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Title	Purification, characterization, and distribution of an ovarian dipeptidyl peptidase II from pregnant sows that preferentially cleaves prolyl bonds
Author	Eisenhauer, Donald Alan
Date	1984
College	College of Graduate Studies
Department	Biochemistry
Extent	viii, 110 leaves : ill. plates; 29 cm
Subject.MeSH	Dipeptidyl Peptidases--isolation & purification
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Digitization Specifications	CanoScan 8400, Archival files are tiffs.
Date Digital	2015
Rights	Copyright is held by the author.
Language	English
SC County	Charleston County (S.C.)
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd1984_eisenhauer_da
Abstract	An unusual species form of dipeptidyl peptidase II (DPP II) was identified in sow ovaries on the basis of its 8-fold greater rate of dipeptide release (at pH 6.0) from Phe-Pro-2-naphthylamide (NNap) relative to Lys-Ala-NNap, a substrate typically hydrolyzed faster than Phe-Pro-NNap by DPP II seen in other species. Phe-Pro-NNap, which is hydrolyzed optimally at pH 6.0, was used in the standard fluorometric assay of porcine DPP II (Km 45 μM, pH 6.0, 37°C). The enzyme was purified 1400-fold, to a specific activity of 28 units/mg protein, from an aqueous extract of pregnant sow ovaries by ammonium sulfate fractionation, molecular exclusion chromatography, chromatofocusing (pH 6.5-4.0), affinity chromatography on con A-Sepharose 4B, and ion exchange on high performance liquid chromatography. Polyacrylamide gels (non-denaturing conditions) showed an apparently homogeneous DPP II. Electrophoresis of the reduced, SDS-treated enzyme revealed the presence of 2 identical Mr-54,OOO subunits. This compares with a Mr of 110,000 obtained by molecular exclusion chromatography under nondenaturing conditions. DPP II contained about 2% carbohydrate, primarily mannose, and had a pI of 5.0. Specificity studies revealed a strong preference for naphthylamide substrates containing penultimate prolyl residues, e.g., Phe-Pro-NNap (100%), Lys-Pro-NNap (43%), Arg-Pro-NNap (26%), and Lys-Ala-NNap (12%). No action occurred on N-terminal-blocked substrates or Pro-NNap. Among oligopeptide substrates, significant rates were seen only on tripeptides, i.e., Phe-Pro-Ala (100%), Lys-Ala-Ala (35%), Ala-Ala-Ala (33%), and Gly-Pro-Ala (26%). No action occurred on Z-Gly-Pro-Ala. Porcine DPP II belongs to the serine class of proteases as judged by its sensitivity to a number of serine protease inhibitors that included diisopropylfluorophosphate (DFP). Thiol-blocking and metal binding agents had little or no effect. DPP II was shown to have a lysosomal localization in fresh porcine ovaries, as determined from assays done on subcellular (organelle) fractions obtained from sucrose density gradients following equilibrium-density centrifugation. The fractionation studies demonstrated 2 populations of lysosomes (light" and "heavy) in the porcine ovary. DPP II activity was measured in both pools and its activity profile in the fractionated gradient coincided with two established lysosomal enzymes, acid phosphatase and DPP I. The "heavy" lysosomes were contaminated with mitochondria. Ovarian DPP II was visualized histochemically by light microscopy using Fast Blue B and the 4-methoxy-2-naphthylamide derivatives of Lys-Ala, Phe-Pro, and Lys-Pro. Bright red azo dye reaction product was localized in granules associated with regressing follicles (atretic follicles) and in the basal cells of the follicle wall. DPP II activity was dispersed in the granulosa cells and stroma. These areas of DPP II activity were coincident with gelatinase activity detected by incubating fresh-frozen sections on gelatin membranes at pH 5.0. Aqueous extracts (10%, w/v) were prepared from ten sow ovaries (n=10 for each stage) collected at three stages of the estrous cycle (postovulatory, luteal, and proestrous). Levels of DPP II were highest in the ovary during proestrous. Proestrous is the stage when the collagenous tissues of the follicle are being rapidly degraded to facilitate release of the ovum. In summary, DPP II of porcine ovaries is a homodimeric glycoprotein with a lysosomal localization. Unlike most exopeptidases, it belongs to the serine catalytic class. It catalyzes the release of dipeptides from dipeptide derivatives, in particular those containing a penultimate prolyl residue. The protease is localized primarily in the basal cells of the follicle wall and in granules of atretic follicles. Maximal levels of DPP II activity are seen in the ovary during the proestrous phase of the estrous cycle. These findings support a probable contribution by DPP II to the late stages of intralysosomal collagen degradation.

Purification, characterization, and distribution of an ovarian dipeptidyl peptidase II from pregnant sows that preferentially cleaves prolyl bonds

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