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Research ArticleDEVELOPMENT AND HORMONE ACTION
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Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis

Angus S. Murphy, Karen R. Hoogner, Wendy Ann Peer, Lincoln Taiz
Angus S. Murphy
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Karen R. Hoogner
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Wendy Ann Peer
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Lincoln Taiz
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Published March 2002. DOI: https://doi.org/10.1104/pp.010519

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  • Fig. 1.
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    Fig. 1.

    Purification of NPA-binding proteins with Tyr-AP activity from Arabidopsis PM vesicles. A, Silver stained SDS-PAGE gels of Tyr-AP peak fractions (1 μg of total protein per lane) from each purification stage. PM, Solubilized PM vesicle proteins; S300, Sephacryl S300 gel permeation chromatography peak fraction; Q, Q resin anion-exchange peak fraction; NPA, NPA affinity fraction (350 mm KCl). B, Tyr-aminofluoromethylcoumarin (AFC) enzyme overlay visualization of AP activity of NPA-binding proteins analyzed by native PAGE. C, Periodic acid-Schiff's reagent (PAS) staining of NPA-binding glycoproteins. D, Elution profiles (50–300 mmKCl) of proteins from NPA affinity beads. Dotted line,A 280; solid line, Tyr-AP activity assayed fluorometrically with Tyr-AFC substrate. Protein peak fractions identified with Roman numerals. E, Silver-stained 4% to 20% (w/v) SDS-PAGE gel of combined NPA affinity fractions II and III (100 ng of total protein).

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    Fig. 2.

    Flavonoid binding of NPA affinity chromatography peak fractions (Fig. 1D). Aglycone flavonoid binding of solubilized PM and NPA affinity fraction proteins was detected fluorometrically with DPBA. Values shown are means and sd of three independent experiments.

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    Fig. 3.

    Multiple sequence alignments of AtAPM1 and AtAPP1 with orthologous animal proteins. Consensus sequences are shadowed in black; conserved sequences are in white boxes. Putatative catalytic sites are indicated by solid lines. Glycosylation sites are indicated by triangles. A, AtAPM1. Mouse PSA, Mouse puromycin-sensitive microsomal AP (Q11011); DmAPM, Drosophila melanogasterpuromycin-sensitive AP (AAG48733). B, AtAPP1. HuAPP1, Human cytosolic APP1 (AAH05126). Human membrane-bound APP2 (AAG28480) used to determine consensus alignment is not shown.

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    Fig. 4.

    AtAPM1 and AtAPP1 expression in adult and seedling tissue. A, Northern blots of adult tissue. Total RNA (7.26 μg) was extracted from roots (R), rosette leaves (RL), cauline leaves (CL), lower inflorescence stems (LI), upper inflorescence stems (UI), young flowers (YF), mature flowers (MF), and siliques (S). Blots were probed and visualized with digoxygenin (DIG)-labeled AtAPM1and AtAPP1 probes. rRNA is shown as gel loading control. B, Northern-blot analysis of AtAPM1 and AtAPP1 mRNA in 2- to 10-d-old seedlings. Ten micrograms of total RNA was extracted and probed with DIG-labeled probes.

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    Fig. 5.

    Immunolocalization of AtAPM1 and AtAPP1 in Arabidopsis. Western blots with polyclonal anti-AtAPM1 and AtAPP1 antibodies. In western blots utilizing the anti-AtAPP1 polyclonal antibodies, only protein bands that did not cross-react with pre-immune antisera are shown. A, Cellular partitioning of the native AtAPM1 and AtAPP1 proteins. Total protein (10 μg) from 5-d-old light-grown seedlings was used in all cases except lane P5.5, which used protein from 5.5-d-old seedlings. D, Cellular debris; S, soluble; M, microsomal; P5, PM proteins from 5-d-old seedlings; P5.5, PM proteins from 5.5-d-old seedlings. B, Microsomal preparations (10 μg of total protein) from adult tissues. R, Roots; L, rosette leaves; UI, upper inflorescence; LI, lower inflorescence; F, flowers. C, Western-blot analysis of Tyr-AP peak fractions from each purification stage with anti-AtAPM1 and AtAPP1 polyclonal antibodies. All samples are from 5-d-old light-grown Arabidopsis seedlings. Ten micrograms of total protein was used in lanes M, S, and Q. Two hundred nanograms of total protein was used in lane NPA. M, Microsomal; S, Sephacryl S-300; Q, Q anion-exchange; NPA, NPA affinity.

Tables

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    Table I.

    Specific AP and NPA amidase activities of PM proteins at each step of purification

    SubstratePMSolubilizedS300Q ResinNPA Peak IINPA Peak IIINPA Peak II + III
    nmol min−1 mg−1
    Tyr-AFC31.616.678.1226.859.37.7392.4
    Ala-Pro-AFC17.613.515.722.489.51.966.3
    Trp-AFC20.217.13.34.72.21.74.7
    Pro-AFC16.715.627.931.527.15.946.1
    Ala-AFC2.62.416.162.827.27.584.6
    Leu-AFC10.39.418.219.72.53.12.1
    Ala-NA2.11.62.731.632.118.445.7
    NPA6.34.45.825.117.19.542.1

    Fractions (100 ng) from each purification step were assayed for activity after dialysis against AP assay buffer for 1 h at 4°C. AFC conjugate concentrations were 20 μm. Ala-β-naphthylamide (NA) and NPA concentrations were 100 μm. PM, PM-enriched membranes; Solubilized, detergent-solubilized PM proteins; S300, Sephacryl S-300 PMSF-insensitive peak fraction; Q resin, Q anion-exchange PMSF-insensitive peak fraction; NPA peaks I through III, fractions (or combinations of fractions) corresponding to NPA affinityA 280 peaks (Fig. 1D). All assays were repeated at least three times with similar results.

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      Table II.

      Effects of inhibitors on AP activities of PM NPA-binding proteins

      SubstrateControl SANPA (50 μm)EGTA (2 mm)PMSF (100 μm)Bestatin (20 μm)Amastatin (20 μm)Apstatin (20 μm)Puromycin (20 μm)PAQ22 (20 μm)Dithiothreitol (DTT; 5 mm)Vanadate (5 mm)Thiorphan (50 μm)
      nmol min−1 mg−1 %
      Tyr-AFC381 ± 17.5655878417253614810895101
      Ala-Pro-AFC71 ± 7.77684718487268798699397
      Ala-AFC89 ± 5.35420873637945334969798
      Pro-AFC44 ± 9.28179795888616699739097

      Proteins from NPA affinity fractions II and III (Fig. 1D) were reconstituted by dialysis in AP assay buffer, then assayed fluorometrically for AP activity with AFC conjugates (20 μm) in the presence or absence of inhibitors. Values shown for inhibitor assays are percent control-specific activities (SA). Values calculated are derived from means and sd of three independent experiments. Table entries marked with an asterisk were significantly different from their control (P < 0.05) when analyzed by Student-Newman-Keuls ANOVA.

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        Table III.

        Identification of PM AP-associated proteins purified by NPA affinity chromatography

        Purified ProteinPost-translational ModificationsInternal Fragment SequencedGenBank Protein Identification No.Amino Acids (Predicted)Characterized OrthologSimilarity
        %
        Aminopeptidases
         FII-103 (AtAPM1)GP (E,P)(K)TLEVPTDLVALSN CAB36783 879PS aminopeptidase M (mouse)/IRAP53
         FII-70 (AtAPP1)GP (E,P)(K)VSDEANSYFNGLG CAB16823 634Aminopeptidase P1 (human)60
         FIII-42 (AtAPM1Δ)GP (E,P)(R)VATVVAHELAHQWF CAB36783 UnknownTruncated form of AtAPM1∼68
        AP-associated proteins
         FIII-68 (AtMyA1)GP (P)(R)SVFASAALGK AAB63637 619Myrosinase-associated protein (Brassica napus)62
         FII-55 (AtHSP70p)GP (E,P)(K)DVLK-(R)LVEHFAADEFNK CAB10440 484HSP70 (Arabidopsis)∼68
         FII-44/66 (AtFAGP2)GPI; GP (E,P)(K)AFSDILKSTGADK CAB40990 403Fasciclin-like arabinogalactan protein 2 (Arabidopsis)59
         FIII-40 (ATAPL1)GP (E,P)(K)LFVFGDSYADTGNI CAB82926 359Anther-specific Pro-rich protein (Arabidopsis)46
         FIII-24 (AtGSTF2)GP (P)(K)NISQYAIMAIQ P46422 212Auxin-binding GST II (Arabidopsis)100

        Amino acid sequences of tryptic fragments from PM proteins contained in NPA affinity peaks II and III were obtained as described (see “Materials and Methods”). Protein identifications were assigned by NPA affinity fraction (FII or FIII) and SDS-PAGE apparent molecular mass. GenBank protein identification nos. were assigned if there was a unique database match with a similar predicted molecular mass. GP, Glycoprotein; GPI, glycosyl-phosphatidylinositol anchor; E, experimental evidence; P, predicted. K or R, Inferred lysine or arginine residues.

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          Table IV.

          Enzymatic assays of AtAPM1 and AtAPP1 produced by in vitro translation

          Substrate/InhibitorAtAPM1AtAPP1
          nmol min−1 mg−1
          Tyr-AFC59 ± 5.836 ± 4.9
          Tyr-AFC + kaempferol32 ± 3.1∗ 30 ± 5.5
          Tyr-AFC +PAQ2217 ± 5.4∗ 47 ± 3.1∗
          Tyr-AFC + apstatin54 ± 6.915 ± 4.7∗
          Tyr-AFC +EGTA16 ± 6.4∗ 19 ± 2.2∗
          Ala-Pro-AFC6 ± 4.274 ± 3.1
          Ala-AFC35 ± 4.25 ± 4.8
          Pro-AFC18 ± 5.329 ± 6.1
          Trp-AFC4 ± 3.220 ± 1.7
          Leu-AFC5 ± 3.17 ± 1.9
          NPA8 ± 3.43 ± 1.4

          AP and amidase assays with substrates and inhibitors indicated. Inhibitor concentrations used were as in Table II. Units are nmol AFC or β-NA released min−1 mg−1. Values reported are means and sd of three independent experiments. Student-Newman-Keuls ANOVA indicates that the Tyr-AFC-related values reported are different from a normal distribution (P < 0.001). Values marked with an asterisk differ from controls in pair-wise comparisons (P < 0.05).

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            Table V.

            Specific [3H]NPA binding of PM vesicles, affinity fractions, and in vitro translation products of AtAPM1 and AtAPP1

            Sample[3H]NPA Bound
            pmol mg−1
            PM0.1 ± 0.03
            Fraction I0.0 ± 0.03
            Fraction II7.1 ± 1.67
            Fraction III8.2 ± 1.25
            Fraction IV17.2 ± 3.07
            Fraction II+ III17.8 ± 2.52
            Fraction II + III +APAb5.9 ± 3.55∗
            Fraction IV +APAb16.8 ± 3.25
            In vitro AtAPM13.2 ± 1.07
            In vitro AtAPP11.9 ± 1.6

            Specific activity is calculated as [3H]NPA binding − [3H]NPA binding in the presence of 1,000× cold NPA. Fractions I through IV, NPA affinity fractions I–IV; APAb, mixed polyclonal anti-AtAPM1 and AtAPP1 antibodies (see “Materials and Methods”); in vitro, in vitro translation products. Values reported are means and sd of three independent experiments. Student-Newman-Keuls ANOVA indicates that the values reported are different from a normal distribution (P < 0.001). Values with an asterisk differ from their control in pairwise Student's t tests (P < 0.01).

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            Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis
            Angus S. Murphy, Karen R. Hoogner, Wendy Ann Peer, Lincoln Taiz
            Plant Physiology Mar 2002, 128 (3) 935-950; DOI: 10.1104/pp.010519

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            Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis
            Angus S. Murphy, Karen R. Hoogner, Wendy Ann Peer, Lincoln Taiz
            Plant Physiology Mar 2002, 128 (3) 935-950; DOI: 10.1104/pp.010519
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