Characterization of Heat-Stable Immunoactive in Abalone (Haliotis discus)

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Project title

Code/計畫編號

NSC84-2321-B019-007

Translated Name/計畫中文名

鮑魚耐熱性免疫活化因子的構造與功能解析

Project Coordinator/計畫主持人

Zwe-Ling Kong

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=161598

Year

1995

Start date/計畫起

01-08-1994

Expected Completion/計畫迄

01-07-1995

Bugetid/研究經費

500千元

ResearchField/研究領域

食品科技(工)

將市售冷凍新鮮飽魚(Haliotis discus)以100.degree.C沸水萃取30min,經80%硫銨沈澱分劃,研究所得之萃取物分劃(AHE)對Hybridoma cell line HB4C5及SI102細胞之影響。結果顯示,針對於抗體產生、細胞增殖及細胞凝集測試方面,於蛋白質最終濃度20.mu.g/ml時,即可提高2-3倍活性。AHE分劃分別經pH4-10之處理後,以pH8時之活性最高,pH4則失去活性。在耐熱性方面,經100.degree.C加熱60min仍維持80%以上的細胞增殖活性,但抗體產生活性則下降至15%;另經121.degree.C, 20min處理後,活性變化和100.degree.C加熱60min處理結果相同。而以Trypsin處理24hr後,仍保有90%活性。將細胞以Oligosaccharyltransferase抑制劑Tunicamycin(0.1.mu.g/ml)處理48小時後,再添加AHE測試,增殖效果消失,而以Lysosomal .alpha.-mannosidase抑制劑Swainsonine(2.mu.g/ml)處理細胞48小時後,再添加AHE,雖於低濃度時無活性檢出,但當AHE濃度達20.mu.g/ml時,則仍具高活性。另以糖基抑制作用分析,結果顯示Glucose、Lactose、Sucrose、Galactose及Mannose於濃度50mM時,皆會抑制活性。由以上實驗顯示,AHE活性成分可能須經由細胞膜受體之Glycosylation作用,並證實Mannose及Glucose和細胞膜受體糖鍵結相關,推測為AHE生理活性之重要因素。在作用活性機制上,添加Protein kinase抑制劑Staurosporine(13.67nM)會抑制HB4C5細胞之抗體產生及細胞生長,但於此濃度同時添加AHE(25.mu.g/ml)時,細胞增生可以不受影響,而抗體產生則仍然被抑制;而鈣離子螯合劑EGTA(0.62mM)則會抑制AHE所具之兩種活性,依此推測AHE促使抗體產生須經Classic protein kinase C(PKC)之活化,而對於細胞增生則可能經由PKC-independent pathway,例如Ca/sup 2+//Calmodulin-dependent kinase之活性途徑。將飽魚萃取液經Hydroxylaptite、Phenyl-TOYOPEARL、Mono Q、Phenyl superose及Superdes 75管柱分離後,以Native-PAGE分析得一分子量約為33kDa之純化物,再經SDS-PAGE分析得知此純化物由17.4kDa及16.4kDa兩個次單元所構成。 Abalone (Haliotis discus) heat water extracts (AHE) induced cell proliferation, immunoglobulin production by 2-3 fold at 25.mu.g/ml and cell aggregation on two human hybridoma cell line (HB4C5 and SI102) and can activated two macrophage-like cell line (UM .phi. and J774.1). AHE showed highest stability at pH8.0 treatment and almost lost its activity when below pH5.0. Besides, AHE remained about 80% cell proliferation activity when after treated at 100.degree.C for 60 min, but on the other hand, immunoglobulin production activity, will decreased to about 20% of original. HB4C5 and SI102 cells pretreated with oligosacharyltransferase inhibitor (tumucamycin 0.1.mu.g/ml) showed not activated by AHE, indicated that AHE stimulated cell may via glycosylation of cell membrane receptor. Preincubated cells with lysosomal .alpha.-mannosidase inhibitor (swainsonine 2.mu.g/ml) showed not inhibit activity of AHE (20.mu.g/ml). Moreover, AHE activity will inhibited by 50mM sugar competitives as glucose, lactose, sucrose, galactose and mannose, suggested that mannose and glucose seem to be important effectors on AHE and receptor interaction for activation. In mechanism analysis, EGTA (0.62mM) inhibited cell proliferation activity of AHE, but staurosporine (13.67.mu.g/ml) did not. In addition, EGTA and staurosporine both inhibited immunoglobulin production activity of AHE. Those results showed that AHE stimulated cell proliferation was via PKC-independent pathway, such as Ca/sup 2+//Calmodulin-dependent kinase, on the other hand, the effect of AHE on immunoglobulin production may via cell activation of classic PKC. After purified by Hydroxylapatite, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration, we gain a high immunoglobulin production stimulating protein from AHE. The molecular weight of purified fraction was estimated to be about 35kDa by gel filtration, and 33kDa by native-PAGE. This protein may composed by one 17.4kDa and another 16.4kDa subunits that estimated by SDS-PAGE.

Keyword(s)

鮑魚
耐熱性
免疫活性
細胞增殖
免疫球蛋白
細胞凝集
Haliotis discus
Heat resistance
Immunoactive
Cell proliferation
Immunoglobulin
Cell aggregation

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Characterization of Heat-Stable Immunoactive in Abalone (Haliotis discus)

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