Hypoxia induces the synthesis of tropomyosin in cultured porcine pulmonary artery endothelial cells.
The present study examined the effect of hypoxia on protein synthesis by porcine pulmonary artery endothelial cells (PAEC). Hypoxia decreased protein synthesis in PAEC, but two-dimensional gel electrophoresis of [35S]methionine-labeled PAEC proteins demonstrated the increased synthesis of a set of proteins having molecular masses (M(r)) of 35, 36.5, 45, 116, and 205 kDa. The synthesis of the 35-, 36.5-, and 45-kDa proteins was increased in preconfluent and postconfluent cells. The 35- and 45-kDa proteins were not induced by hyperthermia, whereas the 36.5-kDa protein was induced slightly by hyperthermia. Induction of the 36.5- and 45-kDa proteins required a minimum of 8 h of hypoxia, whereas induction of the 35-kDa protein required only 4 h of exposure to hypoxia. The upregulated synthesis of the 35-, 36.5-, and 45-kDa proteins was reversible with return to normoxia. Actinomycin D, an inhibitor of transcription, did not block the hypoxic induction of the 35- and 36.5-kDa proteins but did block induction of the 45-kDa protein. The partial amino acid sequence of the 35-kDa protein obtained from cyanogen bromide cleavage of the molecule was Asp-Ala-Ile-Lys-Lys-Lys-Met-Gln-Met-Leu-Lys-Leu-Asp-Lys-Glu. This partial sequence of the 35-kDa protein identically matches the sequence of tropomyosin. Amino acid composition data and the isoelectric point (4.8) were also typical of tropomyosin. Finally, specific cross-reactivity was detected between the 35-kDa protein and a monoclonal antibody to chicken gizzard tropomyosin on immunoblot. Thus hypoxia induces the synthesis of tropomyosin, a major microfilament-associated protein, in porcine PAEC in monolayer culture.
Rao
U J
UJ
Department of Medicine, University of Florida College of Medicine, Gainesville.
Denslow
N D
ND
Block
E R
ER
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000096:biosynthesis; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D013552:Swine; D014335:Tropomyosin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D009124:Muscle Proteins; D014335:Tropomyosin