chymostatin and Leukemia--Erythroblastic--Acute

We investigated and characterized the ATP-dependent protease in human erythroleukemia, K562 cells. The succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in a K562 lysate at pH 9 rose more than 10-fold with the addition of 1 mM ATP. The effect of ATP on the protease activity was dose-dependent and inhibited by the addition of ADP. This activity was not inhibited by EDTA, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o)butane or leupeptin, but was strongly inhibited by chymostatin and diisopropylfluorophosphate. The protease activity was eluted just after the void volume from a G3000SW HPLC column. The above results suggest that this protease is identical to the high-molecular-mass protease, ingensin, previously reported by us. The ATP-dependent increase in the protease activity was due to prevention of the inactivation of the protease by ATP, and not to activation of the protease itself in the reaction mixture at 37 degrees C. The depressed succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in the ATP-depleted lysate was restored to the same level by the detergent, SDS. Therefore, we conclude that the inactivation of ingensin occurring on preincubation is not irreversible.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Chromatography, Gel; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Edetic Acid; Enzyme Activation; Enzyme Reactivators; Humans; Hydrogen-Ion Concentration; Hydrolysis; Isoflurophate; Kinetics; Leukemia, Erythroblastic, Acute; Multienzyme Complexes; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Sodium Dodecyl Sulfate; Substrate Specificity; Tumor Cells, Cultured

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.

Topics: alpha 1-Antichymotrypsin; Amino Acids; Binding, Competitive; Chymotrypsin; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Endopeptidases; Humans; Isoflurophate; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Oligopeptides; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Endopeptidases; Substrate Specificity