Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Dec;78(12):7737–7741. doi: 10.1073/pnas.78.12.7737

Immunochemical studies on cultured fibroblasts from patients with inherited methylmalonic acidemia.

J F Kolhouse, C Utley, W A Fenton, L E Rosenberg
PMCID: PMC349345  PMID: 6121323

Abstract

We developed a radioimmunoassay to quantitate material crossreacting immunochemically with human methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2), and have applied this assay to extracts of fibroblasts from controls and from 32 patients with methylmalonic acidemia due to inherited deficiencies in mutase activity. Four control lines had an average of 237 ng of crossreacting material (CRM) per mg of cell protein (range, 193-297 ng/mg). Mutant lines from each of the four cbl complementation groups of inherited methylmalonic acidemia, which have normal amounts of mutase activity in vitro, contained quantities of CRM comparable to those of control lines. On the other hand, 28 cell lines from the mut complementation group, which express mutations at the structural gene locus for the mutase apoenzyme, contained widely diverse amounts of CRM. Each of seven lines from the mut- subgroup, whose residual mutase activity reflects the presence of a structurally altered mutase protein with reduced affinity for cofactor, had detectable CRM ranging from 20% to 100% of control. The 21 lines from the mut0 group, which have no detectable mutase activity in vitro, fell into two populations with regard to CRM: 9 lines had detectable CRM ranging from 3% to 40% of control; 12 others had no detectable CRM (limit of detectability, less than 1% of control). These results emphasize the wide range of mutations at a single structural gene locus that can result in deficient enzyme activity.

Full text

PDF
7737

Images in this article

7738Image
on p.7738

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker M. A., Kostel P. J., Meyer L. J., Seegmiller J. E. Human phosphoribosylpyrophosphate synthetase: increased enzyme specific activity in a family with gout and excessive purine synthesis. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2749–2752. doi: 10.1073/pnas.70.10.2749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dreyfus J. C., Alexandre Y. Immunological studies on glycogen storage diseases type 3 and V. Demonstration of the presence of an immunoreactive protein in one case of muscle phosphorylase deficiency. Biochem Biophys Res Commun. 1971 Sep 17;44(6):1364–1370. doi: 10.1016/s0006-291x(71)80236-x. [DOI] [PubMed] [Google Scholar]
  3. Fenton W. A., Rosenberg L. E. Genetic and biochemical analysis of human cobalamin mutants in cell culture. Annu Rev Genet. 1978;12:223–248. doi: 10.1146/annurev.ge.12.120178.001255. [DOI] [PubMed] [Google Scholar]
  4. Ghangas G. S., Milman G. Radioimmune determination of hypoxanthine phosphoribosyltransferase crossreacting material in erythrocytes of Lesch-Nyhan patients. Proc Natl Acad Sci U S A. 1975 Oct;72(10):4147–4150. doi: 10.1073/pnas.72.10.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gray G. M., Conklin K. A., Townley R. R. Sucrase-isomaltase deficiency. Absence of an inactive enzyme variant. N Engl J Med. 1976 Apr 1;294(14):750–753. doi: 10.1056/NEJM197604012941403. [DOI] [PubMed] [Google Scholar]
  6. Kolhouse J. F., Allen R. H. Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase. Proc Natl Acad Sci U S A. 1977 Mar;74(3):921–925. doi: 10.1073/pnas.74.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kolhouse J. F., Utley C., Allen R. H. Isolation and characterization of methylmalonyl-CoA mutase from human placenta. J Biol Chem. 1980 Apr 10;255(7):2708–2712. [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Mahoney M. J., Hart A. C., Steen V. D., Rosenberg L. E. Methylmalonicacidemia: biochemical heterogeneity in defects of 5'-deoxyadenosylcobalamin synthesis. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2799–2803. doi: 10.1073/pnas.72.7.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Morrow G., 3rd, Mahoney M. J., Mathews C., Lebowitz J. Studies of methylmalonyl coenzyme A carbonylmutase activity in methylmalonic acidemia. I. Correlation of clinical, hepatic, and fibroblast data. Pediatr Res. 1975 Aug;9(8):641–644. doi: 10.1203/00006450-197508000-00006. [DOI] [PubMed] [Google Scholar]
  11. Osborne W. R., Chen S. H., Giblett E. R., Biggar W. D., Ammann A. A., Scott C. R. Purine nucleoside phosphorylase deficiency. Evidence for molecular heterogeneity in two families with enzyme-deficient members. J Clin Invest. 1977 Sep;60(3):741–746. doi: 10.1172/JCI108826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schneider R. J., Burger R. L., Mehlman C. S., Allen R. H. The role and fate of rabbit and human transcobalamin II in the plasma transport of vitamin B12 in the rabbit. J Clin Invest. 1976 Jan;57(1):27–38. doi: 10.1172/JCI108265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tedesco T. A., Mellman W. J. Galactosemia: evidence for a structural gene mutation. Science. 1971 May 14;172(3984):727–728. doi: 10.1126/science.172.3984.727. [DOI] [PubMed] [Google Scholar]
  14. Upchurch K. S., Leyva A., Arnold W. J., Holmes E. W., Kelley W. N. Hypoxanthine phosphoribosyltransferase deficiency: association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein. Proc Natl Acad Sci U S A. 1975 Oct;72(10):4142–4146. doi: 10.1073/pnas.72.10.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Willard H. F., Mellman I. S., Rosenberg L. E. Genetic complementation among inherited deficiencies of methylmalonyl-CoA mutase activity: evidence for a new class of human cobalamin mutant. Am J Hum Genet. 1978 Jan;30(1):1–13. [PMC free article] [PubMed] [Google Scholar]
  16. Willard H. F., Rosenberg L. E. Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin. Biochem Biophys Res Commun. 1977 Oct 10;78(3):927–934. doi: 10.1016/0006-291x(77)90511-3. [DOI] [PubMed] [Google Scholar]
  17. Willard H. F., Rosenberg L. E. Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression. J Clin Invest. 1980 Mar;65(3):690–698. doi: 10.1172/JCI109715. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES