چکیده (انگلیسی):

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 membraneassociated
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.