Determination of membrane protein glycation in diabetic tissue.
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 microg 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.
Zhang
E Y
EY
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 West 12 Ave, Columbus, OH 43210-1291, USA.
Swaan
P W
PW
D000818:Animals; D015153:Blotting, Western; D001894:Borohydrides; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000032:analysis*; D010504:Periodic Acid; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D001894:Borohydrides; D007202:Indicators and Reagents; D008565:Membrane Proteins; D010504:Periodic Acid; C009288:metaperiodate