Please use this identifier to cite or link to this item: http://ahro.austin.org.au/austinjspui/handle/1/9626
Title: Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.
Authors: Smyth, Mark J;Wiltrout, T;Trapani, Joseph A;Ottaway, K S;Sowder, R;Henderson, L E;Kam, C M;Powers, J C;Young, H A;Sayers, T J
Affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.
Issue Date: 5-Dec-1992
Citation: The Journal of Biological Chemistry; 267(34): 24418-25
Abstract: We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.
Internal ID Number: 1447189
URI: http://ahro.austin.org.au/austinjspui/handle/1/9626
URL: http://www.ncbi.nlm.nih.gov/pubmed/1447189
Type: Journal Article
Subjects: Amino Acid Sequence
Animals
Base Sequence
Chromatography, Affinity
Chromatography, Gel
Chromatography, High Pressure Liquid
Cloning, Molecular.methods
Cytoplasmic Granules.enzymology
Electrophoresis, Polyacrylamide Gel
Humans
Killer Cells, Natural.enzymology
Leukemia, Experimental.enzymology.genetics
Molecular Sequence Data
Oligodeoxyribonucleotides
Oligopeptides.metabolism
Polymerase Chain Reaction
Rats
Recombinant Proteins.genetics.isolation & purification
Restriction Mapping
Sequence Homology, Amino Acid
Serine Endopeptidases.genetics.isolation & purification.metabolism
Substrate Specificity
Tumor Cells, Cultured
Appears in Collections:Journal articles

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