Somatic Cell and Molecular Genetics

, Volume 22, Issue 6, pp 489–498 | Cite as

Delivery of cytosolic liver arginase into the mitochondrial matrix space: A possible novel site for gene replacement therapy

  • Paul B. Wissmann
  • Barbara K. Goodman
  • Joseph G. Vockley
  • Rita M. Kern
  • Stephen D. Cederbaum
  • Wayne W. Grody


As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, AI) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.


Ornithine Arginase Urea Cycle Ornithine Transcarbamylase Gene Replacement Therapy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature cited

  1. 1.
    Brusilow, S.W., and Horwich, A.L. (1995). The urea cycle enzymes In: Scriver, C.R., A.L. Beaudet, W.S. Sly, and D. Valle (eds.),The Metabolic and Molecular Bases of Inherited Disease, 7th ed., New York, McGraw-Hill, pp. 1187–1232.Google Scholar
  2. 2.
    Cederbaum, S.D., Shaw, K.N.F., and Valente, M. (1977). Hyperargininemia.J. Pediatr.,90:569–573.PubMedGoogle Scholar
  3. 3.
    Cederbaum, S.D., Shaw, K.N.F., Spector, E.B., Verity, A.M. Snodgrass, J.P., and Sugarman, L.I.G. (1979). Hyperarginimemia with arginase deficiency.Pediatr. Res. 13:827–833.PubMedGoogle Scholar
  4. 4.
    Herzfeld, A., and Raper, S.M. (1976). The heterogeneity of arginases in rat tissues.Biochem. J. 153:469–478.PubMedGoogle Scholar
  5. 5.
    Spector, E.B., Rice, S.C.H., Moedjono, S., Bernard, B., and Cederbaum, S.D. (1982). Biochemical properties of arginase in human adult and fetal tissues.Biochem. Med. 28:165–175.CrossRefPubMedGoogle Scholar
  6. 6.
    Michels, V.V., and Beaudet, A.L. (1978). Arginase deficiency in multiple tissues in argininemia.Clin. Genet. 13:61–67.PubMedGoogle Scholar
  7. 7.
    Spector, E.B., Rice, S.C.H., and Cederbaum, S.D. (1983). Immunologic studies of arginase in tisses of normal human adult and arginase-deficient patients.Pediatr. Res. 17:941–944.PubMedGoogle Scholar
  8. 8.
    Grody, W.W., Dizikes, G.J., and Cederbaum, S.D. (1986). Human arginase isozymes. In:Isozymes: Current Topics in Biological and Medical Research. Vol. 13, New York, Alan R. Liss, pp. 181–214.Google Scholar
  9. 9.
    Carvajal, N., and Cederbaum, S.D. (1986). Kinetics of inhibition of rat liver and kidney arginase by proline and branched chain amino acids.Biochim. Biophys. Acta 870:181–184.PubMedGoogle Scholar
  10. 10.
    Dizikes, G.J., Grody, W.W., Kern, R.M. and Cederbaum, S.D. (1986). Isolation of human liver argmase cDNA and demonstration of nonhomology between the two human arginase genes.Biochem. Biophys. Res. Comm. 141:53–59.CrossRefPubMedGoogle Scholar
  11. 11.
    Vockley, J.G., Jenkinson, C.P., Shukla, H., Kern, R.M., Grody, W.W., and Cederbaum, S.D. (1996). Cloning and characterization of the human type II arginase gene.Genomics 88:118–123.Google Scholar
  12. 12.
    Mezl, V.A., and Knox, W.E. (1977). Metabolism of arginine in lactating rat mammary gland.Biochem. J. 164:105–113.Google Scholar
  13. 13.
    Wang, W.W., Jenkinson, C.P., Griscavage, J.M., Kern, R.M., Arabolos, N.S., Byrns, R.E., Cederbaum, S.D., and Ignarro, L.J. (1995). Co-induction of arginase and nitric oxide synthase in murine macrophages activated by lipopolysaccharide.Biochem. Biophys. Res. Comm. 210:1009–1016.CrossRefPubMedGoogle Scholar
  14. 14.
    Grody, W.W., Argyle,C., Kern, R.M., Dizikes, G.J., Spector, E.B., Strickland, A.D., Klein, D., and Cederbaum, S.D. (1989). Differential expression of the two human arginase genes in hyperargininemia: Enzymatic, pathologic, and molecular analysis.J. Clin. Invest. 83:602–609.PubMedGoogle Scholar
  15. 15.
    Grody, W.W., Kern, R.M., Klein, D., Dodson, A.E., Wissmann, P.B., Barsky, S.H., and Cederbaum, S.D. (1993). Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme.Hum. Genet. 91:1–5.CrossRefPubMedGoogle Scholar
  16. 16.
    Horwich, A., Kalousek, F., Mellman, I., and Rosenberg, L. (1985). A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.EMBO J. 4:1129–1135.PubMedGoogle Scholar
  17. 17.
    Goodman, B.K., Klein, D., Tabor, D.E., Vockley, J.G., Cederbaum, S.D., and Grody, W.W (1994). Functional and molecular analysis of liver arginase promoter sequences from man and Macaca fascicularis.Som. Cell Molec. Genet. 20:313–325.Google Scholar
  18. 18.
    Haggerty, D.F., Spector, E.B., Lynch, M., Kern, R.M., Frank, L.B., and Cederbaum, S.D. (1983). Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and non-hepatic origin.Mol. Cell. Biochem. 53/54: 57–76.CrossRefGoogle Scholar
  19. 19.
    Wissmann, P.B., Goodman, B.K., Vockley, J.G., Kern, R.M., Cederbaum, S.D., and Grody, W.W. (1994). Gene trasfer of liver arginase into different subcellular compartments (Abstract).Am. J. Hum. Genet. 55:A139.Google Scholar
  20. 20.
    Engelhardt, J.F., Steel, G., and Valle, D. (1991). Transcriptional analysis of the human ornithine aminotransferase promoter.J. Biol. Chem. 266:752–758.PubMedGoogle Scholar
  21. 21.
    Selden, R.F., Howie, K.B., Rowe, M.E., Goodman, H.M., and Moore, D.D. (1986). Human growth hormone as a reporter gene in regulation studies employing transtent gene expression.Mol. Cell. Biol. 6:3173–3179.PubMedGoogle Scholar
  22. 22.
    Pedersen, P., Greenawalt, J., Reynafarje, B., Hullihen, J., Decker, G., Soper, J., and Bustamente, E. (1978). Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liverderived tissues.Meth. Cell. Biol. 10:ch. 26.Google Scholar
  23. 23.
    Munujos, P., Coll-Canti, J., Gonzalez-Sastr, F., and Gella, F.J. (1993). Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor.Anal. Biochem. 212:506–509.CrossRefPubMedGoogle Scholar
  24. 24.
    Spector, E.B., Keirnan, M., Bernard, B., and Cederbaum S.D. (1980). Properties of fetal and adult red blood cell arginase: A possible prenatal diagnostic test for arginase deficiency.Am. J. Hum. Genet. 32:79–87.PubMedGoogle Scholar
  25. 25.
    Schimke, R.T. (1964). Enzymes of arginine metabolism in mammalian cell culture. I. Repression of argininosuccinate synthetase and argininosuccinase.J. Biol. Chem. 239:136–144.PubMedGoogle Scholar
  26. 26.
    Chirgwin, J.J., Przbyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry 18:5294–5299.CrossRefPubMedGoogle Scholar
  27. 27.
    Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. (1977). RNA molecular weight determinations by gel electrophoresis under denaturing conditions: A critical reexamination.Biochemistry 16:4743.CrossRefPubMedGoogle Scholar

Copyright information

© Plenum Publishing Coporation 1996

Authors and Affiliations

  • Paul B. Wissmann
    • 2
  • Barbara K. Goodman
    • 2
  • Joseph G. Vockley
    • 2
  • Rita M. Kern
    • 2
  • Stephen D. Cederbaum
    • 2
  • Wayne W. Grody
    • 1
    • 2
  1. 1.Divisions of Medical Genetics and Molecular PathologyLaboratory Medicine and Pediatrics, UCLA School of MedicineLos Angeles
  2. 2.Department of PathologyLaboratory Medicine and Pediatrics, UCLA School of MedicineLos Angeles

Personalised recommendations