The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99 kDa. Pullulanase is a member of the α-amylase family. In addition to a characteristic catalytic (β/α)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.
Skip Nav Destination
Article navigation
May 1998
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
May 01 1998
cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide
Andreas RENZ;
Andreas RENZ
*Max-Planck-Institut für molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, D 14476 Golm, Germany
Search for other works by this author on:
Stephanie SCHIKORA;
Stephanie SCHIKORA
†Lehrstruhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstrasse 30, D 95440 Bayreuth, Germany
Search for other works by this author on:
Roland SCHMID;
Roland SCHMID
‡Abteilung Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D 49076 Osnabrück, Germany
Search for other works by this author on:
Jens KOSSMANN;
Jens KOSSMANN
*Max-Planck-Institut für molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, D 14476 Golm, Germany
Search for other works by this author on:
Erwin BECK
Erwin BECK
1
†Lehrstruhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstrasse 30, D 95440 Bayreuth, Germany
1To whom correspondence should be addressed (e-mail erwin.beck@uni-bayreuth.de).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
September 22 1997
Revision Received:
December 08 1997
Accepted:
January 26 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 331 (3): 937–945.
Article history
Received:
September 22 1997
Revision Received:
December 08 1997
Accepted:
January 26 1998
Citation
Andreas RENZ, Stephanie SCHIKORA, Roland SCHMID, Jens KOSSMANN, Erwin BECK; cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide. Biochem J 1 May 1998; 331 (3): 937–945. doi: https://doi.org/10.1042/bj3310937
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.