Biosynthesis of Proteokeratan Sulfate in the Bovine Cornea. 1) Isolation and Characterization of a Keratan Sulfotransferase and the Role of Sulfation for the Chain Termination

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Abstract
Bovine corneal keratan sulfotransferase and chondroitin sulfotransferase were enriched 244-fold and 255-fold, respectively, from corneal stroma via tissue homogenizing, sequential centrifugation, gel chromatography and DEAE-cellulose chromatography. Sulfotransferase activity was detected in the microsomal fraction and in the cytosol. Keratan sulfotransferase and chondroitin sulfotransferase activity purified from the cytosol could not be separated from each other. The temperature optimum was found at 12.degree. C for keratan sulfate and at 12.degree. C and 25.degree. C for chondroitin sulfate, the pH optimum at pH 6.0 and 8.6 for keratan sulfate and at pH 6.6 and 8.6 for chondroitin sulfate, as substrates. Both enzyme activities exhibit a Km value of 2.5 .times. 10-5 M. The molecular mass was determined by gel chromatography to be 240,000 Da [daltons]. Both enzymes are activated by Mn2+, Mg2+, Zn2+ and Co2+ and inhibited by Cu2+ at concentrations above 0.1 mM as well as at ATP, ADP and AdoPS [5''-adenylylsulfate (adenosine 5''-phosphosulfate)] concentrations above 0.08 mM. 2''-AMP, 3''-AMP, 5''-AMP and cAMP have less inhibitory effects. All adenine nucleotides investigated inhibited the 3''-phosphoadenylyl-sulfate hydrolase activity at concentrations higher than 0.08 mM. Iodoacetamide, iodoacetic acid, dithioerythritol, mercaptoethanol, cysteinium chloride and oxidized glutathion have no effect on the sulfotransferase activity at concentrations 0.08-5.0 mM. These substances strongly inhibit the 3''-phosphoadenylyl-sulfate hydrolase activity. Sulfate transfer by the purified enzyme could be detected in the case of bovine and porcine corneal keratan sulfates, and bovine corneal chondroitin sulfate but not in the case of hyaluronate, over-sulfated chondroitin sulfate from shark cartilage, glycosaminoglycan polysulfate (Arteparon), dermatan sulfate from porcine skin, and heparin from lung and mucosa as substrates. The purified enzyme transferred only into the 6-position of chondroitin sulfate. The enzyme activity decreased with increasing molecular mass and sulfation degree of the substrate keratan sulfate. A mathematical model was postulated, which describes in the case of corneal proteokeratan sulfate biosynthesis, how the biocatalysts deteriorate their own substrate during the synthesis of a sulfated chain by increasing sulfation.

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