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A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues

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Summary

N-Methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas contain high levels of a novel leupeptin-sensitive serine endopeptidase. Its properties apparently differ from those of other similar endopeptidases reported to be present in various normal and malignant mammalian tissues. The same leupeptinsensitive serine endopeptidase was also detected in normal rat mammary tissues, but at levels approximately 20 times lower than those in MNU-induced mammary tumors.

This enzyme, which is a trypsin-like serine endopeptidase, preferentially hydrolyzes various synthetic endopeptidase substrates at the carboxyl side of an arginyl residue. It has an apparent Mr of approximately 160,000 and a Stokes radius of 49\rA, as determined by gel filtration. Its isoelectric points range from 4.5 to 4.8, and it has a pH optimum of approximately 7.0. The enzyme is stable from pH 4.0 to 7.0, but is extremely unstable above pH 7.0. Besides leupeptin, its activity is inhibited by antipain, aprotinin, Nα-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, but is not inhibited by soybean trypsin inhibitor. Many other potential inhibitors or activators such as 2-mercaptoethanol, p-hydroxymercuribenzoic acid and EDTA have no effect on its activity. The enzyme is adsorbed to p-aminobenzamidine agarose affinity beads at pH 6.5 and elutes at pH 4.0.

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Abbreviations

BAPA:

α-N-benzoyl-DL-arginine-4-nitroanilide (all other optically active amino acids in the substrates or inhibitors are of the L-configuration except for the valyl residue in DL-Val-Leu-Arg-4NA)

4NA:

4-nitroanilide

Bz:

α-N-benzoyl

Cbz:

α-N-benzyloxycarbonyl

Suc:

α-N-succinyl

Tos:

p-toluenesulfonyl

CH2Cl:

chloromethyl ketone

DMBA:

7,12-dimethylbenz(a)anthracene

MNU:

N-methyl-Nnitrosourea

Mr:

apparent relative molecular mass

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Eto, I., Bandy, M.D. A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues. Mol Cell Biochem 94, 19–36 (1990). https://doi.org/10.1007/BF00223559

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