Kume T, Taguchi R, Tomita M, Tokuyama S, Morizawa K, Nakachi O, Hirano J, Ikezawa H
Faculty of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Chem Pharm Bull (Tokyo). 1992 Aug;40(8):2133-7. doi: 10.1248/cpb.40.2133.
The properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail. The enzyme was extremely thermostable in 0.1% bovine serum albumin and retained 73% of its activity at 100 degrees C for 10 min, while it was labile in the absence of albumin. The enzymatic activity was inhibited by HgCl2 or p-chloromercuriphenylsulfonic acid and restored by dithiothreitol. The kinetic parameters (Km and Vmax) of PI-PLC were determined for the mixed micelle of yeast phosphatidylinositol (PI)/Triton X-100 or sodium deoxycholate. Four PIs having different acyl chains: dilauroylphosphatidylinositol (DLPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI) and dioleoylphosphatidylinositol (DOPI) were synthesized from yeast PI through the processes of deacylation and reacylation, identified by infrared (IR) and Fourier transform nuclear magnetic resonance (FT-NMR) spectra, and subjected to the action of PI-PLC. All the synthetic PIs were hydrolyzed by this enzyme, with DLPI and DMPI being the best substrates. PI-PLC did not catalyze the hydrolysis of the phosphatidylnucleosides 5'-phosphatidylcytidine, 5'-phosphatidyluridine, 5'-phosphatidylthymidine, 5'-phosphatidyladenosine and 5'-phosphatidyl-2'-deoxyadenosine.
对苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶C（PI-PLC）的性质进行了详细研究。该酶在0.1%牛血清白蛋白中具有极高的热稳定性，在100℃下保温10分钟后仍保留73%的活性，而在无白蛋白的情况下则不稳定。酶活性受到HgCl2或对氯汞苯磺酸的抑制，并可被二硫苏糖醇恢复。针对酵母磷脂酰肌醇（PI）/ Triton X-100或脱氧胆酸钠的混合胶束测定了PI-PLC的动力学参数（Km和Vmax）。通过脱酰基和再酰化过程从酵母PI合成了四种具有不同酰基链的PI：二月桂酰磷脂酰肌醇（DLPI）、二肉豆蔻酰磷脂酰肌醇（DMPI）、二棕榈酰磷脂酰肌醇（DPPI）和二油酰磷脂酰肌醇（DOPI），通过红外（IR）和傅里叶变换核磁共振（FT-NMR）光谱进行鉴定，并使其受到PI-PLC的作用。所有合成的PI均被该酶水解，其中DLPI和DMPI是最佳底物。PI-PLC不催化磷脂酰核苷5'-磷脂酰胞苷、5'-磷脂酰尿苷、5'-磷脂酰胸苷、5'-磷脂酰腺苷和5'-磷脂酰-2'-脱氧腺苷的水解。
