Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1989 May;90(1):41–44. doi: 10.1104/pp.90.1.41

A Cerulenin Insensitive Short Chain 3-Ketoacyl-Acyl Carrier Protein Synthase in Spinacia oleracea Leaves

Jan G Jaworski 1,2, Richard C Clough 1,2, Susan R Barnum 1,2
PMCID: PMC1061673  PMID: 16666765

Abstract

A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryl- and hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated.

Full text

PDF
44

Images in this article

43Figure 2
on p.43
44Figure 3
on p.44

Selected References

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

  1. Bloch K., Vance D. Control mechanisms in the synthesis of saturated fatty acids. Annu Rev Biochem. 1977;46:263–298. doi: 10.1146/annurev.bi.46.070177.001403. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Clough R. C., Barnum S. R., Jaworski J. G. Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate. Anal Biochem. 1989 Jan;176(1):82–84. doi: 10.1016/0003-2697(89)90276-5. [DOI] [PubMed] [Google Scholar]
  4. Cronan J. E., Jr, Klages A. L. Chemical synthesis of acyl thioesters of acyl carrier protein with native structure. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5440–5444. doi: 10.1073/pnas.78.9.5440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. D'Agnolo G., Rosenfeld I. S., Awaya J., Omura S., Vagelos P. R. Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of beta-ketoacyl-acyl carrier protein synthetase. Biochim Biophys Acta. 1973 Nov 29;326(2):155–156. doi: 10.1016/0005-2760(73)90241-5. [DOI] [PubMed] [Google Scholar]
  6. Jackowski S., Rock C. O. Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria. J Biol Chem. 1987 Jun 5;262(16):7927–7931. [PubMed] [Google Scholar]
  7. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  8. Omura S. Cerulenin. Methods Enzymol. 1981;72:520–532. [PubMed] [Google Scholar]
  9. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  10. Rutkoski A., Jaworski J. G. An improved synthesis of malonyl-coenzyme A. Anal Biochem. 1978 Nov;91(1):370–373. doi: 10.1016/0003-2697(78)90854-0. [DOI] [PubMed] [Google Scholar]
  11. Shimakata T., Stumpf P. K. Fatty Acid Synthetase of Spinacia oleracea Leaves. Plant Physiol. 1982 Jun;69(6):1257–1262. doi: 10.1104/pp.69.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Shimakata T., Stumpf P. K. Isolation and function of spinach leaf beta-ketoacyl-[acyl-carrier-protein] synthases. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5808–5812. doi: 10.1073/pnas.79.19.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shimakata T., Stumpf P. K. The purification and function of acetyl coenzyme A:acyl carrier protein transacylase. J Biol Chem. 1983 Mar 25;258(6):3592–3598. [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES