THE AMINOPEPTIDASE ACTIVITY IN THE HUMAN T-CELL LYMPHOMA LINE (JURKAT) IS NOT AT THE CELL-SURFACE AND IS NOT AMINOPEPTIDASE-N (CD-13)

Although lymphocytes are CD-13-negative and therefore should not expre ss 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) an d in HL60 cells (a CD-13-positive myeloid leukaemia line) and compared the activities with those of purified pig AP-N and human renal microv illar membranes. Jurkat cell aminopeptidase activity doubled on disrup ting the cells and the sensitivity to amastatin increased. When the ce lls were fractionated only 4% of the activity was recovered in the mem brane fraction, compared with 87% recovery for alkaline phosphatase. T he profile of activities for intact Jurkat cells was Leu > Ala > Lys > Arg, changing in the cytosolic fraction to Lys greater than or equal to Arg > Leu = Ala; the profiles for intact HL60 cells and AP-N were i dentical, namely Ala > Leu > Arg > Lys. The K-m values for the hydroly sis of Ala-NMec and Leu-NMec by Jurkat cells were 65 mu M and 11 mu M, in each case some 6-fold lower than those for AP-N. The pH-activity c urves for the hydrolysis of Ala-NMec by Jurkat cells and human renal m icrovillar membranes were displaced by almost 1 pH unit and the activi ty was not sensitive to the anionic composition of the buffers. Howeve r, a 3-fold activation of the cytosolic activity by 0.1 M NaCl was obs erved with Arg-NMec as substrate. With Ala-NMec as substrate, the sens itivity of the aminopeptidase activity to inhibitors increased markedl y after disrupting the cells, but still differed from that observed wi th 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 mu M (43 mu M), probestin, 100 nM (< 10 nM), puromycin, 3 0 mu M (> 1 mM). Anion exchange chromatography on Mono Q revealed two activities: that of peak I preferentially hydrolysed Arg-NMec, was act ivated by NaCl and was insensitive to amastatin; while that of peak II was strongly inhibited by amastatin and had a broad specificity. Jurk at cells hydrolysed [Leu(5)]enkephalin, the activity increasing 4-fold on cell disruption, of which 89% was recovered in the cytosolic fract ion and less than 3% in the membrane pellet, contrasting with HL60 cel ls for which most of the activity was recovered in the 100000 g pellet . We conclude that there is no evidence for the presence of AP-N in Ju rkat cells and that the cytosolic activity comprises two aminopeptidas es, one resembling aminopeptidase B. The other, which accounted for 90 % of the activity with Ala-NMec, was sensitive to amastatin, but not t o metal chelators and was not activated by Cl-. The differences in pro perties between the activities of intact and disrupted cells are expla ined by a permeability barrier to entry of substrates and inhibitors.