Two cytosolic puromycin-sensitive aminopeptidase isozymes in chicken brain: molecular homology to brain-specific 14-3-3 protein.
Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co(2+)-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.
Hui
K S
KS
Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962.
Saito
M
M
Hui
M
M
Saito
M
M
Lajtha
A
A
Yamamoto
K
K
Osawa
T
T
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010449:Peptide Mapping; D011691:Puromycin / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D011691:Puromycin; D014446:Tyrosine 3-Monooxygenase; D000626:Aminopeptidases; D014357:Trypsin