Comparative studies of human recombinant 74- and 54-kDa L-histidine decarboxylases.
We have expressed and characterized human recombinant 74-kDa (rHDC74) and 54-kDa (rHDC54) L-histidine decarboxylases (HDCs) in Sf9 cells. By immunoblot analysis, rHDC74 and rHDC54 were shown to be localized predominantly in the particulate and soluble fractions, respectively. rHDC74 exhibited histamine-synthesizing activity equivalent to that of rHDC54. The existence of 74- and 54-kDa HDCs was also confirmed in the particulate and supernatant fractions of the cell lysate, respectively, from the human basophilic leukemia cell line KU-812-F. The ratio of HDC activity to immunoreactivity was similar for the two forms of the enzyme. The specific activity of purified rHDC54 (1.12 mumol/mg/min) was comparable to those of HDCs from other mammalian tissues or cells. The purified rHDC54 was eluted as a monomer form from a Superdex-200 column; the molecular mass of the enzyme was approximately 54 kDa on SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol. The HDC activity of rHDC54 significantly decreased on dialysis against buffer without pyridoxal 5'-phosphate; addition of pyridoxal 5'-phosphate to the dialysate readily increased in the enzyme activity to the original activity. Taken together, these results suggest that human HDC functions as both 74- and 54-kDa forms having equivalent HDC activity, which are localized in the particulate and soluble fractions, respectively, and that the latter form exhibits its activity as a monomer form.
Pharmaceutical Basic Research Laboratories, Japan Tobacco, Inc., Japan.
K
K
Yatsunami
M
M
Tsuchikawa
M
M
Kamada
K
K
Hori
T
T
Higuchi
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006640:Histidine Decarboxylase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D006640:Histidine Decarboxylase