Biochemical and immunohistochemical studies on tropomyosin and glutamate dehydrogenase in the chicken liver.
Recently, we have reported a novel tropomyosin (TM) -binding protein, glutamate dehydrogenase (GDH) and demonstrated by affinity column chromatography that chicken liver TM interacts with GDH in an ATP-dependent manner. To elucidate the physiological roles of the interaction between TM and GDH, we performed co-sedimentation assays of TM and GDH with F-actin, because it is known that TM exerts its physiological functions by associating with actin filaments. The results showed that TM and GDH co-pelleted with F-actin. GDH alone also co-precipitated with F-actin, but the amount of GDH sedimenting with F-actin was increased in the presence of chicken liver TM, suggesting that GDH is involved in the regulation of the actin cytoskeleton. We also prepared crude GDH from the nuclear and mitochondrial fractions obtained by subcellular fractionation of the chicken liver cells. Semi-nondenaturing 2D-PAGE revealed that partially purified GDH from the nuclear fraction was associated with TM, but not GDH from the mitochondrial fraction, suggesting preferential binding of TM to GDH. We determined the nucleotide sequence of chicken GDH cDNA and showed that the GDH transcript was widely expressed in the chicken organs. We examined the localization of TM and GDH by immunohistochemistry and revealed that they were distributed in the cytoplasm of the adult chicken liver. From these results, we propose two hypotheses on the physiological roles of the interaction between TM and GDH in nonmuscle cells.
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Satonari
S
Akutsu
Jun-Ichi
J
Miyazaki
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014335:Tropomyosin / Q000032:analysis*; D014461:Ultracentrifugation
D000199:Actins; D012333:RNA, Messenger; D014335:Tropomyosin; D005969:Glutamate Dehydrogenase