Intracellular localization of the 74- and 53-kDa forms of L-histidine decarboxylase in a rat basophilic/mast cell line, RBL-2H3

Satoshi Tanaka, Ken Ichi Nemoto, Eriko Yamamura, Atsushi Ichikawa

Research output: Contribution to journalArticle

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Abstract

To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/ mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40% of HDC activity accompanied by over 90% of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SD-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.

Original languageEnglish
Pages (from-to)8177-8182
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number14
DOIs
Publication statusPublished - Apr 3 1998
Externally publishedYes

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Histidine Decarboxylase
Histidine
Mast Cells
Rats
Cells
Cell Line
Digitonin
Protein Disulfide-Isomerases
Post Translational Protein Processing
Microsomes
Electrophoresis
L-Lactate Dehydrogenase
Organelles
Cytosol
Trypsin
Fluorescent Antibody Technique
Immune Sera
Polyacrylamide Gel Electrophoresis
Digestion
Fusion reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Intracellular localization of the 74- and 53-kDa forms of L-histidine decarboxylase in a rat basophilic/mast cell line, RBL-2H3. / Tanaka, Satoshi; Nemoto, Ken Ichi; Yamamura, Eriko; Ichikawa, Atsushi.

In: Journal of Biological Chemistry, Vol. 273, No. 14, 03.04.1998, p. 8177-8182.

Research output: Contribution to journalArticle

Tanaka, Satoshi ; Nemoto, Ken Ichi ; Yamamura, Eriko ; Ichikawa, Atsushi. / Intracellular localization of the 74- and 53-kDa forms of L-histidine decarboxylase in a rat basophilic/mast cell line, RBL-2H3. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 14. pp. 8177-8182.
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abstract = "To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/ mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40{\%} of HDC activity accompanied by over 90{\%} of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SD-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.",
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