Inhibition of alkaline phosphatase activity by glucose.

Non-enzymatic glycosylation (NEG) of alkaline phosphatase (AP) was studied after short- and long-term incubation with glucose and other carbohydrates. Glucose and amino sugars clearly inhibited the enzyme activity; this was in contrast to reducing and non-reducing disaccharides, which had an enhancing effect. After AP had been incubated with 18 nmol/l glucose for 180 minutes (short-term incubation), a subsequent extensive dialysis revealed full recovery of the enzymatic activity. This, plus the demonstration of a [3H]sodium borohydride-reducible glucose-protein adduct, indicated that initially a labile aldimine (Schiff base) had been formed. Binding experiments with [14C]glucose and failure of dialysis to achieve a recovery of enzymatic activity after long-term incubation suggested that subsequently a stable ketoamine product had been formed. This was further confirmed by the thiobarbituric acid test, which revealed 0.65 nmol 5-hydroxymethylfurfural/mg protein for glycosylated AP compared to 0.11 for the non-glycosylated control. Preliminary results further suggest that NEG of AP also occurs in vivo. Streptozotocin diabetic rats had significantly lower serum AP activities than did non-diabetic controls (mean +/- SD: 153.7 +/- 28.4 vs. 760.5 +/- 95.7 U/l; p less than 0.001). Blood glucose levels and serum AP activity, which had been determined simultaneously during an oral glucose tolerance test, showed without exception an inverse relationship in each of 32 healthy children studied. The biological significance of these findings remains to be established.

Reduced susceptibility of glycosylated hemoglobin to proteolytic cleavage.

Four different experimental designs were selected to study, whether glycosylation of hemoglobin alters susceptibility against proteolytic degradation. We compared the resistance of different hemoglobin fractions against degradation by pure proteolytic enzymes, by liver cell culture, by live organ culture and the resistance of in vitro glycosylated vs. "non-glycosylated" hemoglobin solutions. The hemolysates were characterized by high performance liquid chromatography. The degree of glycosylation of hemoglobin was further estimated by the thiobarbituric-acid assay. From the data presented it is concluded, that the glucose moiety linked to the N-terminal valine and lysine residues of the alpha- and beta chain of the hemoglobin exerts a protective effect against proteolytic cleavage. This could explain the extended lag phase in response of glycosylated hemoglobin to an improved metabolic control in diabetic patients and the discrepancy between the rate of synthesis and the rate of catabolism.

Proteolytic degradation of the glomerular basement membrane and immunochemical characterization of split products.

Glomerular basement membranes (GBM) were isolated and subjected to enzymatic degradation with the protease trypsin (Serva), chymotrypsin (Serva), papain (Sigma), pepsin (Serva) and collagenase (Worthington) as well as a lysosomal preparation from glass adherent rat blood and peritoneal exudate cells. Split products were characterized by immunoelectrophoresis and cellulose acetate electrophoresis. Urine was obtained from healthy rats and rats with Masugi's experimental glomerulonephritis, dialyzed and concentrated and applied on immunoelectrophoresis, using anti-GBM antibody from rabbit. Urinary GBM split products from healthy and nephrotic rats showed two precipitation lines like digestion products obtained after chymotrypsin degradation. This finding was supported by characterizing individual antigenic degradation products obtained after inhibition of GBM degradation by the lysosomal preparation with ethylenediaminetetraacetic acid and Trasylol alone and in combination, as well as with o-phenanthrolin. It is concluded that GBM-antigens excreted into urine indicate limited digestion of GBM by chymotrypsin-type protease.