{"title":"用硫代磷酸盐底物类似物连续分光光度法测定哺乳动物磷酸肌醇特异性磷脂酶Cδ1","authors":"H.Stewart Hendrickson","doi":"10.1016/S0005-2760(98)00025-3","DOIUrl":null,"url":null,"abstract":"<div><p>1,2-Dimyristoyloxypropane-3-thiophospho(1<span>d</span>-1-<em>myo</em>-inositol) (<span>d</span>-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a <em>Δ</em>(1–132) deletion mutant of mammalian PI-PLC<em>δ</em><sub>1</sub> is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca<sup>2+</sup>, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk <em>K</em><sub>m</sub> for PI-PLC with <span>d</span>-thio-DMPI under these conditions is about 6 <em>μ</em>M. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.</p></div>","PeriodicalId":100162,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1998-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00025-3","citationCount":"3","resultStr":"{\"title\":\"Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cδ1 with a thiophosphate substrate analog\",\"authors\":\"H.Stewart Hendrickson\",\"doi\":\"10.1016/S0005-2760(98)00025-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>1,2-Dimyristoyloxypropane-3-thiophospho(1<span>d</span>-1-<em>myo</em>-inositol) (<span>d</span>-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a <em>Δ</em>(1–132) deletion mutant of mammalian PI-PLC<em>δ</em><sub>1</sub> is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca<sup>2+</sup>, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk <em>K</em><sub>m</sub> for PI-PLC with <span>d</span>-thio-DMPI under these conditions is about 6 <em>μ</em>M. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.</p></div>\",\"PeriodicalId\":100162,\"journal\":{\"name\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1998-05-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00025-3\",\"citationCount\":\"3\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0005276098000253\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0005276098000253","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cδ1 with a thiophosphate substrate analog
1,2-Dimyristoyloxypropane-3-thiophospho(1d-1-myo-inositol) (d-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a Δ(1–132) deletion mutant of mammalian PI-PLCδ1 is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca2+, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk Km for PI-PLC with d-thio-DMPI under these conditions is about 6 μM. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.
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