Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity

• Published in: The Journal of biological chemistry 1991-01, Vol.266 (1), p.98-103
• Main Authors: Poe, M, Blake, J T, Boulton, D A, Gammon, M, Sigal, N H, Wu, J K, Zweerink, H J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cytoplasmic Granules - enzymology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; granzyme B; Granzymes; Humans; Hydrolases; killer cells; Kinetics; Molecular Sequence Data; Protease Inhibitors - pharmacology; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Substrate Specificity; T-Lymphocytes, Cytotoxic - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52407-8

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.