A defect in the metabolic activation of sulfate in a patient with achondrogenesis type IB.

Achondrogenesis type I is a perinatally lethal, short-limb chondrodysplasia. Two types, IA and IB, have been distinguished by radiographic and histological criteria; both types appear to be inherited as autosomal recessive traits. The underlying molecular defects are not known, but histochemical studies have suggested that in achondrogenesis type IB, cartilage matrix is deficient in sulfated proteoglycans. We have studied cartilage extracts of one newborn with achondrogenesis type IB and found that proteoglycans were quantitatively reduced, and, unlike in control cartilage, they did not stain with toluidine blue and did not bind to DEAE. Impaired synthesis of sulfated proteoglycans was observed also in fibroblast cultures of the achondrogenesis IB patient. Radioactive labeling and immunoprecipitation studies indicated that core protein and side chains of proteoglycans were synthesized normally but were not sulfated. Analysis of sulfate metabolism in fibroblast cultures showed, in the patient's cells, normal intracellular levels of free sulfate but markedly reduced levels of the two intermediate compounds in the sulfate activation pathway, adenosine-phosphosulfate and phosphoadenosine-phosphosulfate. The results can be explained by deficient activity of one of the enzymes responsible for the biologic activation of sulfate, possibly similar to that observed in cartilage (but not in skin) of the recessive, nonlethal mouse mutant brachymorphic and leading to defective sulfation of macromolecules. Expression of the sulfation defect in cultured fibroblasts may offer a diagnostic tool for the disorder.

Radioactive-electrophoretic assay of adenosine 5'-triphosphate sulfurylase activity in crude extracts with sulfate or selenate as a substrate.

An assay method for ATP sulfurylase is presented which employs Na2(35)SO4 as a substrate and measures the production of labeled adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate by low-voltage, hanging paper strip electrophoresis. The method is applicable to crude bacterial or mammalian extracts and accurately measures picomole amounts of product(s). Na2(75SeO4 can also be employed as a substrate, if the unstable radioactive product, adenosine 5'-phosphoselenate, is converted to elemental 75Se degrees by inclusion of reduced glutathione in the reaction mixture. The same paper strip electrophoretic technique can then be used to separate 75Se degrees from the radiolabeled substrate. The method also has utility for measuring any direct reduction by crude microbial extracts of radioactive selenate to selenite, independent of ATP sulfurylase.

Assay of adenosine 3'-phosphate 5'-sulphatophosphate in hepatic tissues.

A fluorimetric assay, based on the ability of boiled hepatic extracts to support the sulphO-conjugation of harmol, was used to demonstrate and quantify PAdoPS (adenosine 3'-phosphate 5'-sulphatophosphate) present in liver. A stoicheiometric relationship was established between the sulphate conjugate formed and the 'active sulphate' utilized. Guinea-pig, rat, mouse and rabbit livers contain 3.3, 2.9, 0.8 and 0.5 mumol of PAdoPS/100 G wet wt. respectively.