A novel membrane-bound serine esterase in human T4+ lymphocytes immunologically reactive with antibody inhibiting syncytia induced by HIV-1. Purification and characterization.
A novel membrane-bound serine esterase, named tryptase TL2, which is immunologically reactive with the antibody inhibiting induction of syncytia by human immunodeficiency virus-1 (HIV-1) (Hattori, T., Koito, A., Takatsuki, K., Kido, H., and Kutunuma, N. (1989) FEBS Lett., 248, 48-52), has been purified from a human T4+ lymphocyte clone. The enzyme has a molecular mass of 198 +/- 15 kDa, as judged by gel-permeation liquid chromatography, and is composed of two subunits of 32 kDa and four subunits of 28 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Studies with model peptide substrates showed that the enzyme preferentially recognized L-arginine and cleaved Boc-Gln-Gly-Arg-4-methyl-coumaryl-7-amide and Boc-Gln-Ala-Arg-4-methyl-coumaryl-7-amide with high efficiency at a pH optimum of 8.5. The enzyme was strongly inhibited by the envelope glycoprotein gp 120 of HIV-1, by synthetic peptides with the sequence GPGR in their center, which corresponds to the principal neutralizing epitope of the gp 120s of various HIV-1 strains, by Kunitz-type inhibitors with the sequence GPCR in their active site, such as trypstatin, HI30, and [Arg15, Glu52]aprotinin and by the microbial inhibitors leupeptin and antipain. Studies on the subcellular distribution of tryptase TL2, immunohistochemical analysis, and cell surface radioiodination indicated that the enzyme is mainly localized in the plasma membrane.
Kido
H
H
Division of Enzyme Chemistry, University of Tokushima, Japan.
Fukutomi
A
A
Katunuma
N
N
D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology*; D002459:Cell Fusion; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D004950:Esterases; D015699:HIV Envelope Protein gp120 / Q000494:pharmacology; D015658:HIV Infections / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011233:Precipitin Tests; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D053802:Tryptases; D014407:Tumor Cells, Cultured
D015699:HIV Envelope Protein gp120; D004950:Esterases; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D012697:Serine Endopeptidases; D053802:Tryptases; C070201:tryptase TL(2)