Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).
We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.
Sayers
T J
TJ
Biological Carcinogenesis and Development Program, PRI/DynCorp., Frederick, MD 21702-1201.
Wiltrout
T A
TA
Sowder
R
R
Munger
W L
WL
Smyth
M J
MJ
Henderson
L E
LE
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000737:chemistry; D006131:Growth Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006493:Heparin / Q000378:metabolism; D007694:Killer Cells, Natural / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D006131:Growth Inhibitors; D014409:Tumor Necrosis Factor-alpha; D006493:Heparin; D012697:Serine Endopeptidases