Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column.
The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.
Jiang
S
S
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.
Hasselkus-Light
C S
CS
Ojcius
D M
DM
Young
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification*; D053804:Granzymes; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017385:Sequence Homology; D012697:Serine Endopeptidases / Q000737:chemistry; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*
D004950:Esterases; C022635:serine esterase; D053804:Granzymes; C056485:Gzmf protein, mouse; D012697:Serine Endopeptidases