Abstract
To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.
Highlights
To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful
In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio of 5:l
Hyaluronic acid is a ubiquitous nonbranching acid mucopolysaccharide composed of @ 1-4-linked repeating disaccharide units of glucuronic acid p l-3 linked to N-acetylglucosamine
Summary
314, octyl-@-D-glucopyranoside, CHAPSO’, and 98% pure digitonin were purchased from Calbiochem-. The organism was grown in 15 liters of chemically defined medium to mid. Na2HP04/KH2P04, pH6.1, 0.5 mMMgC12, 0.5 mM dithioerythritol phosphate buffer, pH 5.3, with 0.15 M NaCl was added to hyaluronic containing 30%raffinose, phage lysin (10%v/v), and DNase The bacterial preparation was incubated for 1 h detergent extracts(0.025mg of protein/ml) was carried out in 0.05 M at 37 "C, checked by Gram stain for complete protoplast formation, phosphate buffer, pH 7.4, with 0.15 M NaCl. The reaction mixture and sedimented a t 7,500 X g for 8 min at 22 "C. The membranes were Markwell reagent was adjusted to 4% sodium dodecyl sulfate for aliquoted and stored a t 3-5 mg/ml of protein at -70 "C. assaying samples containing Triton X-100.
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