A novel metalloendopeptidase from rat bone.

Abstract

A soluble metalloendopeptidase was extracted from rat bone. This enzyme can be considered novel, since it is different from all other soluble metalloendopeptidases reported in the literature. The enzyme was strongly inactivated by metal chelating agents and p-hydroxymercuribenzoic acid, indicating that the enzyme may be a metalloendopeptidase and that SH-groups may be necessary for full activity of the enzyme. The enzyme degraded several natural and synthetic peptides and favored the hydrolysis of the X-Y bond in substrates with the sequence of -Pro-X-Y-Pro-, where either X or Y or both are hydrophobic residues. Experiments using commercial collagen preparations indicate that the enzyme is also capable of degrading protein(s) present in these preparations. Using a polyclonal, monospecific antibody against the purified enzyme, several rat tissues were screened with Western blotting for the presence of the enzyme. The enzyme was found in all tissues studied, and was mostly present in the same molecular weight form (60 kDa) as in bone. The endopeptidase was localized in bone tissue using the indirect or sandwich immunolocalization technique with colloidal gold-labelled antibody. Most of the immunostain was found intracellularly in osteoblasts, osteocytes, bone marrow and blood cells. The intracellular localization of the enzyme suggests that it is involved in protein degradation at neutral pH within the cell. The exact physiologic role of the enzyme is unknown, but in view of its hydrolytic capacities, its widespread tissue distribution and its ultrastructural location in bone, it can be speculated that this enzyme is involved in intracellular protein degradation in bone and other rat tissues.