Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily.
A protease in the culture medium of Tetrahymena pyriformis was purified to homogeneity. The purified protease had an apparent molecular mass of 28 kDa on SDS/PAGE. The amino acid sequences of the N-terminal and internal peptides of the protease showed complete identity with those of tetrain, an enzyme previously reported as a Tetrahymena cysteine protease but not characterized in detail. Two overlapping cDNA clones for tetrain were sequenced, and the nucleotide sequence predicts that these clones encode a 330-amino acid protein composed of a 16-residue N-terminal signal sequence followed by a 103-residue propeptide and a 211-residue mature protease. The primary structure and enzymatic properties support the conclusion that tetrain belongs to the cathepsin L subfamily. Immunoblotting analyses showed that mature tetrain was found exclusively in the culture medium. Immunofluorescence microscopy demonstrated that tetrain was concentrated in or around the food vacuoles of cells in the late logarithmic phase, but the staining of food vacuoles was not obvious in the stationary phase. These results suggest that tetrain is synthesized at the logarithmic phase and is secreted into the culture medium as a mature form.
Suzuki
K M
KM
Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima, Japan.
Hayashi
N
N
Hosoya
N
N
Takahashi
T
T
Kosaka
T
T
Hosoya
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry*; D002455:Cell Division / Q000502:physiology; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013768:Tetrahymena / Q000201:enzymology*
D003470:Culture Media; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C106859:tetrain; D056668:Cathepsin L