Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein Víctor Castro, Pablo Fuentealba, Adolfo Henríquez, Alejandro Vallejos, José Benítez, Marcela Lobos, Beatriz Díaz, Nelson Carvajal, Elena Uribe ⇑ Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile article info Article history: Received 14 April 2011 and in revised form 6 May 2011 Available online 12 May 2011 Keywords: Agmatinase Polyamines LIM domain abstract We recently cloned a rat brain agmatinase-like protein (ALP) whose amino acid sequence greatly differs from other agmatinases and exhibits a LIM-like domain close to its carboxyl terminus. The protein was immunohistochemically detected in the hypothalamic region and hippocampal astrocytes and neurons. We now show that truncated species, lacking the LIM-type domain, retains the dimeric structure of the wild-type protein but exhibits a 10-fold increased k cat , a 3-fold decreased K m value for agmatine and altered intrinsic tryptophan fluorescent properties. As expected for a LIM protein, zinc was detected only in the wild-type ALP (2 Zn 2+ /monomer). Our proposal is that the LIM domain functions as an autoinhibitory entity and that inhibition is reversed by interaction of the domain with some yet undefined brain protein. Ó 2011 Elsevier Inc. All rights reserved. Introduction Agmatine, a decarboxylated derivative of L-arginine, has been associated to several important biological processes in mammals, including neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain [1]. This primary amine is packed into synaptic vesicles in the brain and spinal cord and acts on transmembrane receptors (a2-adrenergic, imidazoline and gultamatergic NMDA receptors) [2]. At physiological concentra- tions, agmatine is inhibitory to the neuronal nitric oxide synthase [3] and participate in modulation of insulin release from pancreatic cells [4,5] and renal sodium excretion [6]. A fine control of agma- tine levels is evidently required, thus justifying the interest in the enzymological aspects of agmatine synthesis and degradation in mammals. Agmatine result from decarboxylation of arginine by arginine decarboxylase [7,8] and it is converted to putrescine and urea by agmatinase [9]. With regard to agmatinase, there are reports estab- lishing its presence in the brain [9,10], although the study of their enzymic properties has been complicated by the lack of appropri- ately active preparations of the enzyme. This includes two reported recombinant forms of human agmatinase which are very poorly active under in vitro conditions [11,12]. In one of these reports, the functionality of the cloned human gene was deduced from a functional complementation test with a yeast strain which contains a disruption in the gene encoding ornithine decarboxylase and thus requires exogenous polyamines for growth [11]. In contrast with these reports, we have recently cloned and expressed a rat brain protein which is significantly active as agmatinase in vitro, although its amino acid sequence is only about 12% iden- tical with the human and bacterial enzymes [13]. In any case, cer- tain degree of structural homology was revealed by its reaction with a polyclonal antibody raised against Escherichia coli agmatin- ase. We refer to this protein as agmatinase-like protein (ALP). Like E. coli agmatinase, ALP was shown to require Mn 2+ for cat- alytic activity and to be maximally active at pH 9–9.5 [13]. By RT-PCR and immmunohistochemical methods, the protein was de- tected in the hypothalamus in glial cells and arcuate nucleus neu- rons, and also in hippocampus astrocytes and neurons, but not in brain cortex [14]. Considering that, in general, this localization coincides with that described for its substrate agmatine, ALP was suggested to be involved in the regulation of intracellular concen- trations of this neurotransmitter/neuromodulator. One especially interesting finding was a terminal segment that includes a LIM-type domain in the sequence of ALP [13]. The LIM do- main is a zinc-coordinating domain, consisting of two tandemly re- peated zinc fingers, generally involved in cellular differentiation and control of cellular growth, gene expression, interactions with cyto- skeleton, auto-inhibitory effects and possibly as biosensors that mediate communication between the cellular and nuclear compart- ments [15,16]. The classic LIM consensus sequence includes a CX 2 CX 16–23 HX 2 CX 2 CX 2 CX 16–21 CX 2 (CH/D) sequence, associated with a highly variable sequence in the remainder of the domain that con- fers functional specificity [17]. As a part of a continuing effort aimed to characterize the molec- ular and functional aspects of ALP, we have now turned our atten- tion to the LIM-type domain, by examining the consequences of its 0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2011.05.003 ⇑ Corresponding author. Fax: +56 41 2239687. E-mail address: auribe@udec.cl (E. Uribe). Archives of Biochemistry and Biophysics 512 (2011) 107–110 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi