Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells

• Published in: FEBS letters 1994-03, Vol.342 (1), p.53-56
• Main Authors: Yamada, Masatoshi, Sukenaga, Yoshikazu, Fujii, Hideji, Abe, Fuminori, Takeuchi, Tomio
• Format: Article
• Language: English
• Subjects: 4-methylcoumaryl-7-amide; 7-amino-4-methyl-coumarin; absorbance unit full scale; AMC; Amino Acid Sequence; Aminopeptidase B-like enzyme; Aminopeptidases - antagonists & inhibitors; Aminopeptidases - chemistry; Aminopeptidases - isolation & purification; Aminopeptidases - metabolism; Analytical, structural and metabolic biochemistry; AUFS; Biological and medical sciences; Cations, Divalent - pharmacology; Chlorides - pharmacology; cluster of differentiation; CNBr; cyanogen bromide; dimethyl slufoxide; DMSO; EDTA; EGTA; Enzymes and enzyme inhibitors; ethylene glycol bis (β-aminoethylether)-N,N,N',N',-tetra acetic acid; ethylenediaminetetraacetic acid; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Hydrolases; K562 cell; kDa; kilodaltons; Leucine - analogs & derivatives; Leucine - pharmacology; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - enzymology; MCA; Molecular Sequence Data; Molecular Weight; p-chloromercuribenzoic acid; PCMB; phenylmethylsulfonyl fluoride; phenylthiohydantoin; PMSF; PTH; SDS-PAGE; sodium dodecyl polyacrylamide gel electrophoresis; Substrate Specificity; TFA; trifluoroacetic acid; Tumor Cells, Cultured
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(94)80583-0

A ubenimex-sensitive aminopeptidase B-like enzyme was purified from the non-membrane-bound fraction of K562 cells by a series of chromatographic procedures and slab-gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS-PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn 2+, Cu 2+, Cd 2+, and p-chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N-terminus of the protein was not determined. Partial amino acid sequencing revealed that the N-terminus of this aminopeptidase B-like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid-releasing reaction showed this enzyme to be a new aminopeptidase.