Characterization of Deficient Heme Synthase Activity in Protoporphyria with Cultured Skin Fibroblasts

Abstract

A B S T R A C T Heme synthase (ferrochelatase) activity, as determined by the chelation of ferrous iron to protoporphyrin or deuteroporphyrin, is reduced to 10-25% of normal in tissues of patients with protoporphyria. With cultured skin fibroblasts from seven patients with protoporphyria and six normal individuals, the present studies examined the enzymatic defect.Heme synthase activity in normal and protoporphyria fibroblasts had the same pH optimum, showed similar inhibition by divalent metals, and had the highest specific activity in the mitochondrial-enriched fraction. The ultrastructural features and other biochemical parameters of mitochondria were normal in protoporphyria cells, excluding a general mitochondrial defect. Measurement of the rate of deuteroheme formation at different concentrations of substrate demonstrated a significant reduction in the apparent Km for deuteroporphyrin in detergent-treated sonicates of protoporphyria fibroblasts compared to normal (7.5 ±0.9 gM, mean±SEM, vs. 17.4+1.8), as well as a decrease in the velocity of reaction (mean level was 21% of normal). Studies with intact cells, in which heme synthase activity was estimated indirectly, also indicated that the apparent Km for porphyrin substrate was significantly lower in protoporphyria lines.These data show that heme synthase in protoporphyria fibroblasts has markedly reduced catalytic activity despite an increased affinity for porphyrin substrate. This could be caused by either a change in the enzyme protein, or an alteration of its microenvironment.

Studies in porphyria: functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria.

“…Ferrochelatase from human sources has not to date been purified to homogeneity. Therefore it is not possible to conclude that the enzyme in EPP cells is structurally different from the normal enzyme, although this has been suggested (19).…”

“…Finally, only 4 d are required for the technique described in this method, a time which is much shorter than that required for assays using cultured skin fibroblasts (6,10,26). The lymphocyte culture system is thus considerably more convenient for detecting EPP gene carriers than the fibroblast system.…”

The synthesis of murine ferrochelatase in vitro and in vivo

“…In the genetic disorder protoporphyria, ferrochelatase activity is decreased to about 20 % of normal (Bloomer, 1980). In cattle the disorder is genetic recessive (Bloomer et al, 1982) and appears to be attibutable to production of normal amounts of a modified, less active, form of the enzyme (Bloomer et al, 1987 (Dailey & Fleming, 1983), other alternatives must be proposed.…”

A Radiochemical Assay for Heme Synthase Activity

“…Human liver specimens and human cultured skin fibroblasts are prepared as pre viously described [3,7].…”

“…Distilled water oxidized spectrum was recorded from 600 to 500 nm. The mesoheme formed was calcu lated [3] by using (3 ml) was added and the solution neutralized with 1 N HC1 to approximately pH 8. The solution was buffered with Tris (300 pi, 0.2 mol/1, pH 8.2) and detergent added (1% w/v Tween 80, 1.2 ml).…”