Jacobson P W, Wiesenfeld P W, Gallo L L, Tate R L, Osborne J C
Department of Biochemistry, George Washington University Medical Center, Washington, D.C. 20037.
J Biol Chem. 1990 Jan 5;265(1):515-21.
Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.
胰腺胆固醇酯酶（CEase）调节膳食胆固醇的吸收，在三羟基胆汁盐存在时被激活，而在单羟基胆汁盐存在时保持无活性。大鼠胰腺中的CEase已通过硫酸铵沉淀、羟基磷灰石色谱以及在串联的Sephacryl S - 200/S - 300柱上进行凝胶过滤进行纯化，其均一性和相对分子质量（Mr，55,418 ± 288）已通过沉降平衡离心法测定。通过圆二色光谱偏振法研究了三羟基、二羟基和单羟基胆汁盐对缓冲溶液和体外测定系统中纯化酶构象的影响。溶液中酶的CD光谱显示出一种暗示α - 螺旋性的曲线形状，但低平均残基椭圆率（MRE）值可能表明β - 转角有重要贡献。三羟基胆汁盐胆酸钠在胆汁盐浓度为70 - 100 nM时会导致溶液中酶的负MRE值降低，在高达1 mM的浓度下没有进一步的光谱变化。高于1 μM的胆酸钠浓度在活性测定条件下也会导致酶的负MRE值增加，一旦反应达到平衡，该值会恢复到其原始值。后一种变化被解释为底物与酶结合后诱导的，反应达到平衡后底物部分耗尽。二羟基胆汁盐脱氧胆酸钠在缓冲溶液和活性测定条件下会导致CEase的MRE值不稳定地短暂增加和减少。这些变化取决于胆汁盐浓度，可能反映了蛋白质的自缔合。单羟基胆汁盐牛磺石胆酸钠不影响CEase的CD光谱，二羟基和单羟基胆汁盐均不激活该酶。
