Expression and characterization of recombinant mouse mastocytoma histidine decarboxylase.
The possibility of post-translational processing of mouse mastocytoma histidine decarboxylase (HDC; EC 4.1.1.22) was investigated. The molecular mass of the recombinant HDC expressed in Sf9 cells using HDC cDNA from mouse mastocytoma cells was determined to be 74 kDa by SDS-PAGE. In contrast to the native HDC from mastocytoma cells, the recombinant 74 kDa HDC was essentially inactive and precipitable in Sf9 cells. On the other hand, deletion mutants of the recombinant HDC lacking a C-terminal region equivalent to 10 (64 kDa) or 20 kDa (54 kDa) in size were present as active forms in the soluble fraction of Sf9 cells. To examine the C-terminal deletion of the 74 kDa species yielding the 53 kDa species by means of the immunoblotting analysis, two peptides (corresponding to residues 323-337 and 572-586 of the recombinant 74 kDa HDC peptide) were synthesized, and rabbit antiserum specific for each peptide was prepared. On immunoblotting analysis, anti-peptide 323-337 antiserum recognized both the recombinant 74 kDa and native enzyme subunit peptides, but anti-peptide 572-586 antiserum recognized only the recombinant 74 kDa peptide, i.e., not the native enzyme subunit peptide. Furthermore, HDC activity in the crude extract from Sf9 cells was not precipitable with antipeptide 572-585 antiserum. These results strongly suggest that the 53 kDa subunit peptide of native mastocytoma HDC is derived from the unidentified inactive 74 kDa HDC peptide, probably by post-translational processing of HDC in its C-terminal region.
Yamamoto
J
J
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Fukui
T
T
Suzuki
K
K
Tanaka
S
S
Yatsunami
K
K
Ichikawa
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006640:Histidine Decarboxylase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012515:Mast-Cell Sarcoma / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016296:Mutagenesis; D016376:Oligonucleotides, Antisense; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D006640:Histidine Decarboxylase