluoride.. Immunology 1993 Dec;80(4):633-9.
- Mauco G, Dangelmaier CA, Smith JB. Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.. Biochem J 1984 Dec 15;224(3):933-40.
- Ritter JM. Prostanoid synthesis by aortic rings in human blood: selective increase of prostacyclin mediated by a serum factor.. Br J Pharmacol 1984 Oct;83(2):409-18.
- Lee KY, Ryu SH, Suh PG, Choi WC, Rhee SG. Phospholipase C associated with particulate fractions of bovine brain.. Proc Natl Acad Sci U S A 1987 Aug;84(16):5540-4.
- Ritter JM, Frazer CE, Taylor GW. pH-dependent stimulation by Ca2+ of prostacyclin synthesis in rat aortic rings: effects of drugs and inorganic ions.. Br J Pharmacol 1987 Jun;91(2):439-46.
- Moscat G, Moreno F, Iglesias S, Garcia-Barreno P, Municio AM. Ionophore A23187 induces a refractory state in thrombin-activated release of inositol phosphates.. Biochem J 1986 Sep 15;238(3):709-14.
- Burch RM, Luini A, Axelrod J. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells.. Proc Natl Acad Sci U S A 1986 Oct;83(19):7201-5.
- Banno Y, Nozawa Y. Characterization of partially purified phospholipase C from human platelet membranes.. Biochem J 1987 Nov 15;248(1):95-101.
- Nunn DL, Watson SP. A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.. Biochem J 1987 May 1;243(3):809-13.
- Manne V, Kung HF. Characterization of phosphoinositide-specific phospholipase C from human platelets.. Biochem J 1987 May 1;243(3):763-71.
- Hrbolich JK, Culty M, Haslam RJ. Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5'-[gamma-thio]triphosphate and by thrombin in the presence of GTP.. Biochem J 1987 Apr 15;243(2):457-65.
- Gusovsky F, Hollingsworth EB, Daly JW. Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions.. Proc Natl Acad Sci U S A 1986 May;83(9):3003-7.
- Fluri GS, Rüdisüli A, Willi M, Rohr S, Weingart R. Effects of arachidonic acid on the gap junctions of neonatal rat heart cells.. Pflugers Arch 1990 Oct;417(2):149-56.
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