Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells
| Autor: | Dominique Lafont, Frédéric Carrière, Cécilia Eydoux, Josiane De Caro, Alain De Caro, Paul Boullanger, René Laugier, Francine Ferrato |
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| Přispěvatelé: | Centre National de la Recherche Scientifique (CNRS), Laboratoire Enzymologie Interfaciale et de Physiologie de la Lipolyse - UPR9025 (EIPL), Université de la Méditerranée - Aix-Marseille 2-Université de Provence - Aix-Marseille 1-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2007 |
| Předmět: |
Surface Properties
[SDV]Life Sciences [q-bio] medicine.medical_treatment QD415-436 Phospholipase Biology Biochemistry Paraoxon Pichia Glycerides Pichia pastoris Lactones chemistry.chemical_compound Endocrinology Phosphatidylcholine Pressure medicine Humans Colipases Lipase ComputingMilieux_MISCELLANEOUS Orlistat chemistry.chemical_classification Taurodeoxycholic Acid Protease Molecular mass galactolipase Galactolipids Cell Biology Hydrogen-Ion Concentration biology.organism_classification inhibition Recombinant Proteins Enzyme chemistry Phospholipases biology.protein galactolipid Carboxylic Ester Hydrolases constitutive expression lid domain |
| Zdroj: | Journal of Lipid Research Journal of Lipid Research, 2007, 48 (7), pp.1539-1549. ⟨10.1194/jlr.M600486-JLR200⟩ Journal of Lipid Research, Vol 48, Iss 7, Pp 1539-1549 (2007) |
| ISSN: | 0022-2275 |
| DOI: | 10.1194/jlr.m600486-jlr200 |
| Popis: | Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5–7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media. |
| Databáze: | OpenAIRE |
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