ZymographyによるLipase, Lipoprotein Lipase (LPL) の研究
第三報, LPL活性に及ぼすヘパリン, プロタミンおよびイオン濃度による影響
1983 年 12 巻 1 号 p. 24-29
詳細
1983 年 12 巻 1 号 p. 24-29
A new method of lipase-electrophoresis was developed and two types of LPL (lipoprotein lipase) were separated from tissue-extracts by zymography as previously reported 7-8). The one was namedb-LPL. lt was binding LPL with heparh-like glycosaminoglycan whlch might be existing on the cell surface. Another type was named f-LPL. It was free-LPL, which was not bound with glycosaminoglycan. ln the present paper, a method of fractionated-determination of these two types of LPL have been established utilizing their different properties.
LPL was purified from postheparin plasma by heparin affinity column chromatographic technique. The fractionated b-LPL and f-LPL were prepared respectively by addition of heparin (25u/ml) or protamine sulfate (100μg/ml).Each LPL were shown different enzymatic activity one another at different ionic strength in assay mixture. Namely, the b-LPL had not enzymatic activity at low ionic strength, but was shown a maximal activity at higher ionic strength.On the contrary, f-LPL type was shown a maximal activity at lower ionic strenght.
On the other hand, several lipases other than two types of LPL were contained in clude extract from tissue, however, two types of LPL are distinguishedl from these lipases by: dependence for activity on a serum cofactor and inhibitiion by 1 mol/l NaCl concentrations. Furthermore, the serum activated-lipase which was determined at low ionic strength in assay mixture was not b-LPL, but f-LPL type. Since the b-LPL type was converted to f-LPL type by addition of protamine sulfate to enzyme mixture, the serum activated-lipase (total LPL activity) was equal to sum of f-LPL and b-LPL activity. The b-LPL activity, consequently, enable to be estimated by subtracting the f-LPL activity determined at low ionic strength from total LPL activity.From these results, it was emphasized to benecessary to Pay much attention to ionic strength in assay mixture for determination of LPL activity.
