J. Ken McDonald

Primary structure of the B-chain of porcine relaxin

The sequence of the B-chain of relaxin, and ovarian peptide hormone isolated from ovaries of pregnant sows, has been shown to have the following primary structure: PCA-Ser-Thr-Asn-Asp-Phe-Ile-Lys-Ala-Cys-Gly-Arg-Glu-Leu-Val-Arg-Leu-Trp-Val-Glu-Ile-Cys-Gly-Val-Trp-Ser (2820 daltons). The heterogeneity of relaxin observed during purification procedures is likely to be due to variations in the C-terminal region of the B-chain, in particular the substitution of Gln for Glu20, and the possible addition of arginine or serylarginine at the C terminus. The B-chain exhibited a distribution of sulfhydryl residues relative to one another that is identical to that found in the B-chain of insulin. A similar analogy has already been demonstrated for the A-chains of relaxin and insulin.

Demonstration of a pyroglutamyl residue at the N terminus of the B-chain of porcine relaxin.

Abstract The ovarian peptide hormone relaxin consists, like insulin, of one A- and one B-chain linked by two disulfide bonds. A peptide, isolated from a tryptic digest of the purified B-chain by high-pressure liquid chromatography (HPLC), was examined with the aid of carboxypeptidase C and a pyrrolidonecarboxylyl peptidase. In conjunction with amino acid analysis it could be demonstrated that pyrrolidonecarboxylic acid occupies the N-terminal position of a peptide with the amino acid composition Asp2, Ser, Thr, Phe, Ile, Lys. The appearance of a pyroglutamyl residue in a two-chain hormone is an interesting and unusual feature which has not yet been reported in a similar structure.

Cathepsin L: a latent proteinase in guinea pig sperm

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.

Mammalian lens dipeptidyl aminopeptidase III

Abstract An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K m for this substrate was estimated to be 2.83 × 10 −5 M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.

Dipeptidyl peptidase III of human cataractous lenses. Partial purification.

A partial purification of dipeptidyl peptidase III has been achieved from human cataractous lens. The specific activity was increased 45.5-fold over that of the original aqueous extract. The exopeptidase exhibited a marked preference for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 8.8 and 37 degrees. The Km for this substrate was estimated to be 6.061 X 10(-3). Lens DPP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, puromycin and DFP. The preparation contained leucyl aminopeptidase and a neutral endopeptidase as contaminating proteases.

Glu-Glu Dipeptidase

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 829 is Glu-Glu Dipeptidase.

PEPTIDASES IN CONNECTIVE TISSUE DEGRADATION AND REMODELLING IN REPRODUCTIVE AND INVASIVE TISSUES

An outline is given of the properties and substrate specificities of three lysosomal peptidases, and of efforts to elucidate their potential role in the intracellular degradation of collagen. Use of specific fluorogenic substrates such as Gly-Phe-NNap*, Lys-Ala-NNap and Gly-Pro-Met-NMec allows direct assay of DPP I, DPP II and TPP I respectively. They may hydrolyze macromolecules as exemplified by insulin A and B chains and by poly(Gly-Pro-Ala) as a model collagen α-chain. These are extensively hydrolyzed at acidic pH by the coupled action of the three exopeptidases, which could explain the degradation of proline-rich, collagen-derived polypeptides within lysosomes lacking any known endopeptidases active at prolyl bonds.