A new physiologic function of tissue kallikrein family enzymes and processing of growth factor precursors

1.从小鼠颌下腺中制备了四种组织激肽释放酶家族酶(MK1、MK9、MK13、MK22)。经鉴定，MK13是一种蛋白原转换酶。MK9是一种与EGF结合的蛋白，被发现具有水解原肾素的活性，从而产生活性肾素。MK22(β-NGF内肽酶)作用于肾素，产生肾素和精氨酸肾素两种产物。另一方面，真正的激肽释放酶Mk1根本不处理肾素。结果提示，组织激肽释放酶家族酶具有较高的激动素释放活性，具有较低的原肾素转化活性，反之亦然。这些酶可能在组织肾素-血管紧张素系统中起到生理作用。从DBA/2N小鼠颌下腺中分离纯化了组织激肽释放酶家族的基因，并对其进行了序列分析。克隆了该酶的全长基因，并测定了其核苷酸序列。该酶与原蛋白转换酶(PRECE)MK13/MK26高度同源。它实际上具有从其前体原肾素中产生肾素的活性。该酶和MK13/mK26在DBA/2N和ICR小鼠中的存在和缺失表明，纯化的酶是prece(prece^b)的等位酶。抗MK1抗血清是用纯化的组织激肽释放酶吸附抗血清制备的。然后用该抗血清对MK1进行免疫组织化学定位。MK1定位于分离的颗粒曲管细胞(GCT细胞)分泌颗粒中。这种马赛克染色图案在男性和女性身上都能看到。阳性反应的细胞类型不同，男性呈细长的GCT细胞，女性呈典型的锥体细胞。在男性的GCT细胞中，约有5%的细胞表达Mk1，而在女性的这些节段中，有65%的细胞呈阳性反应。

1. Four tissue kallikrein family enzymes (mK1, mK9, mK13, mK22) were prepared from mouse submandibular gland. mK13 was ientified to be a prorenin converting enzyme. mK9, a binding protein for EGF,was found to have an activity to hydrolyze prorenin to give an active renin. Acting on prorenin, mK22 (beta-NGF endopeptidase) gave two products renin and arginyl renin. On the other hand, mK1, a true kallikrein did not process renin at all. The results suggest that tissue kallikrein family enzyme bearing higher kinin-releasing activity have lower prorenin-converting activity and vice versa. These enzymes may possibly have a physiologic role in the tissue rein-angiotensin system2. From the submandibular gland of DBA/2N mice, and enzyme of tissue kallikrein family was purified and sequenced. A cDNA of this enzyme was also cloned and nucleotide sequence was determined. The enzyme was highly homologous to mK13/mK26, a prorenin converting enzyme (PRECE). It actually had the activity to give renin from its precursor prorenin. The presence and absence of the present enzyme and mK13/mK26 in DBA/2N and ICR mice suggested that the purified enzyme is an allozyme of PRECE (PRECE^b).3. Anti-mK1 antiserum specific to, its antigen was prepared by absorption of antiserum with purified tissue kallikrein enzymes. Immunohistochemical localization of mK1 was then invetigated using the specific antiserum. mK1 localized in the secretory granules of segregated number of granular convoluted tublar cells (GCT cellls). Such mosaic staining pattern was seen in both males and females. The cell type that shows possitive reaction was different between males and females ; slender GCT cells were stained in males whereas typical pyramydal cells were stained in females. About 5% of GCT cells in males were immunoreactive for mK1, whereas 65% of these segments were positive in females.