Masuda I, Cardenal A, Ono W, Hamada J, Haas A L, McCarty D J
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA.
J Rheumatol. 1997 Aug;24(8):1588-94.
To identify the molecular forms of ectonucleotide pyrophosphohydrolase (NTPPHase) in human synovial fluid (SF).
We examined synovial fluids from 32 patients with various joint diseases [10 calcium pyrophosphate dihydrate (CPPD) deposition disease; 7 osteoarthritis (OA); 6 rheumatoid arthritis (RA); 3 after total knee arthroplasty (TKA); 6 olecranon bursa] and 3 normal joint fluids. Joint fluids were analyzed after sequential centrifugation for NTPPHase activity and by Western blot using polyclonal antibodies against 127 kDa porcine articular cartilage vesicle-associated NTPPHase and against PC-1 and 58 kDa, 2 other ecto-NTPPHases. Lysate from human synoviocytes, porcine chondrocytes, and their conditioned media were examined using antibodies to these ecto-NTPPHases. Radiographs of joints from which fluid was obtained were graded for degenerative changes 0-4 using a standard method.
NTPPHase activity was found in all pathological and normal SF tested and correlated with the degree of radiographic degeneration (r = 0.55, p < 0.05). NTPPHase specific activity in ultracentrifugation pellets was highest in CPPD deposition disease fluids (p < 0.05). 127 kDa enzyme was found in both sedimentable and soluble fractions from CPPD, OA, TKA, and normal fluids, and was extensively degraded in all inflammatory fluids. Intact 115 kDa PC-1 was found only in the 2 CPPD fluids with the highest NTPPHase activity. 58 kDa enzyme was found in most fluids, predominantly in the soluble fraction. 127 kDa protein was identified in human synoviocyte conditioned media but not in cell lysate, while PC-1 and 58 kDa proteins were found in the cell lysate but not in the conditioned media.
There was no disease specific association with any one ecto-NTPPHase. Total enzyme activity correlated with the degree of degenerative change. The specific activity of pelletable 127 kDa enzyme was higher in fluids containing CPPD crystals. All 3 ecto-NTPPHases or their presumed degradation products were detectable in some pathologic and normal fluids. A 200 kDa reactive band often accompanied reactivity to the 127 kDa enzyme. PC-1 and 127 kDa proteins were extensively degraded in inflammatory SF, while 58 kDa protein was not. The relative contribution of each of these enzymes to inorganic pyrophosphate production by human joint tissues remains unclear.
鉴定人滑液（SF）中外核苷酸焦磷酸水解酶（NTPPHase）的分子形式。
我们检测了32例患有各种关节疾病患者的滑液[10例二水焦磷酸钙（CPPD）沉积病；7例骨关节炎（OA）；6例类风湿关节炎（RA）；3例全膝关节置换术（TKA）后；6例鹰嘴滑囊炎]以及3份正常关节液。对关节液进行连续离心后分析NTPPHase活性，并使用针对127 kDa猪关节软骨囊泡相关NTPPHase以及针对PC - 1和另外两种胞外NTPPHase（58 kDa）的多克隆抗体进行蛋白质印迹分析。使用针对这些胞外NTPPHase的抗体检测人滑膜细胞、猪软骨细胞的裂解物及其条件培养基。对获取滑液的关节进行X线片检查，采用标准方法将退变程度分为0 - 4级。
在所有检测的病理和正常滑液中均发现NTPPHase活性，且与X线退变程度相关（r = 0.55，p < 0.05）。在CPPD沉积病滑液中，超速离心沉淀中的NTPPHase比活性最高（p < 0.05）。在CPPD、OA、TKA和正常滑液的可沉淀和可溶部分均发现127 kDa酶，且在所有炎性滑液中均有广泛降解。完整的115 kDa PC - 1仅在NTPPHase活性最高的2份CPPD滑液中发现。在大多数滑液中发现58 kDa酶，主要存在于可溶部分。在人滑膜细胞条件培养基中鉴定出127 kDa蛋白，但在细胞裂解物中未发现，而在细胞裂解物中发现PC - 1和58 kDa蛋白，但在条件培养基中未发现。
未发现与任何一种胞外NTPPHase存在疾病特异性关联。总酶活性与退变程度相关。在含有CPPD晶体的滑液中，可沉淀的127 kDa酶的比活性更高。在一些病理和正常滑液中均可检测到所有3种胞外NTPPHase或其推测的降解产物。一条200 kDa的反应带常伴随对127 kDa酶的反应性。PC - 1和127 kDa蛋白在炎性滑液中广泛降解，而58 kDa蛋白未降解。这些酶各自对人关节组织无机焦磷酸产生的相对贡献仍不清楚。
