The aminopeptidase activity in the human T-cell lymphoma line (Jurkat) is not at the cell surface and is not aminopeptidase N (CD-13)

Abstract

Although lymphocytes are CD-13-negative and therefore should not express the ectoenzyme aminopeptidase N (AP-N), there have been a number of reports suggesting the presence of a cell-surface aminopeptidase with many similarities to AP-N. We have determined aminopeptidase activity with 4-methyl-7-coumarylamide (NMec) derivatives of alanine, leucine, lysine and arginine in Jurkat cells (a human T-cell lymphoma line) and in HL60 cells (a CD-13-positive myeloid leukaemia line) and compared the activities with those of purified pig AP-N and human renal microvillar membranes. Jurkat cell aminopeptidase activity doubled on disrupting the cells and the sensitivity to amastatin increased. When the cells were fractionated only 4% of the activity was recovered in the membrane fraction, compared with 87% recovery for alkaline phosphatase. The profile of activities for intact Jurkat cells was Leu > Ala > Lys > Arg, changing in the cytosolic fraction to Lys > or = Arg > Leu = Ala; the profiles for intact HL60 cells and AP-N were identical, namely Ala > Leu > Arg > Lys. The Km values for the hydrolysis of Ala-NMec and Leu-NMec by Jurkat cells were 65 microM and 11 microM, in each case some 6-fold lower than those for AP-N. The pH-activity curves for the hydrolysis of Ala-NMec by Jurkat cells and human renal microvillar membranes were displaced by almost 1 pH unit and the activity was not sensitive to the anionic composition of the buffers. However, a 3-fold activation of the cytosolic activity by 0.1 M NaCl was observed with Arg-NMec as substrate. With Ala-NMec as substrate, the sensitivity of the aminopeptidase activity to inhibitors increased markedly after disrupting the cells, but still differed from that observed with purified pig AP-N; the concentrations giving 50% inhibition were as follows (values for AP-N in parentheses): amastatin. 28 nM (150 nM); bestatin, 12 microM (43 microM), probestin, 100 nM (< 10 nM), puromycin, 30 microM (> 1 mM). Anion exchange chromatography on Mono Q revealed two activities: that of peak I preferentially hydrolysed Arg-NMec, was activated by NaCl and was insensitive to amastatin; while that of peak II was strongly inhibited by amastatin and had a broad specificity.(ABSTRACT TRUNCATED AT 400 WORDS)