Identification of holocarboxylase synthetase (HCS) proteins in human placenta.
Holocarboxylase synthetase (HCS) is a key enzyme in biotin utilization in eukaryotic cells. In a previous work from our laboratory, we described the cloning and sequencing of a full-length human HCS cDNA. Due to the presence of three candidate sites for initiation of translation, the identification of full-length HCS proteins remains uncertain. Using antibodies directed against human HCS sequences, we have identified, in human placenta, three cytosolic HCS proteins, of 86, 82 and 76 kDa. Similar results were observed in lysates of cells transfected with an HCS expression vector, as well as with human HCS cDNA transcribed and translated in a cell-free system. When anti-HCS antibodies were tested for their ability to inhibit HCS enzymatic activity, only the antibody directed against a region of HCS from Ile128 to Pro398, and not the antibodies against more proximal N-terminal regions inhibited HCS activity, suggesting that the sequence from Ile128 to Pro398 is essential for the catalytic activity of this enzyme. HCS synthesized in a cell-free system was not translocated into rat liver mitochondria. These results suggest that our human HCS cDNA encodes the cytosolic forms of the enzyme. These results also suggest that mRNA encoding cytosolic HCS can be translated from all three translation initiation codons, Met1, Met7 and Met58.
Hiratsuka
M
M
Department of Biochemical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan. mhiratsuka@ped.med.tohoku.ac.jp
Sakamoto
O
O
Li
X
X
Suzuki
Y
Y
Aoki
Y
Y
Narisawa
K
K
D000818:Animals; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D018387:Codon, Initiator; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007118:Immunoassay; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D014176:Protein Biosynthesis; D051381:Rats; D014158:Transcription, Genetic
D018387:Codon, Initiator; D018076:DNA, Complementary; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases