Use of purified amyloglucosidase for the specific determination of total carbohydrate content of rat liver homogenate in a single step. |
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Authors: | J A Johnson R M Fusaro |
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Affiliation: | 1. Department of Dermatology, University of Nebraska Medical Center, Omaha, Nebraska, 68105 USA;1. Department of Biochemistry, University of Nebraska Medical Center, Omaha, Nebraska, 68105 USA |
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Abstract: | In prior studies, we employed a crude amyloglucosidase preparation, in conjunction with glucose oxidase reagent, to determine total carbohydrate in liver and muscle homogenates by a two-step procedure. Glycogen content was determined by subtracting tissue glucose (determined separately). By use of a purified amyloglucosidase, we have now verified the accuracy of two-step assay of rat liver homogenate with crude amyloglucosidase. The reliability of glucose oxidase detection of glucose in amyloglucosidase hydrolysates was established by comparing results with those obtained with hexokinase/glucose 6-phosphate dehydrogenase reagent. The availability of purified amyloglucosidase made it feasible to assay total carbohydrate content of homogenate in a single step, by incubating aliquots in the presence of both amyloglucosidase and glucose oxidase reagents. Results obtained with this one-step assay agreed with those of two-step analysis. To our knowledge, this is the first report of direct enzymic assay of total carbohydrate in rat liver homogenate, in a single step. |
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