A major serine protease in rat skeletal muscle: Evidence for its mast cell origin

Abstract

The physical, chemical, and immunologic properties of a protease from rat skeletal muscle, proposed to function in the degradation of certain intracellular enzymes, are identical to those of a chymotrypsin-like serine protease isolated from peritoneal mast cells. The results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea indicate that the two rat proteases have identical mobilities corresponding to a molecular weight of 26,000. The relative amino acid compositions of the proteases are nearly identical. Immunodiffusion tests for crossreaction between the muscle protease and antisera directed toward mast cell protease indicate that the former is immunologically identical to mast cell protease. The first 35 amino-terminal residues of the two enzymes are identical and indicate homology of these proteins to other mammalian serine proteases. The sequence analysis of the protease from muscle was extended -for an additional 16 positions, and comparison of this amino-terminal sequence with that of a similar enzyme from small intestine showed approximately 75% sequence identity. In contrast, only 40% of the residues in this region of bovine chyrnotrypsin A were found at corresponding loci in rat muscle protease. It is concluded that the protease from muscle or mast cells is closely related to the enzyme from small intestine which recently was localized in the "atypical" mast cells of gut mucosa Katunuma and coworkers (1, 2) have isolated several chymotrypsin-like serine proteases from various rat tissues and proposed that these enzymes initiate the degradation of several intracellular pyridoxal phosphate-dependent enzymes such as ornithine aminotransferase. The apo forms of these enzymes were inactivated by the proteases, but not the holo enzymes nor the apo forms of enzymes requiring other cofactors.Recently, an improved method was developed to purify a serine protease from rat skeletal muscle (unpublished data). During that study it was observed that the relative amino acid composition of this enzyme and that of the chymotrypsin-like protease (chymase) of peritoneal mast cells (3) were similar. The present study shows that the amino-terminal sequences and the immunologic properties of these two proteases are identical. EXPERIMENTAL Materials. For the purpose of developing specific antisera, a small amount of the chymotrypsin-like protease of rat peritoneal mast cells was purified as described by Lagunoff and Pritzl (4). Larger quantities of enzymes were prepared by a method which includes affinity adsorption chromatography on ovoinhibitor immobilized on Sepharose 4B (unpublished data). The protease from skeletal muscle was purified by the method of Sanada et al. (5) or by the affinity adsorption method described above. Rabbit antisera directed toward mast cell protease or the "atypical" mast cell protease of small intestine were prepared and their specificities were determined as described (3).Methods. Proteins were reduced and pyridylethylated (6) before sequence a...

Structure, specificity and localization of the serine proteases of connective tissue

“…The amino acid composition (table 1) of this enzyme is clearly different from that of the atypical mast cell protease, but it is remarkably similar to that of the skeletal muscle protease [28]. The amino-terminal sequences of the mast cell protease and the skeletal muscle protease are identical for the first 35 residues [28].…”

“…The amino-terminal sequences of the mast cell protease and the skeletal muscle protease are identical for the first 35 residues [28]. Immunodiffusion tests for cross-reactivity using anti-rat mast cell protease indicated that the mast cell and skeletal muscle proteases are immunologically identical [28]. Finally, immunofluorescent localization of skeletal muscle protease showed that this enzyme was in fact derived from mast cells present in the connective tissue of muscle (N. Katunuma, personal communication).…”

“…The amino acid composition (table 1) of this enzyme is clearly different from that of the atypical mast cell protease, but it is remarkably similar to that of the skeletal muscle protease [28]. The amino-terminal sequences of the mast cell protease and the skeletal muscle protease are identical for the first 35 residues [28]. Immunodiffusion tests for cross-reactivity using anti-rat mast cell protease indicated that the mast cell and skeletal muscle proteases are immunologically identical [28].…”

Proteolytic enzymes of rat liver mitochondria. Evidence for a mast cell origin

“…Furthermore, the presence of cytochrome c oxidase in the high density fraction containing the proteinase-carboxypeptidase activities strongly suggested an inner mitochondrial membr~e location. It was dif~cult, however, to explain the high density of the material (P = 1.25 g/ cm3) which sedimented through the layer of 1.7 M sucrose compared to an average density of 1.20 g/cm3 measured for mitochondria [ 161. We have speculated that the high density proteinase represents the 'tight junction' within mitochondria, sites, where inner and outer membrane are in close proximity [ 11. A reevaluation of the localization of the serine proteinase and carboxypeptidase was initiated by the demonstration [8,9] that the 'group-specific enzymes' [5] isolated from rat small intestine and skeletal muscle were of mast cell origin. Since it is not generally known that rat liver contains mast cells, it was necessary to demonstrate their presence in liver ( fig.1).…”

“…The proteinase in the inner membrane of mitochondria has also been localized [6,7]. Recent experiments [8,9] have shown that the group specific proteinases isolated from small intestine [8] and from skeletal muscle [9] are of mast cell origin. In the light of these findings it became important to examine this possibility, although mast cells have not been described as an integral part of the liver [lo].…”