Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells

K F Ng,N B Schwartz

doi:10.1016/s0021-9258(18)80133-8

Abstract

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.

Highlights

Cells withhyaluronidase prior to extraction with digi- Prehm (8) has presented evidence that the growing hyalurotonin resulted in a %fold increase in solubilized syn- nate chain is bound covalently to the enzyme through the thetase activity
Hyaluronate is widely Inthis report we present evidence on the first successful distributedin both vertebrateandinvertebrate connective solubilization of functional hyaluronate synthetase from a tissues and is found as a component of the cell wall of eucaryotic cell line, mouse oligodendroglioma
Forthis purpose an affinity matrix was solubilizingthe hyaluronate synthetase from glioma cells with prepared by coupling a 60-kDa hyaluronic acid binding do- retention of activity

Summary

RESULTS

Saline and mixed with the appropriate detergent. The membranes were extracted at 4 “C for 1h with gentle stirring. The solubilized enzyme from untreated cells, bycontrast, was eluted from the void volume of a Sepharose CL-GB column with about a 16-fold increase in specific activity (Fig. 4b). Both hyaluronate species have similar profiles; a several high molecular weighptrotein components which were peak consisting of large material which elutes in the void enriched in this preparation. Washed crude membranes were obtained from solubilized enzyme from hyaluronidase-treated was small hyaluronidase-treated and -untreated cells, and a portion of enough to be included by Sepharose CL-GB and eluted as a each preparation was extracted with digitonin and centri- broad peak immediately after the voidvolume

Untreated Proteoglycan-Sepharose 4B Sepharose 4B

Findings

DISCUSSION