The nature of heterogeneous components of extracellular-superoxide dismutase purified from human umbilical cords.
Extracellular-superoxide dismutase (EC. 1.15.1.1., EC-SOD) is a secretory, tetrameric glycoprotein. This enzyme in plasma is heterogeneous with regard to heparin affinity and can be divided into at least three fractions approximately equally large: EC-SOD A, which lacks affinity; EC-SOD B with intermediate affinity; and EC-SOD C with high affinity. In this article, EC-SOD has been purified with a high yield from human umbilical cords. Of the umbilical cord EC-SOD, 0.8% behaved as subtype A, 1.9% as subtype B, and almost all as high heparin affinity subtype C. Purified native EC-SOD (n-EC-SOD C) showed a single band with enzymatic activity on polyacrylamide gel electrophoresis. It showed two bands with apparent molecular masses of 29.3 and 32.0 kDa on SDS-PAGE, while recombinant EC-SOD C (r-EC-SOD C) showed only one band with 32.0 kDa. By western blotting analysis with anti r-EC-SOD C antibody, two bands of n-EC-SOD C were detected at the same positions as in the gel stained with Coomassie blue. The appearance of two monomeric components with different molecular masses does not reside in the carbohydrate moiety, because the difference between the two components was not abolished by glycopeptidase F treatment; however, both bands were shifted to lower molecular weight ranges by this treatment. The two components could be clearly separated from each other by C4 reverse-phase high-performance liquid chromatography (HPLC).(ABSTRACT TRUNCATED AT 250 WORDS)
Ohta
H
H
Department of Pharmaceutics, Gifu Pharmaceutical University, Japan.
Adachi
T
T
Hirano
K
K
D000581:Amidohydrolases / Q000494:pharmacology; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology*; D005609:Free Radicals; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014470:Umbilical Cord / Q000201:enzymology*
D005609:Free Radicals; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006493:Heparin; D013482:Superoxide Dismutase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase