Bimmler D, Graf R, Scheele G A, Frick T W
Pankreatitis-Forschungslabor, Departement Chirurgie, Universitätsspital, Zürich, Switzerland.
J Biol Chem. 1997 Jan 31;272(5):3073-82. doi: 10.1074/jbc.272.5.3073.
Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name "lithostathine" is therefore inappropriate, and the protein's key function remains to be elucidated.
除了消化酶外，胰液还含有几种不直接参与消化的蛋白质。其中一种，即抑石素，据报道在体外具有方解石晶体抑制活性。由于胰液相对于碳酸钙是过饱和的，因此推测抑石素可稳定胰液。抑石素会被痕量的胰蛋白酶切割，产生一个C端多肽和一个N端十一肽，后者已被确定为抑石素在晶体抑制方面的活性位点。我们在杆状病毒表达系统中制备了大鼠抑石素。为了测试其功能活性，使用非变性多步程序对该蛋白质进行了纯化。在低微摩尔范围内，重组大鼠抑石素在体外表现出方解石晶体抑制活性，证实了早期的报道。对重组抑石素进行了有限的胰蛋白酶水解，并分离了两种切割产物；C端多肽通过离心沉淀，N端十一肽通过高效液相色谱纯化。只有C端肽显示出可测量的方解石晶体抑制活性。此外，与大鼠或人类抑石素的N端十一肽序列相同的合成十一肽没有活性。然而，在相同的体外试验中进行测试时，其他胰腺或胰腺外蛋白质在与抑石素相同的浓度范围内显示出抑制活性，无机磷酸盐也具有活性。基于这些发现，抑石素似乎不太可能是一种生理相关的方解石晶体抑制剂。因此，“抑石素”这个名称并不恰当，该蛋白质的关键功能仍有待阐明。
