Fast and sensitive simultaneous staining method of Q-enzyme, α-amylase, R-enzyme, phosphorylase and soluble starch synthase separated by starch-polyacrylamide gel electrophoresis

Rammesmayer, Gerald; Praznik, Werner

doi:10.1016/0021-9673(92)80384-7

Cited by 13 publications

(12 citation statements)

References 31 publications

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“…This method is based on native electrophoretic division of proteins in the presence of starch, followed by reaction of starch-utilizing enzymes with the starch present in the gel. Starch forms a blue background in the gel after staining, α-amylase gives a transparent band corresponding to utilized starch, and a red band corresponds to glycogen, formed by the migration of a glycogen-branching enzyme to this position (Rammesmayer & Praznik, 1992). As shown in , two distinct glycogen-branching enzymes are present in the WT S. aureofaciens strain.…”

Section: Resultsmentioning

confidence: 99%

“…Glycogen-branching enzyme activity was detected after native starch-PAGE as described by Rammesmayer & Praznik (1992). Samples of S100 fractions (10 μg protein) were loaded on 15% (w/v) acrylamide/1 % (w/v) starch gels and further treated as described by Rammesmayer & Praznik (1992). The zones of glycogen-branching enzyme activity appeared as sharp red bands on the blue-stained background.…”

Section: Methodsmentioning

confidence: 99%

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Disruption of a glycogen-branching enzyme gene, glgB, specifically affects the sporulation-associated phase of glycogen accumulation in Streptomyces aureofaciens

et al. 1996

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~~~In the course of Sfrepfomyces differentiation, glycogen is accumulated in two discrete phases: in substrate hyphae that undergo aerial mycelium formation (phase I), and during septation of aerial hyphae (phase 11). We have disrupted a previously identified gene, g/gB, encoding a putative glycogen-branching enzyme in Streptomyces aureofaciens. Disruption of the gene had no profound effect on sporulation. However, the amount of glycogen-like polysaccharides, compared to wild-type (WT) S-aureofaciens, decreased in the late stage of differentiation of the glgB-disrupted strain. Absorption spectra of polysaccharides extracted from the WT and g/gB-disrupted strains have shown the presence of glycogen in both strains in the first stage of differentiation (aerial mycelium formation), and unbranched glucan was detected in the g/gBdisrupted strain in the late stage of differentiation. The results were confirmed by electron microscopy after silver proteinate staining of glycogen granules. Two distinct glycogen-branching enzymes, which had temporally different expression during differentiation, were detected in WT 5. aureofaciens. The absence of this enzyme activity in the late stage of differentiation in the g/gB mutant suggests that the product of the g/gB gene is responsible for phase II glycogen accumulation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Disruption of a glycogen-branching enzyme gene, glgB, specifically affects the sporulation-associated phase of glycogen accumulation in Streptomyces aureofaciens

et al. 1996

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“…However, we did observe the disappearance of an 88-kD starch hydrolytic activity in all of the sta7-carrying mutants ( Figure 6A). On starch-containing zymograms, the activity bands stained light blue, the hallmark of debranching activities ( Figure 6B; Kakefuda and Duke, 1984;Rammesmayer and Praznik, 1992). We then tested cosegregation in crosses between loss of enzyme activity and the mutant phenotype.…”

Section: Sta7-carrying Mutants Are Defective For An 88-kd Starch Hydrmentioning

confidence: 99%

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants.

et al. 1996

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It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.

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“…Subcellular fractions were analyzed on 10% native-PAGE for activity staining (Rammesmayer and Praznik 1992).…”

Section: Subcellular Fractionation and Native-pagementioning

confidence: 99%

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

Hong

Zhang

2008

Biotechnol Lett

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A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.

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Fast and sensitive simultaneous staining method of Q-enzyme, α-amylase, R-enzyme, phosphorylase and soluble starch synthase separated by starch-polyacrylamide gel electrophoresis

Cited by 13 publications

References 31 publications

Disruption of a glycogen-branching enzyme gene, glgB, specifically affects the sporulation-associated phase of glycogen accumulation in Streptomyces aureofaciens

Disruption of a glycogen-branching enzyme gene, glgB, specifically affects the sporulation-associated phase of glycogen accumulation in Streptomyces aureofaciens

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants.

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

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