Wertz P W, Downing D T
Marshall Dermatology Research Laboratories, University of Iowa College of Medicine, Iowa City 52242.
Biochim Biophys Acta. 1989 Feb 6;1001(2):115-9. doi: 10.1016/0005-2760(89)90136-7.
One of the final steps in epidermal differentiation is the conversion of glucosylceramides to ceramides, which presumably is mediated by a beta-glucosidase activity. In the present manuscript, it is demonstrated that pig epidermis contains beta-glucosidase activity which is 3.3-times greater than alpha-glucosidase and 5-times greater than beta-galactosidase. This beta-glucosidase was found to be maximally active between pH 3.0 and essentially inactive at pH 9.0. In a standard assay, a disk of epidermis (8 mg dry weight) was submerged in 1 ml of 50 mM acetate buffer (pH 4.7) containing 150 mM NaCl and 15 mM p-nitrophenyl-beta-D-glucopyranoside at room temperature. Reaction was stopped by addition of 4 ml of 100 mM (pH 9.0) borate buffer and the supernatant was transferred to a separate tube. The nitrophenylate anion was then measured spectrophotometrically at a wavelength of 405 nm. Under these conditions, product formation was linear for at least 90 min and an apparent Km of 244 microM was estimated for the synthetic substrate. When the amount of epidermis in the assay was varied, the formation of product per unit of time remained proportional to the amount of epidermis. The level of beta-glucosidase activity was enhanced slightly by the inclusion of sodium taurocholate.
表皮分化的最后步骤之一是将葡萄糖神经酰胺转化为神经酰胺，这可能是由β-葡萄糖苷酶活性介导的。在本论文中，已证明猪表皮含有β-葡萄糖苷酶活性，其活性比α-葡萄糖苷酶高3.3倍，比β-半乳糖苷酶高5倍。发现这种β-葡萄糖苷酶在pH 3.0时活性最高，在pH 9.0时基本无活性。在标准测定中，将一片表皮（干重8毫克）在室温下浸入1毫升含有150毫摩尔氯化钠和15毫摩尔对硝基苯基-β-D-吡喃葡萄糖苷的50毫摩尔醋酸盐缓冲液（pH 4.7）中。通过加入4毫升100毫摩尔（pH 9.0）硼酸盐缓冲液终止反应，并将上清液转移至另一管中。然后在405纳米波长下用分光光度法测量硝基苯阴离子。在这些条件下，产物形成至少90分钟呈线性，并且估计合成底物的表观Km为244微摩尔。当测定中表皮的量变化时，单位时间内产物的形成与表皮的量保持成比例。加入牛磺胆酸钠会使β-葡萄糖苷酶活性水平略有提高。
