Alter S C, Metcalfe D D, Bradford T R, Schwartz L B
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Biochem J. 1987 Dec 15;248(3):821-7. doi: 10.1042/bj2480821.
Tryptase was previously shown to undergo rapid inactivation under physiological conditions unless stabilized by the presence of heparin. The current study shows that increasing the concentration of free tryptase enhances the preservation of enzymic activity, consistent with dissociation of the tetramer, rather than autodegradation, as the mechanism of inactivation. Heparin glycosaminoglycan fragments of Mr greater than 5700 are necessary for complete stabilization of tryptase activity. This stabilizing effect depends upon negative charge density rather than carbohydrate composition. Thus, keratan sulphate or hyaluronic acid were no better than physiological buffer alone; chondroitin monosulphates and heparan sulphate each prolonged the t1/2 about 20-fold over buffer alone; chondroitin sulphate E prolonged the t1/2 69-fold; and dextran sulphate and heparin provided complete stabilization of tryptase activity for 120 min. Poly-D-glutamic acid prolonged the t1/2 55-fold. In each case the loss of tryptase activity followed apparent first-order kinetics. Increasing the NaCl concentration from 0.01 M to 1.0 M increased the stability of free tryptase. In contrast, increasing the NaCl concentration in the presence of stabilizing polysaccharides decreased the stability of tryptase until dissociation of tryptase from each polysaccharide presumably occurred; thereafter tryptase stability increased as did that of free tryptase. The effect of salt concentration on heparin-stabilized tryptase activity (as opposed to stability) was also evaluated. The mast cell proteoglycans heparin and chondroitin sulphate E, by virtue of containing the naturally occurring glycosaminoglycans of highest negative charge density, may play a major role in the regulation of mast cell tryptase activity in vivo.
先前的研究表明，在生理条件下，除非有肝素存在使其稳定，否则类胰蛋白酶会迅速失活。当前的研究表明，增加游离类胰蛋白酶的浓度可增强酶活性的保留，这与四聚体的解离而非自身降解作为失活机制是一致的。分子量大于5700的肝素糖胺聚糖片段是类胰蛋白酶活性完全稳定所必需的。这种稳定作用取决于负电荷密度而非碳水化合物组成。因此，硫酸角质素或透明质酸并不比单独的生理缓冲液效果更好；硫酸软骨素单硫酸盐和硫酸乙酰肝素各自使半衰期比单独的缓冲液延长约20倍；硫酸软骨素E使半衰期延长69倍；硫酸葡聚糖和肝素可使类胰蛋白酶活性完全稳定120分钟。聚-D-谷氨酸使半衰期延长55倍。在每种情况下，类胰蛋白酶活性的丧失均遵循明显的一级动力学。将氯化钠浓度从0.01 M增加到1.0 M可提高游离类胰蛋白酶的稳定性。相反，在存在稳定多糖的情况下增加氯化钠浓度会降低类胰蛋白酶的稳定性，直到类胰蛋白酶可能从每种多糖上解离；此后，类胰蛋白酶的稳定性增加，游离类胰蛋白酶的稳定性也增加。还评估了盐浓度对肝素稳定的类胰蛋白酶活性（与稳定性相反）的影响。肥大细胞蛋白聚糖肝素和硫酸软骨素E由于含有天然存在的负电荷密度最高的糖胺聚糖，可能在体内肥大细胞类胰蛋白酶活性的调节中起主要作用。
