title: Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.
abstract: A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.
authors.affiliation: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark.
authors.forename: Kira Løw
authors.initials: KL
authors.lastname: Larsen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark.
authors.forename: Rokhsana J
authors.initials: RJ
authors.lastname: Andersen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark. Electronic address: jebk@novozymes.com.
authors.forename: Jesper
authors.initials: J
authors.lastname: Brask
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D010407:Penicillium / Q000201:enzymology; D010504:Periodic Acid / Q000737:chemistry; D010712:Phosphatidic Acids / Q000138:chemical synthesis / Q000378:metabolism; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000032:analysis* / Q000378:metabolism
chemical_list: C033523:4-nitrophenylphosphorylcholine; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010504:Periodic Acid; D010767:Phosphorylcholine; C009288:metaperiodate; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase
keywords: Enzyme kinetics; Periodate-coupled assay; Phospholipase C (PLC); Sphingomyelinase; Synthetic assay substrate