INTERFACIAL BINDING OF HUMAN GASTRIC LIPASE TO LIPID MONOLAYERS, MEASURED WITH AN ELISA

被引:28
作者
AOUBALA, M [1 ]
IVANOVA, M [1 ]
DOUCHET, I [1 ]
DECARO, A [1 ]
VERGER, R [1 ]
机构
[1] CNRS, LIPOLYSE ENZYMAT LAB, IFRC, UPR 9025, F-13402 MARSEILLE 20, FRANCE
关键词
D O I
10.1021/bi00034a011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
TWO sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin-peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN . m(-1) were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mu mol/(min . mg of enzyme)]. At a given Lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers. Given the low tensioactivity of the mAbs of the IgG isotype [Ivanova et al. (1993) Colloids Surf. BI, 17-22], we also investigated the effects of five anti-HGL mAbs (mAbs 4-3, 25-4, 35-2, 83-15, and 218-13) on the catalytic activity as well as on the interfacial binding of HGL to lipid/water interfaces. Four out of these five mAbs (mAbs 4-3, 25-4, 35-2, and 83-15) were found to significantly reduce the lipolytic activity of HGL. Moreover, three of the four inhibitory mAbs (mAbs 4-3, 25-4, and 35-2) were found to reduce the specific activity of HGL, while mAb 83-15 had no effect on the specific activity. These results clearly indicate that the latter mAb (83-15) complexed with HGL mainly affects the binding of the enzyme to the lipid/water interface, while the other three inhibitory mAbs (mAbs 4-3, 25-4, and 35-2) affect both the binding and the catalytic steps of HGL.
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页码:10786 / 10793
页数:8
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