Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP.
The family M1 of Zn-dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA-library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn-binding motif and displays high homology to the other M1 family members except for its N-terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS-7 and BHK cells. Northern-blot analysis revealed ubiquitous expression of a major 3. 1-kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl beta-naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin-insensitive leucyl-specific aminopeptidase is a signal-sequence-bearing member of family M1 and may be another example of the small subset of substrate-specific peptidases.
Schomburg
L
L
Max-Planck-Institut für Experimentelle Endokrinologie, Hannover, Germany. lutz.schomburg@mail.uni-wuerzburg.de
Kollmus
H
H
Friedrichsen
S
S
Bauer
K
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002413:Cations, Divalent; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D015723:Gene Library; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008647:Mesocricetus; D008969:Molecular Sequence Data; D005810:Multigene Family; D009928:Organ Specificity; D010618:Phenanthrolines / Q000494:pharmacology; D010902:Pituitary Gland / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000737:chemistry; D011691:Puromycin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; D002614:Chelating Agents; D018076:DNA, Complementary; D010618:Phenanthrolines; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011691:Puromycin; D004492:Edetic Acid; D007931:Leucyl Aminopeptidase; C025205:1,10-phenanthroline