HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, C.-a. A. Articles by Valle, D. Search for Related Content PubMed PubMed Citation Articles by Hu, C.-a. A. Articles by Valle, D. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 10, 6754-6762, March 5, 1999

Molecular Enzymology of Mammalian ¹-Pyrroline-5-carboxylate Synthase
ALTERNATIVE SPLICE DONOR UTILIZATION GENERATES ISOFORMS WITH DIFFERENT SENSITIVITY TO ORNITHINE INHIBITION

Chien-an A. Hu, Wei-Wen Lin^¶, Cassandra Obie, and David Valle

From the  Howard Hughes Medical Institute, Department of Pediatrics and Institute of Genetic Medicine and the ^¶ Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

¹-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to ¹-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine. We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by "exon sliding," a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the -glutamyl kinase active site of P5CS. The long form predominates in all tissues examined except gut. We also isolated the corresponding long and short murine P5CS transcripts. To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in -glutamyl kinase- and -glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms. We found that both confer P5CS activity and that the short isoform is inhibited by L-ornithine with a K_i of ~0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by L-ornithine.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Lv, Y. Yang, H. Yin, F. Chu, H. Wang, W. Zhang, Y. Zhang, and Y. Jin Molecular Determinants and Evolutionary Dynamics of Wobble Splicing Mol. Biol. Evol., May 1, 2009; 26(5): 1081 - 1092. [Abstract] [Full Text] [PDF]

				M. Hiller, K. Szafranski, R. Sinha, K. Huse, S. Nikolajewa, P. Rosenstiel, S. Schreiber, R. Backofen, and M. Platzer Assessing the fraction of short-distance tandem splice sites under purifying selection RNA, April 1, 2008; 14(4): 616 - 629. [Abstract] [Full Text] [PDF]

				Y. Alnouti and C. D. Klaassen Tissue Distribution, Ontogeny, and Regulation of Aldehyde Dehydrogenase (Aldh) Enzymes mRNA by Prototypical Microsomal Enzyme Inducers in Mice Toxicol. Sci., January 1, 2008; 101(1): 51 - 64. [Abstract] [Full Text] [PDF]

				D. T. Johnson, R. A. Harris, P. V. Blair, and R. S. Balaban Functional consequences of mitochondrial proteome heterogeneity Am J Physiol Cell Physiol, February 1, 2007; 292(2): C698 - C707. [Abstract] [Full Text] [PDF]

				Z. Liu, H. Lu, Z. Jiang, A. Pastuszyn, and C.-a. A. Hu Apolipoprotein L6, a Novel Proapoptotic Bcl-2 Homology 3-Only Protein, Induces Mitochondria-Mediated Apoptosis in Cancer Cells Mol. Cancer Res., January 1, 2005; 3(1): 21 - 31. [Abstract] [Full Text] [PDF]

				M. Lin, M. Zhang, M. Abraham, S. M. Smith, and J. L. Napoli Mouse Retinal Dehydrogenase 4 (RALDH4), Molecular Cloning, Cellular Expression, and Activity in 9-cis-Retinoic Acid Biosynthesis in Intact Cells J. Biol. Chem., March 7, 2003; 278(11): 9856 - 9861. [Abstract] [Full Text] [PDF]

				Y. Morita, S. Nakamori, and H. Takagi L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding {gamma}-Glutamyl Kinase Appl. Envir. Microbiol., January 1, 2003; 69(1): 212 - 219. [Abstract] [Full Text] [PDF]

				M. R. Baumgartner, C.-a. A. Hu, S. Almashanu, G. Steel, C. Obie, B. Aral, D. Rabier, P. Kamoun, J.-M. Saudubray, and D. Valle Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding {Delta}1-pyrroline-5-carboxylate synthase Hum. Mol. Genet., November 1, 2000; 9(19): 2853 - 2858. [Abstract] [Full Text] [PDF]

				Z. Hong, K. Lakkineni, Z. Zhang, and D. P. S. Verma Removal of Feedback Inhibition of Delta 1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress Plant Physiology, April 1, 2000; 122(4): 1129 - 1136. [Abstract] [Full Text]