Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.
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.
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Triscott
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van de Rijn
D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology*; D013291:Streptococcus / Q000187:drug effects / Q000378:metabolism*
D002250:Carbon Radioisotopes; D003902:Detergents; D010743:Phospholipids; D006820:Hyaluronic Acid; D004072:Digitonin