+ Site Statistics
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on Google+Follow on Google+
Follow on LinkedInFollow on LinkedIn

+ Translate

Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme

Biochemical Journal 331: 929-935
Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme
Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1. 41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1-7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands ('substrate-induced forms', numbered I-III) with different specific activities, dissociation constants of the enzyme-substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.

(PDF same-day service: $19.90)

Accession: 003245826

PMID: 9560324

DOI: 10.1042/bj3310929

Related references

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide. Biochemical Journal 331: 937-945, 1998

Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms. Biochimica et biophysica acta = International journal of biochemistry and biophysics, 1548(2): 175-186, 2001

Kinetic analysis of parallel reactions, the initial reactant of which presents with two interconvertible isomeric forms (hydrolysis and hydroxylaminolysis of 6-phosphogluconolactone). Journal of Theoretical Biology 200(4): 427-434, 1999

Coexistence of monomers and stable oligomerization forms of HIV-1 Tat and requirement of monomeric forms for the transactivating function on the HIV-1 LTR. FASEB Journal 14(6): A935, April 20, 2000

Acid phosphatase from needles of Pinus silvestris L. Purification of two interconvertible enzyme forms and characterization of a low-molecular weight factor associated with the enzyme. Biochimica et Biophysica Acta 660(2): 204-213, 1981

Succinate thiokinase. VI. Multiple interconvertible forms of the enzyme. Journal of Biological Chemistry 248(1): 15-24, 1973

Platelet pyruvate kinase. Two interconvertible forms of the enzyme. Biochimica et Biophysica Acta 522(1): 104-112, 1978

Purification and properties of pig liver prenyltransferase: interconvertible forms of the enzyme. Archives of Biochemistry and Biophysics 183(2): 718-725, 1977

A metabolic switch produced by enzymically interconvertible forms of an enzyme. Febs Letters 187(2): 193-195, 1985

Purification of mitochondrial creatine kinase 2 interconvertible forms of the active enzyme. Biochemical and Biophysical Research Communications 76(3): 950-956, 1977