熱休克蛋白質的純化及其生理功能

Abstract

以生長四十小時後的大豆黃化（etiolated）幼苗為材料，用 3H －白胺酸標誌蛋白質，經高速離心後取去核糖體上清液，以硫酸銨分劃，發現所有的熱休克蛋白質均集濃在70?100％硫酸銨分劃區中。以此分劃區來研究蛋白質的生理功能，可得以下結論：首先，對於熱休克蛋白質本身而言，除了在熱休克過程中對於熱反應具穩定性外，更有保護它種蛋白質在熱休克下不被變性的能力。這種保護效果與所加入熱休克蛋白質的量成正比，同時其所保護的對象則傾向於與膜系統相結合的蛋白質。這些熱休克蛋白質不論是在活體內或試管中均表現出對於熱反應具有穩定性。幼苗合成出熱休克蛋白質後，重新置?正常生長溫度，觀察熱休克蛋白質被保留程度，經四天後發現熱休克蛋白質依然存在。同時也認為在所有的熱休克蛋白質族群中，以15?18KD這群低分子量的熱休克蛋白質，在提供植物獲得熱保護的過程中所做的貢獻遠超過高分子量的熱休克蛋白質。 前人對於熱休克蛋白質的研究，均以其次單位（subunit）為對象，至於這些蛋白質在活體內的可能真正結構（native form），至今仍然沒有任何文獻可供參考。在本論文中，藉由膠體過濾法及離子交換法，在熱休克蛋白質純化過程中，可觀察這些次單位在活體中的可能結合形式。其中以15?18KD會與22KD及70KD等相互地結合在一起，68?92KD這群高分子量則可能結合成為一個群體，至於27KD這群，則不參雜其他次單位，而獨自存在。

All the heat shock (HS) proteins from a postribosomal supernatant of heat shocked soybean seedlings were found to be enriched in a 70-100% (NH4)2SO4 fraction. This HS protein enriched fraction was quite resistant to heat denaturation. Moreover, this fraction, when added to the postribosomal supernatant from control (non-heat shocked) seedlings, showed a significant ability to protect proteins from heat denaturation. Heated at 55℃, some 50% of the control proteins, which were normally denatured after HS treatment, were protected for at least 1 hr when 1 mg of this HS protein fraction was added. The degree of protection was proportional to the amount of HS protein fraction added. What's more, these HS proteins have a tendency to protect membrane system binding proteins. Low molecular weight (LMW) 15-18 kD HS proteins contribute more than high molecular weight (HMW) 68-92 kD varieties to plants in terms of providing thermaltolerance. Gel filtration and ion exchanger chromatography were effective in purification of HS proteins. The native form of HS proteins in vivo were also detected. Such subunits as 15-18kD, 22kD and 70kD HS proteins would associate together as a unit. HMW ranging from 68-92 kD HS proteins would form another one. As for 27kD HS proteins, they would not contaminate with other submits. They exist by themselves.