Plant Sciences Section, School of Agriculture and Forestry, University of Melbourne, 3052, Parkville, Vic., Australia.
Planta. 1978 Jan;138(2):153-60. doi: 10.1007/BF00391172.
In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9-13) 125 ng leaf(-1) day(-1); II (day 15-27) 11 ng leaf(-1) day(-1); III (day 29-49) 22 ng leaf(-1) day(-1). Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.
在小麦幼苗主叶的粗提取物中，Triticum aestivum L.，cv. Olympic，通过测量核酮糖-1,5-二磷酸羧化酶（RuBPCase）释放的氨基酸氮的速率来确定最大蛋白酶活性，只有当提取缓冲液中包含 EDTA 和 L-半胱氨酸时才会获得。在 pH 6.8 下研磨可获得最高的蛋白酶活性，尽管在 pH 5.6 至 8.0 的范围内活性水平相似；该范围也与蛋白质的最大可提取性相吻合。在较低 pH 下提取的 RuBPCase 降解蛋白酶的量较少并不是由于 pH 对酶稳定性的影响。反应的最适温度为 50°C，在该温度下至少 120 分钟内反应速率呈线性。在没有底物的情况下，发现蛋白酶在 30°C 以上的温度下非常敏感，即使短时间暴露也会导致活性迅速丧失。测定 pH 值与 RuBPCase 降解之间的关系表明，降解仅限于酸性蛋白酶组，最适 pH 值为 4.8，在 pH 值大于 6.4 时没有检测到活性。可提取的 RuBPCase 蛋白酶的水平表现出明显的昼夜变化，在光照后期活性增加，然后在关灯后下降。研究了叶片年龄对 RuBPCase、RuBPCase 蛋白酶和总可溶性蛋白水平的影响。播种后 9 天达到最大 RuBPCase 活性和可溶性蛋白水平。获得最大水平后，总可溶性蛋白的模式显示出三个明显的蛋白质损失期：I（第 9-13 天）125 ng 叶-1 天-1；II（第 15-27 天）11 ng 叶-1 天-1；III（第 29-49 天）22 ng 叶-1 天-1。RuBPCase 活性和总蛋白模式的比较表明，RuBPCase 的丢失可能是 I 期蛋白质快速丢失的主要原因。蛋白酶活性在 RuBPCase 活性最快下降期间急剧增加，并且由于 RuBPCase 的比活性也下降，我们得出结论，RuBPCase 比其他蛋白质降解得更快。一旦大部分 RuBPCase 丢失，RuBPCase 蛋白酶活性与总可溶性蛋白丢失率之间似乎没有直接关系，因为蛋白酶在蛋白质丢失最慢的 II 期表现出最大活性，并且在活性下降时，蛋白质丢失率在总蛋白质丢失的第三和最后一期保持稳定。
