title: Purification and characterization of procathepsin L, a self-processing zymogen of guinea pig spermatozoa that acts on a cathepsin D assay substrate.
abstract: Procathepsin L, the precursor to a powerful lysosomal cysteine proteinase, has been purified to apparent homogeneity from guinea pig spermatozoa, a novel and previously unrecognized source of this catalytically active zymogen. In the range of pH 5.0, procathepsin L (39,000 M(r)) readily self-processed yielding a mature, single-chain proteinase (29,000 M(r)) and an intact propeptide (10,000 M(r)) by what appeared kinetically to be an intramolecular reaction mechanism. These characteristics resembled those reported for the "major excreted protein" (MEP) of malignantly transformed mouse fibroblasts-a protein that has been characterized as the precursor to the mouse analog of human cathepsin L (B. R. Troen, S. Gal, and M. M. Gottesman (1987) Biochem. J. 246, 731-735). Other characteristics shared by the guinea pig and mouse zymogens included proteolytic activity at pH 5.0, homologous N-terminal amino acid sequences, and immunological relatedness. It was thus concluded that acrosomal procathepsin L is the guinea pig analog of MEP. Acrosomal procathepsin L had a specific activity on benzyloxy-carbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) of 30 mumol min-1 mg-1 enzyme at pH 3.2 and 37 degrees C. Relative to the assay substrate, rates on other fluorogenic substrates were 90% for Z-Phe-Cit-NMec, 63% for Z-Leu-Leu-Arg-NMec, 43% for D-Phe-Ser(Bzl)-Phe-Phe-Ala-Ala-p-aminobenzoate (a "specific" cathepsin D assay substrate), and 32% for Z-Val-Val-Arg-NMec. No action was detected on Z-Arg-Arg-NMec or Arg-NMec. Mature cathepsin L showed the same relative order of substrate specificity as its proenzyme form, but the absolute rates were about 5-fold greater. Additionally, the mature (single-chain) form of cathepsin L displayed Km and kcat values on Z-Phe-Arg-NMec that yielded an exceptionally high catalytic coefficient (11,600 s-1 mM-1) compared to values reported for two-chain forms of cathepsin L. Self-processing by acrosomal procathepsin L at pH 5.5 was totally inhibited by leupeptin, cystatin C, Ep-475, and Z-Phe-Phe-CHN2 at 1 microM levels. Gossypol (0.1 mM) gave 94% inhibition. Interestingly, dextran sulfate (100 micrograms ml-1) gave a 3.6-fold increase in the rate of self-processing seen at pH 5.5--a phenomenon of potential physiological relevance in view of the high-negative-charge density present within the hyaluronic acid-rich outer layer (cumulus oophorus) of the ovum.
mesh_terms: D000177:Acrosome / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification*; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D051379:Mice; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D004792:Enzyme Precursors; D010455:Peptides; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L