tailieunhanh - Báo cáo khóa học: Mouse spermine oxidase gene splice variants Nuclear subcellular localization of a novel active isoform

Spermine oxidase (SMO) is a flavoenzyme involved in polyamine homeostasis in animal cells. Themouse spermine oxidasegene (mSMO) codes for splicevariants, including the previously reportedmajor active isoform, herein named alfa (a). In the present work, eight additional gene splicing variants were characterized. The heterologous expression and biochemical characterization ofthree recombinant iso-forms (namely mSMOl,-cand -d) revealed that only the recombinant protein mSMOldisplays biochemical charac-. | Eur. J. Biochem. 271 760-770 2004 FEBS 2004 doi Mouse spermine oxidase gene splice variants Nuclear subcellular localization of a novel active isoform Manuela Cervelli 1 Alessandro Bellini1 Marzia Bianchi1. Lucia Marcocci2 Stefania Nocera2 Fabio Polticelli1 Rodolfo Federico1. Roberto Amendola3 and Paolo Mariottini1 1 Dipartimento di Biologia Universita Roma Tre Rome Italy 2Dipartimento di Scienze Biochimiche A. Rossi Fanelli Universita La Sapienza Rome Italy 3Istituto per la Radioprotezione ENEA CR Casaccia Roma Italy Spermine oxidase SMO is a flavoenzyme involved in polyamine homeostasis in animal cells. The mouse spermine oxidase gene mSMO codes for splice variants including the previously reported major active isoform herein named alfa a . In the present work eight additional gene splicing variants were characterized. The heterologous expression and biochemical characterization of three recombinant isoforms namely mSMOti. -y and -Ỗ revealed that only the recombinant protein mSMOti displays biochemical characteristics similar to those of mSMOa the other two recombinant proteins contained no detectable SMO activity. In order to investigate in greater detail the SMO enzyme activity associated with their subcellular localization mSMOa and -Lt V5-tagged proteins were transiently and stably transfected in the murine neuroblastoma cell line N18TG2. Very interestingly the novel active mSMOi isoform was found to be present in both nuclear and cytoplasmic compartments thus providing the first evidence of SMO activity in the nucleus while a cytoplasmic localization was confirmed for the mSMOa isoform. In addition the relative transcription levels ofthe gene splicing variants were evaluated by RT-PCR analysis to verify a relationship with the SMO enzyme activity in various murine organs. Keywords enzyme isoform mouse spermine oxidase subcellular localization. In mammalian cells the maintenance ofpolyamine homeostasis is accomplished by the .