Purification and physicochemical properties of starch phosphorylase from young banana leaves

Kumar, Anil; Sanwal, G.G.

doi:10.1021/bi00260a036

Cited by 22 publications

(13 citation statements)

References 76 publications

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“…The extraction of starch phosphorylase (α-glucan phosphorylase) was based on the method described by Kumar and Sanwal (1982), but with modifications. After extraction, the homogenate was centrifuged at 4°C for 10 min at 16,200 g. The supernatant was incubated at 30°C for 30 min in a 200 µl reaction mixture containing 0.2 M MOPS (pH 7.1), 15 mg ml -1 soluble potato starch, and 16 mM phosphate mix Na 2 HPO 4 /KH 2 PO 4 .…”

Section: Methodsmentioning

confidence: 99%

Maltose Processing and Not β-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis

et al. 2019

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Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways in higher plants and is characterized by high water use efficiency. This mainly relies on major nocturnal CO2 fixation sustained by degradation of storage carbohydrate such as starch to provide phosphoenolpyruvate (PEP) and energy. In contrast to C3 plants where starch is mainly degraded by the hydrolytic route, different observations suggested the phosphorolytic route to be a major pathway for starch degradation in CAM plants. To elucidate the interplay and relevant contributions of the phosphorolytic and hydrolytic pathways for starch degradation in CAM, we assessed diel patterns for metabolites and enzymes implicated in both the hydrolytic route (β-amylase, DPE1, DPE2, maltase) and the phosphorolytic route (starch phosphorylase) of starch degradation in the CAM orchid Phalaenopsis “Edessa.” By comparing the catalytic enzyme activities and starch degradation rates, we showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by β-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.

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Section: Methodsmentioning

confidence: 99%

Maltose Processing and Not β-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis

et al. 2019

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“…The supernatant obtained after centrifugation at 8,000 g for 50 min was considered to be the crude extract. Starch phosphorylase activity was measured in the phosphorolytic and synthesis directions, as described by Kumar and Sanwal (1982). In the phosphorolytic direction, the assay system consisted of 50 mM Mes buffer (pH 6.0), 1% freshly prepared soluble starch, 5 mM sodium fluoride, enzyme extract and 50 mM Na 2 HPO 4 in a total volume of 500 ll.…”

Section: Protein Extraction and Phosphorylase Activitiesmentioning

confidence: 99%

Activity and expression of banana starch phosphorylases during fruit development and ripening

Mota

Cordenunsi

Nascimento

et al. 2002

Planta

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Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanicão) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or alpha-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS-PAGE gels.

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“…Starch phosphorylase activity was measured in phosphorolytic direction, as described by Kumar and Sanwal (1982). The assay system consisted of 50 mM MES buffer (pH 6.0), 1% freshly prepared soluble starch, 5 mM sodium fluoride, enzyme extract and 50 mM Na 2 HPO 4 in a total volume of 500 µL.…”

Section: Methodsmentioning

confidence: 99%

Starch Mobilization and Sucrose Accumulation in the Pulp of Keitt Mangoes During Postharvest Ripening

Silva

Nascimento

Lajolo

et al. 2008

J Food Biochemistry

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Sugar is a determinant for the quality of mangoes, but information about its accumulation is scarce. Although starch can contribute to sugar production during ripening, not much is known about the enzymes involved. This work presents the changes in carbohydrate and enzymes during the development and ripening of Keitt mangoes. Starch disappearance was concomitant to a fivefold increase of sucrose, the most abundant sugar of the ripe fruits. The activities of α‐amylase, β‐amylase, phosphorylase and isoamylase were detected in the pulp, and while α‐amylase increased parallel to the starch content, β‐amylase presented a 20‐fold increase during ripening. On the other hand, high phosphorylase activity was observed when fruits were still accumulating starch, and lowered during ripening. Isoamylase was detected during development and increased slightly during ripening, which would be in agreement to the expected role for isoamylases as acting on both subproduct of starch synthesis and degradation. PRACTICAL APPLICATIONS The present work reinforces our previous works that Keitt mangoes do not ripen when attached to the tree. This fact allowed us to study all the starch degradation after mango harvesting which does not occur with the other cultivars. Data obtained in this work reinforce the role of α‐amylase, β‐amylase and isoamylase rather than the starch phosphorylases on starch granule degradation in mangoes, and the subsequent soluble sugar accumulation.

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Purification and physicochemical properties of starch phosphorylase from young banana leaves

Cited by 22 publications

References 76 publications

Maltose Processing and Not β-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis

Maltose Processing and Not β-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis

Activity and expression of banana starch phosphorylases during fruit development and ripening

Starch Mobilization and Sucrose Accumulation in the Pulp of Keitt Mangoes During Postharvest Ripening

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