TY - JOUR
T1 - Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells
AU - Fedde, Kenton N.
AU - Lane, Carolyn C.
AU - Whyte, Michael P.
N1 - Funding Information:
i Presented in part at the 9th Annual Meeting of the American Society for Bone and Mineral Research, Indianapolis, Indiana, June 6-9, 1987, and supported by NIH Program Project Grant 5 PO1 AC82087-05, by a grant-in-aid from The Shriners Hospitals for Crippled Children, and by an NIH Biomedical Research Support Grant from The Jewish Hospital at Washington University. Dr. Fedde is a recipient of a Research Fellowship from The Shriners Hospitals for Crippled Children. ‘To whom all correspondence should be addressed at the Division of Bone and Mineral Diseases, The Jewish Hospital at Washington University, 216 South Eingshighway, St. Louis, MO 68110. ’ Abbreviations used: ALP, alkaline phosphatase; Hepes, 4-(2-hydroxyethylf-l-piperasineethanesul-fonic acid; 4-MUP, Cmethylumbelliferyl phosphate, PAGE, polyacrylamide gel electrophoresis; PEA,
PY - 1988/8
Y1 - 1988/8
N2 - Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5′-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (μm) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) > 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.
AB - Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5′-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (μm) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) > 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.
UR - http://www.scopus.com/inward/record.url?scp=0023724387&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(88)90305-0
DO - 10.1016/0003-9861(88)90305-0
M3 - Article
C2 - 3165254
AN - SCOPUS:0023724387
SN - 0003-9861
VL - 264
SP - 400
EP - 409
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -