Interaction of glucose and metformin with isolated red cell membrane.
Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.
Freisleben
H J
HJ
Gustav-Embden-Zentrum der Biologischen Chemie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany.
Fürstenberger
H J
HJ
Deisinger
S
S
Freisleben
K B
KB
Wiernsperger
N
N
Zimmer
G
G
D001643:Bridged Bicyclo Compounds; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects; D008565:Membrane Proteins / Q000737:chemistry; D008687:Metformin / Q000494:pharmacology*; D011485:Protein Binding; D012709:Serum Albumin / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013439:Sulfhydryl Reagents
D001643:Bridged Bicyclo Compounds; D007328:Insulin; D008565:Membrane Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008687:Metformin; D005947:Glucose; C028381:monobromobimane