Ronzani N, Hasselbach W, Stephan L
Max-Planck-Institute für Medizinische Forschung, Abteilung für Physiologie, Heidelberg, Federal Republic of Germany.
Eur J Biochem. 1990 Mar 30;188(3):557-65. doi: 10.1111/j.1432-1033.1990.tb15436.x.
The reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium-transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1-100.0 MPa affects trypsin cleavage of the control substrate N-alpha-benzoyl-L-arginine-4-nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium-transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T1 cleavage site [C. J. Brandl et al. (1986) Cell 44, 597-607]. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1-3 in addition to the established peptides A1 and A2. Under pressure and in the presence of calcium, A1 and A2 rapidly disappear indicating the presence of calcium-binding sites in these peptides. In contrast, the B1-3 peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium-dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure-induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20-40 ml/mol and 80-100 ml/mol in the ranges 0.1-40.0 MPa and 40.0-100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium-transport enzyme which activates and modifies tryptic-cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme residues in its E1 state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.
在室温下，压力对肌浆网钙转运酶的抑制作用是可逆的，同时伴随着该酶对胰蛋白酶切割的可及性显著增强，这种增强依赖于钙的存在。以磷酸二硝基苯酯为底物监测钙转运酶的活性。在0.1 - 100.0 MPa范围内的压力，在有钙和无钙的情况下，对对照底物盐酸N-α-苯甲酰-L-精氨酸-4-硝基苯胺的胰蛋白酶切割影响很小。相比之下，在室温下对钙转运酶施加100.0 MPa的压力会加速随后在T2位点而非T1位点的胰蛋白酶切割[C. J. 布兰德l等人（1986年）《细胞》44卷，597 - 607页]。在胰蛋白酶消化过程中施加压力同样仅影响在T2位点的切割，在无钙时该切割进行缓慢，而在有钙时迅速进行。在大气压下无钙时以及在高压下无钙和有钙时，除了已确定的肽段A1和A2外，还会出现新的切割位点，产生新的亚片段B1 - 3。在压力下且有钙存在时，A1和A2迅速消失，表明这些肽段中存在钙结合位点。相比之下，最有可能是B片段衍生物的B1 - 3肽段在有钙和无钙的情况下都会积累。与在大气压下的胰蛋白酶切割不同，在压力下A片段和B片段的胰蛋白酶切割趋于完全。与A片段和B片段的消失同时发生的是，钙依赖性底物水解消失。计算压力诱导的可逆酶抑制和胰蛋白酶切割的活化体积，在0.1 - 40.0 MPa和40.0 - 100.0 MPa范围内分别得到密切相关但符号相反的体积，分别为20 - 40 ml/mol和80 - 100 ml/mol。因此，压力会使钙转运酶产生可逆变化，在有钙存在的情况下，即当酶处于其E1状态时，激活并改变A片段T2位点及其亚片段中位点的胰蛋白酶切割模式。相比之下，压力会加速B片段的胰蛋白酶切割，而与钙的存在与否无关。
