Evidence of gelatinase secretion by the submandibular gland in prepubescent rats

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.