鯉魚腎泌分解?酵素活性之調控

Abstract

腎泌分解?（Nephrosin）是一個分子量約23k的分泌性金屬耦合蛋白?。它是我們實驗室自鯉魚頭腎萃取液中純化得到的。由一級構造推論它屬於astacin家族。它主要分佈於成體鯉魚的腎臟、頭腎和脾臟，是此家族成員中於淋巴造血（Lymphohematopoietic）器官中被發現的首例。細胞膜電位去極化可刺激它自頭腎的分泌（洪1994），分泌到細胞外的腎泌分解?可以透過與細胞問質中的肝制凝（Heparin）素等黏多醣類（Glycosaminoglycans）結合而滯留其中（詹，1997）。細胞外是一個含高濃度鈣離子（與鎂離子）的環境，且許多分泌性金屬耦合蛋白?會受其影響，故本論文進一步研究腎泌分解?、鈣離子、與肝制凝素間的交互作用。 金屬耦合蛋白?的催化中心需一鋅離子作為輔因數。在試管中，有些二價金屬陽離子（如錳、鈷、銅等）可取代鋅離子並具作用。實驗結果顯示，鈣離子不能取代鋅離子成為腎泌分解?催化中的輔因數，但它會增強鋅離子對腎泌分解?的活化。故腎泌分解?除了其催化中心有一鋅離子結合位置，應還有一鈣離子結合位置以媒介鈣離子之作用。它的鋅離子結合位置也可與鎘、鈷、銅、錳、鎳離子結合，並具作用。而鈣離子結合位置也可與鎂、鍶、鈷、銅、錳離子結合並具作用。另一方面，當腎泌分解?由鋅離子活化時肝制凝素會抑制其活性（詹，1997），但以鋅離子與鈣離子共同活化時肝制凝素則失去抑制酵素活性的能力。由上述結果我們推論，貯存於釋放顆粒中的腎泌分解?在分泌前以結合肝制凝素來抑制其酵素活性，當腎泌分解?被釋放至細胞外後，細胞外高濃度之鈣離子和（或）鎂離子可移去肝制凝素之抑制，並更進一步活化腎泌分解?的酵素活性。

Nephrosin is a newly discovered member of the astacin family, a group of secreted or membrane metalloproteinases found in the 1990s. It is the first astacin enzyme found in lymphohematopoietic tissues (kidney, head kidney, and spleen in fish). Carp nephrosin in its secreted form contains 198 amino acid residues whose predicted relative molecular weight is 23k. Four putative heparin binding motifs can be identified by which secreted nephrosin may be localized to the heparan sulphate proteoglycan present in extracellular matrix. In this thesis, we examined the metal ion requirement for the activation of purified nephrosin (apoenzyme). It is concluded that in addition to the catalytic Zn2+ binding site (Zn site), nephrosin contains a Ca2+ binding site since Ca2+ (0.2-0.8mM) can further enhance the activity of nephlosin when it is activated by 100μM Zn2+. Pontential agonists for the Zn site of nephrosin include Zn2+, Mn2+, Co2+, Cu2+, Ni2+ and Cd2+, and potential agonists for the Ca site include Mn2+, Co2+, Cu2+, Ca2+, Sr2+ and Mg2+. It was also observed that heparin reduced nephosin activity when nephrosin was activated by Zn2+ (100μM) but lost its inhibitory activity when nephrosin was co-activated by Zn2+ (100μM) and Ca2+ (500μM). It is postulated that nephrosin is stored in its mature form but its activity is prohibited by binding to heparin or heparin-like glycosaminoglycan. Once nephrosin is secreted into the extracellular fluid, exposure to higher concentrations of extracellular Ca2+ may remove the inhibitory action of heparin and thus convert the inactive form into fully active form.