Title

Purification And Characterization Of The Rat Ovarian Prostaglandin Synthase

Date of Award

1983

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Abstract

Prostaglandin (PG) synthase is a membrane-bound glycoprotein which can be solubilized from membrane preparations using Tween 20. The enzyme is highly unstable during purification but the addition of 5 mM diethyldithiocarbamate (DDC), 1 mM phenol and 2 uM hematin have proven useful in preventing inactivation. The rat ovarian PG synthase was applied to a DEAE-cellulose column and eluted with 0.025-0.050 M KCl. Fractions containing enzyme activity were pooled, applied to a lentil lectin (LL) carbohydrate affinity column, and subsequently eluted with 0.2 M (alpha)-methylmannoside. Under optimum conditions a 1,670-fold purification with 20% recovery of activity was obtained. Non-denaturing PAGE resulted in an additional two-fold purification with 22% recovery of activity. Upon SDS-PAGE, the enzyme purified by non-SDS PAGE demonstrated significant enhancement of two protein bands near M(,r) = 70,000. Chromatofocusing chromatography revealed an apparent pI for the enzyme of 6.1 (+OR-) 0.2. Upon chromatography on Bio-Gel A1.5 M, enzyme activity eluted with protein of M(,r) = 225,000. This result is possibly due to the existence of the enzyme as an aggregate of at least a dimer. Prostacyclin (PGI) isomerase co-purified along with PG synthase during DEAE-cellulose and LL chromatography. The ID(,50) values of aspirin and flufenamic acid for the PG synthase were 18 and 9 uM respectively. DDC or glutathione (GSH) in the presence of 1 mM phenol inhibited PGE and stimulated PGF formation by purified enzyme, but did not affect total PG production. The combination of DDC and GSH stimulated both PGE and PGF formation by the purified enzyme and enhanced total PG production. The stimulatory effect of DDC and GSH on PG formation by solubilized enzyme was even more marked. Quinones inhibited and indolic compounds stimulated activity in the presence of DDC and GSH. Purified enzyme was less subject to inhibition by hematin than solubilized enzyme. These results indicate that isolation of the PG synthase in the presence of hematin, phenol and DDC both stabilizes and activates the enzyme. The enzyme is then less subject to further stimulation by these or other cofactors. The rat ovarian PG synthase did not cross-react with anti-sheep seminal vesicle PG synthase. Partially purified enzyme was antigenic in mice and use of the most highly purified preparations should facilitate production of rat ovarian PG synthase-specific monoclonal antibodies.

Keywords

Biology, General

Link to Full Text

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