Abstract
The glycoprotein glucoamylase (EC 3.2.1.3) G1 from Aspergillus niger was digested with trypsin after 2-pyridylethylation and the resulting peptide fragments were separated by gel filtration followed by reverse phase HPLC. A different set of peptide fragments was obtained from the citraconylated, 2-pyridylethylated enzyme. These were separated by gel filtrations, affinity chromatography on Con A-Sepharose, and reverse phase HPLC. The amino acid sequence of the isolated peptide fragments was determined by automated Edman degradation and digestion with carboxypeptidases Y and B.
The majority of the carbohydrate of glucoamylase G1 was located in a fragment which carried approximately 35 units of neutral sugar linked O-glycosidically to threonine and serine residues, while a minor fraction was located in a different tryptic fragment which contained a single N-glycosylated asparagine residue.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- ca:
-
citraconyl
- CHO:
-
carbohydrate
- Con A:
-
concanavalin A
- DFP:
-
diisopropylfluorophosphate
- DPCC:
-
diphenylcarbamyl chloride
- EDTA:
-
ethylenediaminetetraacetic acid, disodium salt
- SDS:
-
sodium dodecyl sulfate
- HPLC:
-
high pressure liquid chromatography
- PTH:
-
phenylthiohydantoin-
- 2-pe:
-
2-pyridylethyl-
References
Chung, H. &F. Friedberg: Sequence of the N-terminal half of Bacillus amyloliquefaciens α-amylase. Biochem. J. 185, 387–395 (1980)
Dixon, H.B.F. &R.N. Perham: Reversible blocking of amino groups with citraconic anhydride. Biochem. J. 109, 312–314 (1968)
Downs, F. &W. Pigman: Determination of O-glycosidic linkages to L-serine and L-threonine residues of glycoproteins. Meth. Carbohyd. Chem. 7, 200–204 (1972)
Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers &F. Smith: Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356 (1956)
Edelhoch, H.: Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954 (1967)
Edman, P. &A. Begg: A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967)
Fägerstam, L.G.: Cellulases from Trichoderma reesei QM 9414. Enzymatic and structural properties. Dissertation, Acta Universitatis Upsaliensis no. 610, Uppsala 1981
Freedberg, I.M., Y. Levin, C.M. Kay, W.D. McCubbin &E. Katchalski-Katzir: Purification and characterization of Aspergillus niger exo-1,4-glucosidase. Biochim. Biophys. Acta 391, 361–381 (1975)
Friedman, M., J.C. Zahnley &J.R. Wagner: Estimation of the disulfide content of trypsin inhibitors as S-β-(2-pyridylethyl)-L-cysteine. Anal. Biochem. 106, 27–34 (1980)
Gray, W.R.: End-group analysis using dansyl chloride. Meth. Enzymol. 25, 121–138 (1972)
Gum, E.K. &R.D. Brown: Structural characterization of a glycoprotein cellulase, 1,4-β-D-glucan cellobiohydrolase C from Trichoderma viride. Biochim. Biophys. Acta 446, 371–386 (1976)
Hagenbüchle, O., R. Bovey &R.A. Young: Tissue-specific expression of mouse α-amylase genes: nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland. Cell 21, 179–187 (1980)
Innis, M.: Personal communication
Inokuchi, N., T. Takahashi &M. Irie: Purification and characterization of a minor glucoamylase from Aspergillus saitoi. J. Biochem. 90, 1055–1067 (1981)
Johansen, J.T., C. Overballe-Petersen, B. Martin, V. Hasemann &I. Svendsen: The complete amino acid sequence of copper, zinc superoxide dismutase from Saccharomyces cerevisiae. Carlsberg Res. Commun. 44, 201–217 (1979)
Klapper, D.G., C.E. Wilde III &J.D. Capra: Automated amino acid sequence of small peptides utilizing Polybrene. Anal. Biochem. 85, 126–131 (1978)
Klemm, P.: The complete amino-acid sequence of the K88 Antigen, a fimbrial protein from Escherichia coli. Eur. J. Biochem. 117, 617–627 (1981)
Kluh, I.: Amino acid sequence of hog pancreatic α-amylase isoenzyme I. FEBS Letters 136, 231–234 (1981)
Lineback, D.R. &L.A. Aira: Structural characterization of two forms of glucoamylase from Aspergillus niger. Cereal. Chem. 49, 283–296 (1972)
Mahoney, W.C. &M.A. Hermodson: Separation of large denatured peptides by reverse phase high performance liquid chromatography. Trifluoroacetic acid as a peptide solvent. J. Biol. Chem. 255, 11199–11203 (1980)
Mendez, E. &Lai, C.Y.: Regeneration of amino acids from thiazolinones formed in the Edman degradation. Anal. Biochem. 68, 47–53 (1975)
Mort, A.J. &D.T.A. Lamport: Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal. Biochem. 82, 289–309 (1977)
Nagata, Y., S. Suga, O. Kado &B. Maruo: N-terminal amino acid sequence of α-amylase from Bacillus subtilis var. amylosacchariticus: Comparison with that of “liquefying” type α-amylase. Agri. Biol. Chem. 44, 215–216 (1980)
Nunberg, J., V. Schweickart, F. Lawyer, M. Innis, J. Flatgaard, V. Wittman, & J. Mende: Cloning of the Aspergillus awamori glucoamylase gene. Abstract from “Gene manipulation in the exploitation and study of fungi”. Amer. Soc. Microbiol., May 1983, South Bent, Indiana.
Pasero, L., B. Abadie, Y. Chicheportiche, Y. Mazzei, D. Moinier, J.P. Bizzozero, M. Fougereau &G. Marchis-Mouren: Porcine pancreatic α-amylase I: Sequence between the 35th and 410th amino acid. Biochimie 63, 71–76 (1981)
Pazur, J.H., H.R. Knull &A. Cepure: Glucoenzymes: Structure and properties of the two forms of glucoamylase from Aspergillus niger. Carbohyd. Res. 20, 83–96 (1971)
Pazur, J.H., Y. Tominaga, L.S. Forsberg &D.L. Simpson: Glycoenzymes: an unusual type of glycoprotein structure for a glucoamylase. Carbohyd. Res. 84, 103–114 (1980)
Rosenthal, A.L. &J.H. Nordin: Enzymes that hydrolyze fungal cell wall polysaccharides. The carbohydrate constitution of mycodextranase, an endo-α(1→4)-D-glucanase from Penicillium melinii. J. Biol. Chem. 256, 5295–5303 (1975)
Sachdev, O. &F. Friedberg: Sequence of cyanogen bromide fragments D and E of B. amyloliquefaciens alpha amylase. Int. J. Peptide Protein Res. 18, 228–236 (1981)
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)
Svensson, B., T.G. Pedersen, I. Svendsen, T. Sakai &M. Ottesen: Characterization of two forms of glucoamylase from Aspergillus niger. Carlsberg Res. Commun. 47, 55–69 (1982)
Täkkinen, K., R.F. Pettersson, N. Kalkkinen, I. Palva, H. Söderlund &L. Kääriänen: Amino acid sequence of α-Amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J. Biol. Chem. 258, 1007–1013 (1983)
Toda, H., K. Kondo &K. Narita: The complete amino acid sequence of Taka-amylase A. Proc. Japan Acad 58, Ser. B, 208–212 (1982)
Yamaguchi, H., T. Ikenaka &X. Matsushima: The complete sequence of a glycopeptide obtained from Taka-amylase A. J. Biochem. 70, 587–594 (1971)
Yoshino, E. &S. Hayashida: Enzymatic modification of glucoamylase of Aspergillus awamori var. kawachi. J. Ferment. Technol. 56, 289–295 (1978)
Ueda, S.: Fungal glucoamylases and raw starch digestion. Trends Biochem. Sci. 6, 89–90 (1981)
Author information
Authors and Affiliations
Additional information
d signifies deglycosylated. G1 designates the larger of the two forms of glucoamylase from A. niger (31).
Rights and permissions
About this article
Cite this article
Svensson, B., Larsen, K. & Svendsen, I. Amino acid sequence of tryptic fragments of glucoamylase G1 from Aspergillus niger. Carlsberg Res. Commun. 48, 517–527 (1983). https://doi.org/10.1007/BF02908694
Issue Date:
DOI: https://doi.org/10.1007/BF02908694