INACTIVATION OF PANCREATIC LIPASES BY AMPHIPHILIC REAGENTS 5-(DODECYLDITHIO)-2-NITROBENZOIC ACID AND TETRAHYDROLIPSTATIN - DEPENDENCE UPON PARTITIONING BETWEEN MICELLAR AND OIL PHASES

被引:56
作者
CUDREY, C
VANTILBEURGH, H
GARGOURI, Y
VERGER, R
机构
[1] CNRS,CTR BIOCHIM & BIOL MOLEC,ERS 26,GDR 1000,31 CHEMIN J AIGUIER,F-13402 MARSEILLE 20,FRANCE
[2] FAC MED NORD,CNRS,LCCMB,F-13916 MARSEILLE 20,FRANCE
[3] ECOLE NATL INGN SFAX,BIOCHIM LAB,3038 SFAX,TUNISIA
关键词
D O I
10.1021/bi00213a008
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
We have reported previously that Cys103 (SH(II)) of human pancreatic lipase (HPL), unlike the nonessential Cys181 (SH(I)), was buried and inaccessible to classical water-soluble sulfhydryl reagents. The lipolytic activity of HPL was lost after the labeling of the above two SH groups with the amphiphilic sulfhydryl reagent, 5-(dodecyldithio)-2-nitrobenzoic acid (C12-TNB), suggesting that the SH(II) residue may play an important role in the hydrolytic process [Gargouri, Y., Cudrey, C., Medjoub, H., & Verger, R. (1992) Eur. J. Biochem. 204, 1063-1067]. For the present experiments, we selected dog pancreatic lipase (DPL), purifying it for the first time, and recombinant guinea pig pancreatic lipase (r-GPL), which both contain a buried SH(II) group but no accessible SH(I) group. The single SH(II) of DPL and r-GPL reacted only with the amphiphilic SH reagent (C12-TNB), and its labeling was correlated with a rapid lipase inactivation. Although it is spatially remote from the catalytic triad, the SH(II) group of pancreatic lipases, when chemically labeled, was found to be responsible for the loss of their lipolytic activity. The presence of a bulky dodecyl chain, linked by a disulfide bond to the SH(II), may have prevented the critical beta-5 loop (residues 76-85) movement by steric hindrance and consequently disturbed the formation of the oxyanion hole. Thus, pancreatic lipase inactivation by the amphiphilic sulfhydryl reagent can be said to be due to the prevention of a productive induced fit. Tetrahydrolipstatin (THL) is an amphiphilic inactivator reacting with the essential serine of the lipase active site. Comparisons of the partitioning between the micellar and oil phases of THL and C12-TNB were made in order to estimate the hydrophobic-lipophilic balance of each inactivator. Its preferential micellar partitioning makes C12-TNB inefficient in the presence of NaTDC, whereas THL is mostly associated with the triglyceride phase even in the presence of bile salts. The latter physicochemical property is probably a requirement for prototypic lipase inactivators to be effective under physiological conditions, i.e., in the presence of bile and lipids.
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页码:13800 / 13808
页数:9
相关论文
共 33 条
  • [21] LARGE SPECTRAL CHANGES ACCOMPANY THE CONFORMATIONAL TRANSITION OF HUMAN PANCREATIC LIPASE INDUCED BY ACYLATION WITH THE INHIBITOR TETRAHYDROLIPSTATIN
    LUTHIPENG, Q
    WINKLER, FK
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1992, 205 (01): : 383 - 390
  • [22] LUTHIPENG Q, 1992, FEBS LETT, V288, P11
  • [23] MEIER MK, 1989, INT J OBES S1, V13, P134
  • [24] INACTIVATION OF GASTRIC AND PANCREATIC LIPASES BY DIETHYL PARA-NITROPHENYL PHOSPHATE
    MOREAU, H
    MOULIN, A
    GARGOURI, Y
    NOEL, JP
    VERGER, R
    [J]. BIOCHEMISTRY, 1991, 30 (04) : 1037 - 1041
  • [25] INTERFACE-MEDIATED INACTIVATION OF PANCREATIC LIPASE BY A WATER-REACTIVE COMPOUND - 2-SULFOBENZOIC CYCLIC ANHYDRIDE
    MOULIN, A
    FOURNERON, JD
    PIERONI, G
    VERGER, R
    [J]. BIOCHEMISTRY, 1989, 28 (15) : 6340 - 6346
  • [26] INACTIVATION OF PANCREATIC AND GASTRIC LIPASES BY TETRAHYDROLIPSTATIN AND ALKYL-DITHIO-5-(2-NITROBENZOIC ACID) - A KINETIC-STUDY WITH 1,2-DIDECANOYL-SN-GLYCEROL MONOLAYERS
    RANSAC, S
    GARGOURI, Y
    MOREAU, H
    VERGER, R
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1991, 202 (02): : 395 - 400
  • [27] INHIBITION OF PANCREATIC LIPASE BY MIXED MICELLES OF DIETHYL PARA-NITROPHENYL PHOSPHATE AND BILE-SALTS
    ROUARD, M
    SARI, H
    NURIT, S
    ENTRESSANGLES, B
    DESNUELLE, P
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA, 1978, 530 (02) : 227 - 235
  • [28] ACTION DE LA LIPASE PANCREATIQUE SUR LES ESTERS EN EMULSION
    SARDA, L
    DESNUELLE, P
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA, 1958, 30 (03) : 513 - 521
  • [29] INTERACTION OF PHOSPHOLIPASE-A2 WITH MICELLAR INTERFACES - ROLE OF N-TERMINAL REGION
    VANDAMMIERAS, MCE
    SLOTBOOM, AJ
    PIETERSON, WA
    HAAS, GHD
    [J]. BIOCHEMISTRY, 1975, 14 (25) : 5387 - 5394
  • [30] INTERFACIAL ACTIVATION OF THE LIPASE PROCOLIPASE COMPLEX BY MIXED MICELLES REVEALED BY X-RAY CRYSTALLOGRAPHY
    VANTILBEURGH, H
    EGLOFF, MP
    MARTINEZ, C
    RUGANI, N
    VERGER, R
    CAMBILLAU, C
    [J]. NATURE, 1993, 362 (6423) : 814 - 820