PROTEIN-KINASE ACTIVITIES IN RIPENING MANGO, MANGIFERA-INDICA L., FRUIT TISSUE III - PURIFICATION AND CHARACTERIZATION OF A CALCIUM-REGULATED PROTEIN-KINASE
L. Frylinck et Ia. Dubery, PROTEIN-KINASE ACTIVITIES IN RIPENING MANGO, MANGIFERA-INDICA L., FRUIT TISSUE III - PURIFICATION AND CHARACTERIZATION OF A CALCIUM-REGULATED PROTEIN-KINASE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1387(1-2), 1998, pp. 342-354
A calcium-dependent protein kinase (PK-III), not requiring calmodulin
for activity, was purified from extracts of ripening mango fruit tissu
e. Purification was achieved by ammonium sulfate fractionation and seq
uential anion exchange-, hydrophobic interaction-, dye ligand affinity
- and gel filtration chromatography; which allowed a recovery of 1-5%
of the total available kinase activity. The final specific activity in
the presence of 1 mM Ca2+ was consistently 9 nmol min(-1) mg(-1) The
purified enzyme was a monomer with a M-r of 49 000, but was resolved b
y denaturing electrophoresis into two related protein bands of 49 and
45 kDa. Enzyme activity was activated > 30-fold by micromolar amounts
of free calcium and was dependent upon millimolar Mg2+ or Mn2+ concent
rations. Calmodulin (1 M-mu) had no effect on PK-III activity but the
calmodulin antagonists, calmidazolium and chlorpromazine, inhibited PK
-III in a dose-dependent manner over a range of 0 to 100 mu M. The res
ults suggest a regulatory domain that is similar to calmodulin, PK-III
phosphorylated histone III-S and to a lesser extent casein, but did n
ot phosphorylate histone II-S, phosvitin or protamine sulfate. The enz
yme phosphorylated substrate proteins on either serine or threonine bu
t not tyrosine. Some endogenous substrates and the ability to autophos
phorylate were revealed by autoradiographic studies. PK-III displayed
a broad pH optimum (pH 6.6-9.5), and the optimum reaction temperature
with histone III-S as substrate was 35 degrees C. The kinetic reaction
mechanism of PK-III was studied by using casein as substrate. The K(m
)ATP and K(m)casein of PK-III were determined as 10 mu M and 1.0 mg ml
(-1), respectively. (C) 1998 Elsevier Science B.V. All rights reserved
.