Inhibition of Protocollagen Proline Hydroxylase by Dilantin

Abstract

Dilantin (sodium 5,5'-diphenylhydantoinate) is considered to be one of the basic drugs in the treatment of convulsive disorders (1). One of the common side effects of Dilantin is the hyperplastic enlargement of gingivae (2, 3). Several lines of indirect evidence suggest that gingival hyperplasia may involve increased collagen [ for review, see Ref. (4)].In a recent report (S), Ebadi and Scott have reported enhanced synthesis of collagen in carrageenin-induced granulomas in guinea pigs treated with Dilantin. They suggested that increased protocollagen proline hydroxylase (PPH) activity may be involved in the increased synthesis of collagen. We have examined the effect of Dilantin on purified PPH and our observations suggest that Dilantin inhibits PPH, probably by complexing Fe2+ which is required in the hydroxylation reaction (6). Materials and Methods.Dilantin was obtained from Sigma Chemical Co. Protocollagen substrate was prepared from cartilage explants from 10-day-old chick embryos. The tissues were preincubated for 15 min with 1.0 mM, a,a'-bipyridyl in Krebs' phosphate buffered medium (7). 3,4-3H-proline (50 pCi, 4.8 Ci/mmole, New England Nuclear) was added in a final volume of 5.0 ml and incubation continued for 2 hr after which the tissues were washed with chilled water and homogenized in 0.5 N acetic acid and extracted overnight at 4' in 0.5 N acetic acid. The extracts were centrifuged at 100,OOOg fot 1 hr and the supernatant was dialyzed against several changes of 0.05 N acetic acid. Aliquots lsupported by Grants AM 15178 and HD 05812 from the US. Public Health Service. 2Recipient of a Research Career Development Award, DE 41,311 and the author to whom correspondence should be addressed.of the dialyzed protocollagen extract, containing approximately 150,000 dpm were used in the enzyme assay.PPH was purified by an adaptation of the procedure described by Halme, Kivirikko and Simons (8) through the DEAE-cellulose chromatography step. The most purified preparations obtained in this step had a specific activity 330 times greater than that of the 15, OOOg supernatant fraction of chick embryo homogenate used as the starting point. The enzyme assay was conducted as described previously ( 6 ) .Results and Discussion. Increasing concentrations of Dilantin caused increasing inhibition of the enzyme (Table I ) . In order to examine the nature of the interaction between Dilantin and PPH, a Hill plot (Fig. 1 ) (9) was constructed from the inhibition data. The interaction coefficient measured from the slope of the Hill plot was 0.97 suggesting that TABLE I. Inhibition of Protocollagen Proline Hydroxylase by Dilantin. 3HHOb formed % of Addition sa (dpm/ml aliquot) controlb None 3960 100 + Dilantin, I mM 3405 86 + Dilantin, 2mM 2850 72 + Dilantin, 4mM 2495 63 + Dilantin, 8mM 1620 41 + Dilantin, 8 m M + Fe2+, I.0mM 3828 96 a Complete components for the enzyme assay included enzyme, 200 pg; substrate, 108,000 dpm; ascorbic acid, 0.5 mM; a-ketoglutaric acid, 0.1 mM; ferrous ammonium sulfate, 0.1 mM; Tris-HCl buffer, 100 mM...

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