行政院國家科學委員會專題研究計畫 成果報告
利用基因重組方法探討大白鼠 alphaA-水晶體蛋白之功能與 結構
計畫類別： 個別型計畫
計畫編號： NSC91-2113-M-006-014-
執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立成功大學化學系（所）
計畫主持人： 黃福永
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中 華 民 國 92 年 10 月 30 日
行政院國家科學委員會專題研究計畫成果報告
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利用基因重組方法探討大白鼠 α-水晶體蛋白之功能與結構(I)
Functional and Structural Studies of Mutant Rat Lens α-Crystallins (I)
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計劃類別：個別型計畫
計畫編號：NSC91-2113-M-006-014
執行期間：91 年 8 月 1 日 至 92 年 7 月 31 日
個別型計畫：計畫主持人：黃福永
處理方式：▓已送發表
執行單位：國立成功大學化學系 中華民國 92 年 10 月 25 日
1
In the project of “Functional and Structural Studies of Mutant Rat Lens alpha-Crystallin (I)”, we have basically constructed theαins-crystallin and mutant αA-crystallin gene. We are in the process of purifying the expressed proteins, then followed by characterizing the functionality. This is the first experiment doing recombinant DNA in this lab. We did experience the difficulty, however, we have gotten through it. The project progress is a little late, however, during this period we also have completed research related to rat lens crystalline and rice coleoptile trypsin inhibitor. The results have been summarized and written into three papers. Followings are the paper title and the abstract contents.
Paper # 1:
Title: Characterization of Thermal-Induced High Molecular Weight Aggregates of Rat Lens α-Crystallin
Abstract: α -Crystallin and in vivo (native) high molecular weight aggregate
(HMWA) were isolated from four weeks old Rat lenses. α-Crystallin was further heated at various temperatures (50-60℃) to induce HMWA, which was used to make a comparison with native HMWA. Spectroscopic measurements of trytophan (Trp), non- tryptophan (non-Trp), 1-anilino- naphthalene-8-sulfonic acid (ANS), and 2-(4’-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) fluorescence intensities indicated that native HMWA is more hydrophobic than α-crystallin, possibly resulting from different process of partial unfolding when forming HMWA for nativeα-crystallin. Gel filtration chromatography showed that thermal-induced HMWA ofα-crystallin prepared by preheating at 60℃ for an hour has the same molecular weight as that of native HMWA. Trp, ANS, and MIANS fluorescence as well as far-UV CD measurements indicated that thermal-induced HMWA and native HMWA shared structural similarity, which further suggested both HMWAs undergo similar aggregation mechanism. Chaperone-like activity toward dithiothreitol(DTT)-induced insulin B-chain aggregation showed that α-crystallin preheated at 50
℃ has better activity than α-crystallin and native HMWA. With the increase of preheating temperature, the activity of α-crystallin decreased and when preheated at 60℃ the thermal-induced HMWA shows less active than that of native HMWA. The correlation between the ANS fluorescence and the chaperone-like activity suggested that surface hydrophobicity is not the sole determinant for chaperone-like function of α-crystallin. In order to prove whether disulfide bond formation is a cause for the formation of HMWA, cysteine modification of α-crystallin was carried out using 50 mM β-mercaptoethanol and 50 mM iodoacetamide for 12 hours, respectively, then was confirmed by SDS-PAGE for no disulfide bond formed. This modifiedα -crystallin was heated at 60℃ for an hour, then were subjected to gel filtration and SDS-PAGE and found that thermal-induced HMWA with same molecular as that of native HMWA was observed for both modifiedα-crystallin from gel filtration chromatography and no disulfide bond was observed based on SDS-PAGE. These results indicate that disulfide bond formation was not necessary a (main) factor leading to the formation of HMWA, at least in in vitro thermal-induced HMWA formation. Our study suggests that the aggregation mechanism via partial unfolding for thermal-inducedα-crystallin is similar to that for nativeα-crystallin; however, the unfolding process may differ as to show different non-Trp fluorescence, different near-UV CD, and different chaperone-like activity.
Paper #2
Title: Proteomic Study of Galactosemic Rat Lenses
Abstract:
This study was to use one and two dimensional gel electrophoresis to investigate the changes of galactosemic rat lens proteins. Four weeks old female rats were fed with 50% galactose for six weeks. The rat lenses for both the experimental and control groups were taken every two weeks for study. One- and3
two-dimensional gel electrophoretic analyses were performed for lens total proteins and α-, β-, and γ-crystallins obtained from S-300 gel filtration chromatograph.
Following proteins changes were observed. i). For α-crystallin, a new protein spot (t1) was observed, which was identified being a post-translational modified αA-crystallin.
Two modified spots of a2 and a3 of αB-crystallin were observed, which intensity increased with the increase of galactosemic time. Truncation of α-crystallin was also observed. ii). For β-crystallin, it was found that the spot intensity of βB1 and two modified spots of βB3 and βA4 decreased with the increase of galactosemic time;
while the spot intensity of modified βB2 increased with the increase of galactosemic time. iii). For γ-crystallin, three γS-crystallin spots were observed and their spot intensity was decreased with the increase of galactosemic time; while no other γ-crystallins showed decreasing intensity, which indicate that compared to other types of γ-crystallins, γS-crystallins are more sensitive to environmental changes. This proteomic study has shown that galactosemic rats could be resulted in the formation of cataract in short time, which is caused by the modifications and truncations of crystallins, then leading to the precipitation.
Paper #3
Title: A Trypsin Inhibitor from Rice Coleoptiles
Abstract: A rice trypsin inhibitor with molecular of around 18 kDa has been
obtained through cationic and gel filtration column chromatography from coleoptiles grown under submerge condition. This trypsin inhibitor was further characterized toward proteases of chymotrpsin and trypsin. It was found that the inhibition mode when competing with substrate L-N-α-benzoyl-arginine-para-nitroanilide (L-BAPNA) toward chymotrypsin was not a typical competitive mode. However, the inhibition mode when competing with L-BAPAN toward trypsin was found a typicalcompetitive mode as that of soybean trypsin inhibitor. The EI complex dissociation constant, Kd
, for rice trypsin inhibitor, toward trypsin was 4.0 x 10
-7 M, while it was7.4 x 10-7 M for soybean trypsin inhibitor. When the molar ratio of Rice trypsin inhibitor to trypsin was about 0.3, 50% of trypsin activity had been inhibited, while it was about 0.5 for soybean trypsin inhibitor. This study shows that rice trypsin
inhibitor has better inhibition activity than soybean trypsin inhibitor does toward trypsin. Thus, it would be interesting and important to investigate further in the application of this inhibitor in medicinal and food chemistry.
