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Carlsberg Research Communications

, Volume 48, Issue 4, pp 285–305 | Cite as

Barley acyl carrier protein: Its amino acid sequence and assay using purified malonyl-CoA:ACP transacylase

  • Peter Bordier Høj
  • Ib Svendsen
Article

Abstract

Malonyl-CoA:ACP transacylase from barley (Hordeum vulgare L.) has been purified to homogeneity and used in an assay for acyl carrier protein (ACP). The transacylase is an acidic, monomeric protein with a molecular weight of 34,500 very similar to the analogous E. coli enzyme.

A heat and acid stable acyl carrier protein from barley has been purified to homogeneity and its chemical composition determined. The ACP consists of a continuous stretch of the following 72 amino acids H2N-A-A-M-G-E-A-Q-A-K-K-E-T-V-D-K-V-(C?)-M-I-V-K-K-Q-L-A-V-P-D-G-T-P-V-T-A-E-S-K-F-S-E-L-G-A-D-S-L-D-T-V-E-I-V-M-G-L-E-E-E-F-N-I-T-V-D-E-T-S-A-Q-D-I-A72...A87-COOH. A comparison of the primary structure of this plant ACP and bacterial ACP reveals two identical sequences (underlined) in the midregion of the molecule containing the 4′-phosphopantetheine attachment site, while differences occur outside this region. Nine extra residues (italicized) are present at the N-terminal end of the barley protein thereby accounting for its larger size. Identical products are obtained when barley chloroplast fatty acid synthetase is incubated with either barley or E. coli ACP, but the latter is twice as active as the former in fatty acid synthesis. The possible significance of the N-terminal part of the ACP is discussed in relation to the reported differences in biochemical activities of plant and bacterial ACPs.

Keywords

Fatty acid synthetase cyanogen bromide carboxypeptidase Y chromatofocusing structure-function relationship 

Abbreviations

ACP

acyl carrier protein

BSA

bovine serum albumin

CoA

coenzyme A

DTT

1,4-dithiotreitol

fas

fatty acid synthetase

Hepes

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

kD

kilodalton

Mes

2-(N-morpholino)ethanesulfonic acid

Mops

3-(N-morpholino)propanesulfonic acid

Pipes

piperazine-N,N′-bis(2-ethane sulfonic acid)

PTH

phenylthiohydantoin

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tricine

N-(tris(hydroxymethyl) methyl) glycine

u. v.

ultra violet

References

  1. 1.
    Abita, J.P., M. Lazdunski &G.P. Ailhaud: Structure function relationships of the acyl carrier protein of Escherichia coli. Eur. J. Biochem. 23, 412–420 (1971)PubMedCrossRefGoogle Scholar
  2. 2.
    Ailhaud, G.P., P.R. Vagelos &H. Goldfine: Involvement of acyl carrier protein in acylation of glycerol 3-phosphate in Clostridium butyricum. J. Biol. Chem. 242, 4459–4465 (1967)PubMedGoogle Scholar
  3. 3.
    Bartsch, M., M. Kimura &A.-R. Subramanian: Purification primary structure, and homology relationships of a chloroplast ribosomal protein. Proc. Natl. Acad. Sci. USA 79, 6871–6875 (1982)PubMedCrossRefGoogle Scholar
  4. 4.
    Bosch, H. van den, J.R. Williamson &P.R. Vagelos: Localization of acyl carrier protein in Escherichia coli. Nature 228, 338–341 (1970)CrossRefGoogle Scholar
  5. 5.
    Bradford, M.M.: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254 (1976)PubMedCrossRefGoogle Scholar
  6. 6.
    Capaldi, R.A. &G. Vanderkoi: The low polarity of many membrane proteins. Proc. Natl. Acad. Sci. USA 69, 930–932 (1972)PubMedCrossRefGoogle Scholar
  7. 7.
    Caughey, I. &R.G.O. Keckwick: The characteristics of some components of the fatty acid synthetase system in the plastids from the mesocarp of avocado (Persea americana) fruit. Eur. J. Biochem. 123, 553–561 (1982)PubMedCrossRefGoogle Scholar
  8. 8.
    DiNello, R.K. &M.L. Ernst-Fonberg: Purification and partial characterization of an acyl carrier protein from Euglena gracilis. J. Biol. Chem. 248, 1707–1711 (1973)PubMedGoogle Scholar
  9. 9.
    Ernst-Fonberg, M.L., F. Dubinskas &Z.L. Jonak: Comparison of two fatty acid synthetases from Euglena gracilis variety bacillaris. Arch. Biochem. Biophys. 165, 646–655 (1974)PubMedCrossRefGoogle Scholar
  10. 10.
    Frentzen, M., E. Heinz, T.A. McKeon &P.K. Stumpf: Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts. Eur. J. Biochem. 129, 629–636 (1983)PubMedGoogle Scholar
  11. 11.
    Friedman, M., L.H. Krull &J.F. Cavins: The chromatographic determination of cystine and cysteine residues in proteins as S-beta-(4-pyridyl ethyl)-cysteine. J. Biol. Chem. 245, 3868–3871 (1970)PubMedGoogle Scholar
  12. 12.
    Hancock, W.S., G.R. Marshall &P. Roy Vagelos: Acyl carrier protein XX. Chemical synthesis and characterization of analogues of acyl carrier protein. J. Biol. Chem. 248, 2424–2434 (1973)PubMedGoogle Scholar
  13. 13.
    Hirs, C.H.W.: Determination of cystine as cysteic acid. Meth. Enzymol. 11, 59–62 (1962)CrossRefGoogle Scholar
  14. 14.
    Høj, P.B. &J.D. Mikkelsen: Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts. Carlsberg Res. Commun. 47, 119–141 (1982)Google Scholar
  15. 15.
    Høj, P.B. &J.D. Mikkelsen: Partial purification and characterization of fatty acid synthetase from barley chloroplasts. In: Biochemistry and metabolism of plant lipids. J.F.G.M. Wintermans and P.J.C. Kuiper eds., Elsevier/North Holland. Biomedical Press, Amsterdam, 17–20 (1982)Google Scholar
  16. 16.
    Kaplan, N.O.: Concepts and significance of enzyme evolution. In: Homologous enzymes and biochemical evolution. N. van Thoai & J. Roche, eds. Gordon and Breach, New York, London 405–432 (1968)Google Scholar
  17. 17.
    Lee, H.-M. &J.F. Riordan: Does carboxypeptidase Y have intrinsic endopeptidase activity? Biochem. Biophys. Res. Commun. 85, 1135–1142 (1978)PubMedCrossRefGoogle Scholar
  18. 18.
    Machold, O., D.J. Simpson &B.L. Møller: Chlorophyll proteins of thylakoids from wild type and mutants of barley (Hordeum vulgare L.). Carlsberg Res. Commun. 44, 235–254 (1979)CrossRefGoogle Scholar
  19. 19.
    Majerus, P.W., A.W. Alberts &P.R. Vagelos: Acyl carrier protein from Escherichia coli. Methods Enzymol. XIV, 43–50 (1969)Google Scholar
  20. 20.
    Majerus, P.W.: Acyl carrier protein: Structural requirements for function in fatty acid biosynthesis. J. Biol. Chem. 242, 2325–2332 (1967)PubMedGoogle Scholar
  21. 21.
    Majerus P.W.: Acyl carrier protein: Effects of acetylation and tryptic hydrolysis on function in fatty acid synthesis. Science, 159, 428–431 (1968)PubMedCrossRefGoogle Scholar
  22. 22.
    Matsumura, S. &P.K. Stumpf: Fat metabolism in higher plants XXXV. Partial primary structure of spinach acyl carrier protein. Arch. Biochem. Biophys. 125, 932–941 (1968)PubMedCrossRefGoogle Scholar
  23. 23.
    Matsumura, S., D.N. Brindley &K. Bloch: Acyl carrier protein from Mycobacterium phlei. Biochem. Biophys. Res. Commun. 38, 369–377 (1970)PubMedCrossRefGoogle Scholar
  24. 24.
    McKeon, T.A. &P.K. Stumpf: Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem. 257, 12141–12147 (1982)PubMedGoogle Scholar
  25. 25.
    Mikkelsen, J.D.: Structure and biosynthesis of beta-diketones in barley spike epicuticular wax. Carlsberg Res. Commun. 44, 133–147 (1979)CrossRefGoogle Scholar
  26. 26.
    Møller, B.L. &P.B. Høj: A thylakoid polypeptide involved in the reconstitution of photosynthetic ‘oxygen evolution’. Carlsberg Res. Commun. 48, 169–185 (1983)CrossRefGoogle Scholar
  27. 27.
    Ohlrogge, J.B., D.N. Kuhn &P.K. Stumpf: Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc. Natl. Acad. Sci. USA 76 1194–1198 (1979)PubMedCrossRefGoogle Scholar
  28. 28.
    Phillips, G.T., J.E. Nixon, J.A. Dorsey, P.H.W. Butterworth, C.J. Chesterton &J.W. Porter: The mechanism of synthesis of fatty acids by the pigeon liver enzyme system. Arch. Biochem. Biophys. 138, 380–391 (1970)PubMedCrossRefGoogle Scholar
  29. 29.
    Rasmussen, S.K., H.E. Hopp &A. Brandt: Nucleotide sequences of cDNA clones for B1 Hordein polypeptides. Carlsberg Res. Commun. 48, 187–199 (1983)CrossRefGoogle Scholar
  30. 30.
    Rock, C.O. &J.E. Cronan Jr.: Reevaluation of the solution structure of acyl carrier protein. J. Biol. Chem. 254, 9778–9785 (1979)PubMedGoogle Scholar
  31. 31.
    Roncari, D.A.K., R.A. Bradshaw &P.R. Vagelos: Acyl carrier protein. XIX Amino acid sequence of liver fatty acid synthetase peptides containing 4-phosphopantetheine. J. Biol. Chem. 247, 6234–6242 (1972)PubMedGoogle Scholar
  32. 32.
    Rottem, S., O.M. Pered &S. Razin: Acyl carrier protein in Mycoplasmas. J. Bacteriol. 113, 586–591 (1973)PubMedGoogle Scholar
  33. 33.
    Ruch, F.E. &P.R. Vagelos: The isolation and general properties of Escherichia coli malonylcoenzyme A acyl carrier protein transacylase. J. Biol. Chem. 248, 8086–8094 (1973)PubMedGoogle Scholar
  34. 34.
    Sabaitas, J.E. &G.L. Powell: Acyl carrier protein metabolism and regulation of fatty acid biosynthesis by Lactobacillus plantarum. J. Biol. Chem. 251, 4706–4712 (1976)Google Scholar
  35. 35.
    Schreckenbach, T., H. Wobser &F. Lynen: The palmityl binding sites of fatty acid synthetase from yeast. Eur. J. Biochem. 80, 13–23 (1977)PubMedCrossRefGoogle Scholar
  36. 36.
    Schultz, H.: The importance of carboxyl groups for the biological activity of acyl carrier protein of Escherichia coli. Biochem. Biophys. Res. Commun. 46, 1446–1453 (1972)CrossRefGoogle Scholar
  37. 37.
    Shimakata, T. &P.K. Stumpf: Fatty acid synthetase of Spinacia oleracea leaves. Plant. Physiol. 69, 1257–1262 (1982)PubMedGoogle Scholar
  38. 38.
    Shimakata, T. &P.K. Stumpf: The purification and function of acetyl coenzyme A: acyl carrier protein transacylase. J. Biol. Chem. 258, 3592–3598 (1983)PubMedGoogle Scholar
  39. 39.
    Simoni, R.D., R.S. Criddle &P.K. Stumpf: Fat metabolism in higher plants XXXI. Purification and properties of plant and bacterial acyl carrier proteins. J. Biol. Chem. 242, 573–581 (1967)PubMedGoogle Scholar
  40. 40.
    Svendsen, I., B. Martin &I. Jonassen: Characteristics of hiproly barley III. Amino acid sequences of two lysine-rich proteins. Carlsberg Res. Commun. 45, 79–85, (1980)CrossRefGoogle Scholar
  41. 41.
    Vanaman, T., S.J. Wakil &R.L. Hill: The complete amino acid sequence of the acyl carrier protein of Escherichia coli. J. Biol. Chem. 243, 6420–6431 (1968)PubMedGoogle Scholar

Copyright information

© Carlsberg Laboratory 1983

Authors and Affiliations

  • Peter Bordier Høj
    • 1
    • 2
  • Ib Svendsen
    • 3
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagen Valby
  2. 2.Institute of GeneticsUniversity of CopenhagenCopenhagen K
  3. 3.Department of ChemistryCarlsberg LaboratoryCopenhagen Valby

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