Venkatesh B K, Achar Raghu Ram, Sharanappa P, Priya B S, Swamy S Nanjunda
Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Research Foundation, JSS Technical Institutions Campus, Mysore, India.
Department of Studies in Biosciences, University of Mysore, Hemagangothri, Hassan, India.
Pharmacogn Mag. 2015 Oct;11(Suppl 3):S457-61. doi: 10.4103/0973-1296.168987.
Kollamalayaali tribes of South India use latex of Maclura spinosa for milk curdling. This action is implicated to proteases which exhibit strong pharmacological potential in retardation of blood flow and acceleration of wound healing.
To validate the presence of a proteolytic enzyme(s) in Maclura spinosa latex (MSL), and to investigate their probable role in hemostasis.
Processed latex was examined for proteolytic and hemostatic activity using casein and human fibrinogen as substrates, respectively. Caseinoltyic activity was compared with two standard proteases viz., trypsin I and trypsin II. Effect of various standard protease inhibitors viz., iodoacetic acid (IAA), phenylmethylsulfonyl fluoride (PMSF), ethylene glycol tetraacetic acid, and ethylenediaminetetraacetic acid on both caseinolytic and fibrinogenolytic activities were examined. Electrophoretogram of fibrinogenolytic assays were subjected to densitometric analysis.
Proteolytic action of MSL was found to be highly efficient over trypsin I and trypsin II in dose-dependent caseinolytic activity (P < 0.05; specific activity of 1,080 units/mg protein). The Aα and Bβ bands of human fibrinogen were readily cleaved by MSL (for 1 μg crude protein and 30 min of incubation time). Furthermore, MSL cleaved γ subunit in dose- and time-dependent manner. Quantitative correlation of these results was obtained by densitometric analysis. The caseinolytic activity of MSL was inhibited by IAA, PMSF. While, only PMSF inhibited fibrinogenolytic activity.
MSL contains proteolytic enzymes belonging to two distinct superfamilies viz., serine protease and cysteine proteases. The fibrinogenolytic activity of MSL is restricted to serine proteases only. The study extrapolates the use of M. spinosa latex from milk curdling to hemostasis.
Proteolytic enzymes present in latex of Maclura spinosa can be assigned to two different protease superfamilies viz., serine protease and cysteine protease as revealed by the inhibitory studies of caseinolytic activity. Among them, only serine protease can be considered as hemostatically significant as inhibition of fibrinogenolytic action of Maclura spinosa latex protease is shown only by PMSF, a serine protease-specific inhibitor. Abbreviations used: MSL: Maclura spinos Latex, IAA: Iodo Acetic Acid, EDTA: Ethylene Diamine Tetra Acetic Acid, EGTA: Ethylene glycol tetra acetic acid, PMSF: Phenyl methyl sulphonyl fluoride.
印度南部的科拉马拉亚利部落使用无刺柘树的乳胶来使牛奶凝固。这种作用与蛋白酶有关，这些蛋白酶在减缓血流和加速伤口愈合方面具有强大的药理潜力。
验证无刺柘树乳胶（MSL）中是否存在蛋白水解酶，并研究它们在止血中的可能作用。
分别以酪蛋白和人纤维蛋白原为底物，检测处理后的乳胶的蛋白水解活性和止血活性。将酪蛋白水解活性与两种标准蛋白酶即胰蛋白酶I和胰蛋白酶II进行比较。研究了各种标准蛋白酶抑制剂即碘乙酸（IAA）、苯甲基磺酰氟（PMSF）、乙二醇四乙酸和乙二胺四乙酸对酪蛋白水解活性和纤维蛋白原水解活性的影响。对纤维蛋白原水解试验的电泳图谱进行光密度分析。
在剂量依赖性酪蛋白水解活性方面，发现MSL的蛋白水解作用比胰蛋白酶I和胰蛋白酶II高效得多（P < 0.05；比活性为1,080单位/毫克蛋白质）。人纤维蛋白原的Aα和Bβ条带很容易被MSL切割（1微克粗蛋白，孵育30分钟）。此外，MSL以剂量和时间依赖性方式切割γ亚基。通过光密度分析获得了这些结果的定量相关性。MSL的酪蛋白水解活性受到IAA、PMSF的抑制。而只有PMSF抑制纤维蛋白原水解活性。
MSL含有属于两个不同超家族的蛋白水解酶，即丝氨酸蛋白酶和半胱氨酸蛋白酶。MSL的纤维蛋白原水解活性仅局限于丝氨酸蛋白酶。该研究将无刺柘树乳胶从用于牛奶凝固外推至用于止血。
如酪蛋白水解活性抑制研究所揭示的，无刺柘树乳胶中存在的蛋白水解酶可分为两个不同的蛋白酶超家族，即丝氨酸蛋白酶和半胱氨酸蛋白酶。其中，只有丝氨酸蛋白酶可被认为在止血方面具有重要意义，因为只有丝氨酸蛋白酶特异性抑制剂PMSF能抑制无刺柘树乳胶蛋白酶的纤维蛋白原水解作用。缩写词：MSL：无刺柘树乳胶，IAA：碘乙酸，EDTA：乙二胺四乙酸，EGTA：乙二醇四乙酸，PMSF：苯甲基磺酰氟
