Determination of membrane protein glycation in diabetic tissue
Journal Title: The AAPS Journal - Year 1999, Vol 1, Issue 4
Abstract
Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins derived from normal and diabetic tissue. We present a detailed sample preparation protocol based on the borohydride-periodate assay, modified to allow manipulation of animal tissue. Assay noise associated with extraction protocols and nonproteinaceous buffer components was eliminated by the using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) as a membrane detergent, applying desalting columns, and including a protein precipitation step. The glycation level of membrane proteins from diabetic rats is elevated to 4.89 nmol/mg protein (standard deviation [SD] 0.48) compared with normoglycemic control tissue (2.23 nmol/mg protein, SD 0.64). This result is consistent with and correlated to the total glycated hemoglobin levels in diabetic and normoglycemic rats. Using<100 μg protein, the described methods allow further study of protein glycation effects on the function of individual transporter proteins and the role of these modifications in diabetes.
Authors and Affiliations
Eric Y. Zhang, Peter W. Swaan
Keywords
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- EP ID EP682126
- DOI 10.1208/ps010420
- Views 94
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