Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour.
Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family.
Eto
I
I
Department of Nutrition Sciences, University of Alabama, Birmingham 35294.
Grubbs
C J
CJ
D000230:Adenocarcinoma / Q000139:chemically induced / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002850:Chromatography, Gel; D004586:Electrophoresis; D004789:Enzyme Activation; D004795:Enzyme Stability; D005260:Female; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007976:Leupeptins / Q000494:pharmacology*; D008325:Mammary Neoplasms, Experimental / Q000139:chemically induced / Q000201:enzymology*; D008770:Methylnitrosourea; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013379:Substrate Specificity; D014020:Tissue Extracts / Q000494:pharmacology
D007976:Leupeptins; D014020:Tissue Extracts; D008770:Methylnitrosourea; D001586:Benzoylarginine Nitroanilide; D012697:Serine Endopeptidases; C032854:leupeptin