Partial breakdown of glycated alkaline phosphatases mediated by reactive oxygen species.
The lower levels of serum alkaline phosphatase (AP) activity found in patients with diabetes mellitus apparently originate from the selective disappearance or decrease in bone AP activity in the circulation. Hence, we investigated in vitro the effect of glycation on the activities of five AP isozymes. Aseptic incubation with 25 mmol/L of D-glucose and APs rapidly reduced bone and placental AP activities before those of liver, kidney and intestinal enzymes. The resulting bone and placental AP molecules were clearly glycated, according to the result of aminophenylboronic acid affinity chromatography. Furthermore, Western blotting analysis revealed that the placental AP molecule was fragmented, and its partial cleavage took place at Ala154 on the AP molecule. Since glycation of serum proteins causes the generation of reactive oxygen species, the effects of reactive oxygen species on placental AP activity were assayed, and the results indicated that hydroxyl radicals might be a major factor for the specific inactivation of AP activities. The reduction in AP activity by incubation with glucose in vitro was reversed by the further addition of catalase. Furthermore, ferrous ion with hydrogen peroxide, which generates hydroxyl radicals, had an inhibitory effect on AP activities. These findings suggest that the reduced AP activity in diabetic patients might result from partial cleavage of the bone AP molecule by reactive oxygen species induced by glycation.
Koyama
I
I
Department of Medical Technology, Junior College, Saitama Medical School, Japan. koyamaw@saitama-med.ac.jp
Yakushijin
M
M
Goseki
M
M
Iimura
T
T
Sato
T
T
Sonoda
M
M
Hokari
S
S
Komoda
T
T
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D001842:Bone and Bones / Q000201:enzymology; D001897:Boronic Acids; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004386:Duodenum / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000494:pharmacology; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology; D017382:Reactive Oxygen Species / Q000378:metabolism*
D001897:Boronic Acids; D016166:Free Radical Scavengers; D007527:Isoenzymes; D017382:Reactive Oxygen Species; C028592:3-aminobenzeneboronic acid; D000469:Alkaline Phosphatase; D005947:Glucose