Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene.
Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.
Thiede
M A
MA
Department of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.
Yoon
K
K
Golub
E E
EE
Noda
M
M
Rodan
G A
GA
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005796:Genes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000201:enzymology / Q000235:genetics*; D011487:Protein Conformation; D051381:Rats; D012513:Sarcoma, Experimental / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D000469:Alkaline Phosphatase