CHARACTERIZATION OF THE PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C-RELEASED FORM OF RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE AND ITS POSSIBLE SIGNIFICANCE ON ENDOCHONDRAL OSSIFICATION
Jm. Pizauro et al., CHARACTERIZATION OF THE PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C-RELEASED FORM OF RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE AND ITS POSSIBLE SIGNIFICANCE ON ENDOCHONDRAL OSSIFICATION, Molecular and cellular biochemistry, 152(2), 1995, pp. 121-129
Alkaline phosphatase activity was released up to 100% from the membran
e by incubating the rat osseous plate membrane-bound enzyme with phosp
hatidylinositol-specific phospholipase C. The molecular weight of the
released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,0
00 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the
membrane-bound enzyme with phospholipase C did not destroy its ability
to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and P
NPP by phosphatidylinositol-specific phospholipase C-released enzyme e
xhibited 'Michaelian' kinetics with K-0.5=70 and 979 mu M, respectivel
y. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions we
re observed (n=1.4). Magnesium ions were stimulatory (K-0.5=1.5 mM) an
d zinc ions were a powerful noncompetitive inhibitor (K-i=6.2 mu M) of
phosphatidylinositol-specific phospholipase C-released enzyme. Phosph
atidylinositol-specific phospholipase C-released alkaline phosphatase
was relatively stable at 40 degrees C. However, with increasing temper
ature from 40-60 degrees C, the enzyme was inactivated rapidly followi
ng first order kinetics and thermal inactivation constants varied from
5.08 x 10(-4) min(-1) to 0.684 min(-1). Treatment of phosphatydilinos
itol-specific phospholipase C-released alkaline phosphatase with Chell
ex 100 depleted to 5% its original PNPPase activity. Magnesium (K-0.5=
29.5 mu M), manganese (K-0.5=5 mu M) and cobalt ions (K-0.5=10.1 mu M)
restored the activity of Chelex-treated enzyme, demonstrating its met
alloenzyme nature. The stimulation of Chelex-treated enzyme by calcium
ions (K-0.5=653 mu M) was less effective (only 26%) and occurred with
site-site interactions (n=0.7). Zinc ions had no stimulatory effects.
The possibility that the soluble form of the enzyme, detected during
endochondral ossification, would arise by the hydrolysis of the Pl-anc
hored form of osseous plate alkaline phosphatase is discussed.