Enhanced stability of beta-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran.
In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.
D000818:Animals; D002417:Cattle; D003911:Dextrans / Q000378:metabolism*; D004589:Electrophoresis, Disc; D005696:Galactosidases / Q000378:metabolism*; D006358:Hot Temperature; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D012709:Serum Albumin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism*
D003911:Dextrans; D012709:Serum Albumin; D005696:Galactosidases; D001616:beta-Galactosidase