Abstract
Incubation of human serum with D-[6-3H]glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material. Upon chromatography on Sephadex G-200, radioactivity was found associated with each of the major molecular weight classes of serum protein. Purified human serum albumin was also glucosylated in vitro upon exposure to D-[6-3H]glucose in phosphate-buffered saline. The glucosylated and unmodified albumins were separated by ion exchange chromatography. The physiological significance of these observations in vitro was confirmed by the isolation and quantitation of glucosylated albumin from normal human serum. Glucosylated albumin represents approximately 6 to 15% of total serum albumin in normal adults. The post-translational modification appears to occur by a nonenzymatic process analogous to that responsible for glucosylation of hemoglobin A to hemoglobin AIc, i.e. through Schiff base formation and Amadori rearrangement to a ketoamine derivative.
Highlights
Sephadex G-200, radioactivity was found associated with each of the major molecular weight classes of serum protein
We report here the in vitro glucosylation of albumin and several other serum proteins, and document, for the first time, that 6 to 15% of human albumin in serum exists naturally in a glucosylated form
Human serum albumin was purified from fresh human serum by affinity chromatography on Affi-Gel Blue (Bio-Rad) [8], followed by gel chromatography on Bio-Gel P-150 (Bio-Rad). n-[6-‘HIGlucose
Summary
Human serum albumin was purified from fresh human serum by affinity chromatography on Affi-Gel Blue (Bio-Rad) [8], followed by gel chromatography on Bio-Gel P-150 (Bio-Rad). n-[6-‘HIGlucose (34Ci/mmol) was purchased from New England Nuclear Co.Incubations-Albumin solutions and human serum were sterilized by ultrafiltration, and incubated in the dark at room temperature.Albumin solutions were prepared in Dulbecco’s phosphate-buffered saline [9], containing 5 mM glucose. Human serum albumin was purified from fresh human serum by affinity chromatography on Affi-Gel Blue (Bio-Rad) [8], followed by gel chromatography on Bio-Gel P-150 (Bio-Rad). Ci/mmol) was purchased from New England Nuclear Co. Incubations-Albumin solutions and human serum were sterilized by ultrafiltration, and incubated in the dark at room temperature. Albumin solutions were prepared in Dulbecco’s phosphate-buffered saline [9], containing 5 mM glucose. Serum was incubated under an atmosphere of 95% 02, 5% CO1 to maintain pH 7.3 to 7.4. Trace amounts of [6-“HIglucose were added to incubation mixtures to obtain desired specific activity.
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