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Plant Physiol, November 1999, Vol. 121, pp. 921-928
Peroxisomal NADP-Dependent Isocitrate Dehydrogenase.
Characterization and Activity Regulation during Natural
Senescence1
Francisco J.
Corpas,*
Juan B.
Barroso,
Luisa M.
Sandalio,
José M.
Palma,
José A.
Lupiáñez, and
Luis A.
del Río
Departamento de Bioquímica, Biología Celular y
Molecular de Plantas, Estación Experimental del Zaidín,
Consejo Superior de Investigaciones Científicas,
Apartado 419, E-18080 Granada, Spain (F.J.C., L.M.S., J.M.P.,
L.A.d.R.); Departamento de Bioquímica y Biología
Molecular, Facultad de Ciencias Experimentales, Universidad de
Jaén, Paraje "Las Lagunillas" s/n, E-23071 Jaén, Spain
(J.B.B.); and Departamento de Bioquímica y Biología
Molecular, Centro de Ciencias Biológicas, Universidad de Granada,
Avenida Fuentenueva s/n, E-18001 Granada, Spain (J.A.L.)
The peroxisomal localization and
characterization of NADP-dependent isocitrate dehydrogenase (perICDH)
in young and senescent pea (Pisum sativum) leaves was
studied by subcellular fractionation, kinetic analysis, immunoblotting,
and immunoelectron microscopy. The subunit molecular mass for perICDH
determined by immunoblotting was 46 kD. By isoelectric focusing (IEF)
of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved
into four isoforms, perICDH-1 to perICDH-4, with isoelectric points
(pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties
of the NADP-ICDH in peroxisomes from young and senescent pea leaves
were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in
senescent leaf peroxisomes was 11-fold lower than in young leaf
peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not
altered during senescence. The kinetic behavior of this enzyme suggests
a possible fine control of enzymatic activity by modulation of its
Michaelis constant during the natural senescence of pea leaves. After
embedding, electron microscopy immunogold labeling of NADP-ICDH
confirmed that this enzyme was localized in the peroxisomal matrix.
Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these
cell organelles and may be the main system for the reduction of NADP to
NADPH for its re-utilization in the peroxisomal metabolism.
1
This work was supported by the Dirección
General de Enseñanza Superior e Investigación
Científica (grant no. PB95-0004-01) and by the Junta de
Andalucía (research groups nos. CVI 0157 and CVI 0192), Spain,
and the European Union (contract no. CHRX-CT94-0605).
*
Corresponding author; e-mail javier.corpas{at}eez.csic.es; fax
34-958-129600.
© 1999 American Society of Plant Physiologists
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