鯉魚頭腎25kDa耦合金屬水解酵素之純化

Abstract

目前吾已從鯉魚的頭腎(lleadkidney)中純化得到一個25kDa的蛋白質水解酵素；純化的方法是將頭腎研磨均質後取上清液，先通過Dye Reactive Blue 72 agarose affinity液狀層析管柱，經過透析濃縮後，再利用FPLC Mono-P液狀層析管柱分析。從銀染色蛋白質電泳膠片圖的結果顯示其分子量與純度。分析各類蛋白質水解酵素的抑制劑對此25kDa蛋白質水解酵素的抑制能力，發現金屬螫合劑的抑制效果最佳，因此推論它是一個耦合金屬蛋白質水解酵素，命名為MP-25。從染鋅離子分佈的頭腎切片結果中，知道頭腎富含鋅離子，也許可佐證上述事實。在勝?水解實驗中，以substance P和各胺基酸長度的Bradykinin為MP-25的基質，被水解的勝?片斷經HPLC的純化，再由氨端胺基酸序列分析得知，MP-25喜切厭水性胺基酸的位置，例如phenylalanine；另一特色是基質勝?長度要七個以上。接者，以此MP-25為抗原打入天竺鼠中引發對抗MP-25的專一性抗體。再利用西方點墨法分析此MP-25的分佈，結果顯示它存在於頭腎、脾臟和腎臟中。因為鯉魚的器官分化程度與高等哺乳動物不同，前述的那三種器官都兼有免疫功能。而在灌流實驗中，以化學物質刺激頭腎活體組織，分析刺激前後釋放蛋白質種類和數量的變化。經由酵素分佈電泳圖和西方點墨法的結果得之，MP-25以高濃度的鉀離子(50mM)、romutide、腎上腺素刺激下，會自頭腎組織中釋放。從上述結果可知釋放MP-25是刺激免疫組織的效應之一，推論在鯉魚體的免疫系統中MP-25可能扮演某種角色。

We have purified a 25kDa protease from the headkidney of carp by sequential column chromatography on Dye Reactive Blue 72 affinity column and FPLC Mono-P column. Using various inhibitors specific for different classes of proteases, we found metal chelators had better inhibitory effects on the activity of the 25kDa protease. Therefore, we have identified this 25kDa protease as a member of metalloprotease (MP-25). The strong staining of headkidney sections by a histochemical method using sodium selenite and silver has indicated that carp headkidney is rich in zinc ion. Furthermore, we have determined the substrate specificity of this protease by sequence analysis of the digested peptide fragments from Lys-bradykinin and substance P. MP-25 prefers cutting P1 site at hydrophobic amino acid residues, e.g. Phe-Ser and Gly-Phe in Lys- bradykinin, and Gln-Phe and Phe-Phe in substance P. We have also raised a polyclonal antiserum against MP-25 and found that MP-25 is present in the headkidney, kidney and spleen, all of which are responsible for immune functions in the carp. In addition, we have demonstrated that a high concentration of potassium, romutide or epinephrine stimulated the secretion of MP-25 from perfused headkidney. From these results, it is very likely that MP-25 is secreted from the headkidney in response to stimulation of immune system. Therefore, MP-25 may mediate certain functions of fish immune system.