Früh H, Kostoulas G, Michel B A, Baici A
University Hospital, Department of Rheumatology, Zurich, Switzerland.
Biol Chem. 1996 Sep;377(9):579-86. doi: 10.1515/bchm3.1996.377.9.579.
Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.
人髓过氧物酶（白细胞蛋白酶3）与白细胞弹性蛋白酶具有许多生化特性，但在提高酸性溶液的pH值和/或离子强度时，或在玻璃容器中处理时，其酶活性会迅速丧失。这给需要长时间孵育的动力学实验带来了限制。纯化后，髓过氧物酶方便地储存在pH 3.2的甘氨酸/盐酸缓冲液中，而测定则在pH 7.0、离子强度0.11的磷酸钠/钾缓冲液中进行，同时存在0.05%（w/v）的 Triton X-100，并注意避免与玻璃表面接触。在这些条件下，比较了白细胞弹性蛋白酶和髓过氧物酶与肽底物、不可逆抑制剂和糖胺聚糖的动力学参数。甲氧基琥珀酰-L-赖氨酸（2-吡啶甲酰基）丙氨酸-脯氨酸-缬氨酸-4-硝基苯胺是白细胞弹性蛋白酶的优良底物，也被证明是髓过氧物酶的良好底物（Km = 16 μM，kcat/Km = 30,600 M⁻¹s⁻¹）。3,4-二氯异香豆素（ki/Ki = 6,389 M⁻¹s⁻¹）和甲氧基琥珀酰-丙氨酸-丙氨酸-脯氨酸-缬氨酸-氯甲烷（ki/Ki = 579 M⁻¹s⁻¹）对髓过氧物酶的失活是通过两步不可逆的复合机制发生的，其效力分别是白细胞弹性蛋白酶的二分之一和五分之一。硫酸软骨素、硫酸皮肤素和一种硫酸软骨素多糖等糖胺聚糖，在肽底物存在下作为非必需激活剂与髓过氧物酶相互作用（激活倍数高达6.7倍），在弹性蛋白存在下作为部分抑制剂（饱和时约50%抑制）。这一特性使髓过氧物酶与白细胞弹性蛋白酶区分开来，白细胞弹性蛋白酶总是被糖胺聚糖抑制，与底物无关。
