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Limited proteolysis of phospholipase C-gamma 1 indicates stable association of X and Y domains with enhanced catalytic activity.

机译:磷脂酶C-γ1的有限蛋白水解表明X和Y结构域稳定缔合,催化活性增强。

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摘要

Phospholipase C-gamma 1 (PLC-gamma 1) was treated with Staphylococcus aureus V8 protease (V8) and the digestion products were analysed with site-specific antibodies. V8 treatment generated three immunodetectable PLC-gamma 1 fragments of 120, 97, and 39 kDa. The 39 kDa fragment is derived from the C-terminus of PLC-gamma 1 and includes the conserved Y domain present in all PLC isoenzymes. The 120 and 97 kDa fragments are derived from the N-terminus of PLC-gamma 1, possess the conserved X domain common to all PLC isoenzymes, and the src-homology domains unique to PLC-gamma 1 and -gamma 2. It is likely that the 97 kDa fragment is a V8 product of the 120 kDa fragment. As the C-terminal 39 kDa fragment, and either of the N-terminal 120 or 97 kDa fragments, were precipitable with antibody specific to a sequence present in only the 39 kDa fragment, the data indicate co-precipitation of separate polypeptide chains that remain associated after V8 proteolysis. Importantly, V8 treatment increased the activity of PLC-gamma 1 and did not alter the calcium requirement. The influence of other modulators of PLC-gamma 1 activity, however, was lost following V8 treatment. These results suggest the stable association of the X and Y domains within PLC-gamma 1, and demonstrate that proteolysis in the region of PLC-gamma 1 that is subject to tyrosine phosphorylation can enhance catalytic activity.
机译:用金黄色葡萄球菌V8蛋白酶(V8)处理磷脂酶C-γ1(PLC-γ1),并用位点特异性抗体分析消化产物。 V8处理产生了120、97和39 kDa的三个可免疫检测的PLC-γ1片段。 39 kDa片段衍生自PLC-γ1的C末端,包括所有PLC同工酶中存在的保守Y结构域。 120和97 kDa片段衍生自PLC-γ1的N端,具有所有PLC同工酶共有的保守X结构域,以及PLC-γ1和-gamma 2特有的src同源结构域。 97 kDa片段是120 kDa片段的V8产物。由于C端39 kDa片段以及N端120或97 kDa片段中的任何一个都可以用对仅存在于39 kDa片段中的序列具有特异性的抗体沉淀,因此数据表明共沉淀了剩余的单独多肽链V8蛋白水解后相关。重要的是,V8处理可提高PLC-γ1的活性,并且不会改变钙的需求量。但是,在V8处理后,失去了其他PLC-γ1活性调节剂的影响。这些结果表明PLC-γ1中的X和Y域的稳定关联,并证明受酪氨酸磷酸化的PLC-γ1区域中的蛋白水解可以增强催化活性。

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