title: Cloning of two LIMCH1 isoforms: characterization of their distribution in rat brain and their agmatinase activity.
abstract: Agmatine, a precursor for polyamine biosynthesis, is also associated with neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. This molecule results from the decarboxylation of L-arginine by arginine decarboxylase, and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new protein that also hydrolyzes agmatine, agmatinase-like protein (ALP), which was identified through immunohistochemical analysis in the hypothalamus and hippocampus of rats. However, its sequence differs greatly from all known agmatinases and does not contain the typical Mn(2+) ligands associated with the urea hydrolase family of proteins. ALP has a LIM-like domain close to its carboxyl terminus, and the removal of which results in a truncated variant with a tenfold increased k cat value and a threefold decreased K m value for agmatine. Analysis of the gene database revealed several transcripts, denominated LIMCH1 isoforms, with extreme 3' sequences identical to ALP. Limch1 gene products have been described as members of a multi-domain family of proteins with the biggest isoform containing a calponin homology (CH) domain at its N-terminus. Here, we cloned two LIMCH1 transcripts, one of 3177 bp and the other of 2709 bp (ALP contains 1569 bp) and analyzed LIMCH1 expression and distribution in rat brain using RT-PCR, Western blot and immunohistochemical analyses. LIMCH1 was detected mainly in the hypothalamic and hippocampal regions, which is similar to the distribution of ALP and agmatine in brain. In addition, we cloned and expressed both isoforms in E. coli and confirmed that they were catalytically active on agmatine with kinetic parameters similar to ALP. LIM domain-truncated variants of both isoforms moderately increased the k cat and catalytic efficiency. Thus, we propose that LIMCH1 is useful to regulate the intracellular concentrations of the neurotransmitter/neuromodulator, agmatine.
authors.affiliation: Departamento de Ciencias Biológicas y Químicas, Facultad de Ciencias, Universidad San Sebastián, Lientur 1457, Concepción, Chile.
authors.forename: David
authors.initials: D
authors.lastname: García
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Patricio
authors.initials: P
authors.lastname: Ordenes
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: José
authors.initials: J
authors.lastname: Benítez
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Arlette
authors.initials: A
authors.lastname: González
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: María A
authors.initials: MA
authors.lastname: García-Robles
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad Católica del Norte, Coquimbo, Chile.
authors.forename: Vasthi
authors.initials: V
authors.lastname: López
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Nelson
authors.initials: N
authors.lastname: Carvajal
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile. auribe@udec.cl.
authors.forename: Elena
authors.initials: E
authors.lastname: Uribe
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D008297:Male; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014512:Ureohydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D014512:Ureohydrolases; C019625:agmatinase
keywords: Agmatine; Hypothalamus; LIM domain; LIMCH1