Post- and Pre-Natal Assessment of α-l-Iduronidase Deficiency with a Radiolabelled Natural Substrate

Abstract

1. a-L-Iduronidase activity was assayed by incubation of iduronosyl anhydro[ l-3H]mannitol 6-sulphate with homogenates of cultured skin fibroblasts, amniotic cells and leucocytes derived from normal individuals, patients affected with GLiduronidase deficiency disorder (mucopolysaccharidosis type I: Hurler, Scheie and Hurler-Scheie compound) and parents of such patients.2. The assay for cc-L-iduronidase, described for use with these cell types, clearly distinguished affected homozygotes from heterozygotes and normal controls.3. The mean specific activity of a-L-iduronidase in homogenates prepared from cultured skin fibroblasts and leucocytes from more than seven obligate heterozygotes for mucopolysaccharidosis type I was found to be about one-half of the mean of more than 40 normal controls. A number of heterozygotes had a-L-iduronidase activity that identified them as carriers; others had values clearly within the normal range. Thus heterozygote detection of mucopolysaccharidosis type I is not certain.4. With iduronosyl anhydr~[l-~Hlmannitol 6sulphate used as substrate, cell types from five pregnancies at risk for a-L-iduronidase deficiency were examined; each foetus was predicted to be unaffected. For one foetus it was not possible from measurements of total enzyme activity alone to distinguish between the heterozygous carrier state and

Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates

“…Fibroblast cultures were established from skin biopsies taken in, or provided to, this hospital and were harvested as previously described [9]. Typing of MPS patients was consistent with the extent and type ofglycosaminoglycanuria, clinical phenotype and nature of the lysosomal enzyme deficiency [9,10].…”

“…Fibroblast cultures were established from skin biopsies taken in, or provided to, this hospital and were harvested as previously described [9]. Typing of MPS patients was consistent with the extent and type ofglycosaminoglycanuria, clinical phenotype and nature of the lysosomal enzyme deficiency [9,10]. Cells were suspended at a concentration of 2-10 mg of protein/ml in aqueous Triton X-100 (1 mg/ml), and were disrupted by rapid freezing and thawing six times in a solid-CO2/ethanol mixture.…”

Human glucosamine-6-sulphatase deficiency. Diagnostic enzymology towards heparin-derived trisaccharide substrates

“…MPS IIID fibroblasts were obtained from patients with heparansulphaturia and with deficient 6S activity in leucocyte and cultured skin fibroblast homogenates [13,14]. Homogenates of cultured human skin fibroblasts and chorionic villus cells, homogenates of peripheralblood leucocytes and urine supernatants were prepared as described previously [15,16] and dialysed for 16 h at 4°C against 150 mM-NaCl. Protein was determined by the method of Lowry et al [17], with BSA as a standard.…”

“…of buffer C, centrifuged at 25000 g for 30 min as previously described [25] and processed as described above. Cultured normal human skin fibroblasts from four 50 ml culture bottles were harvested at confluence as previously described [15,16], freezethawed six times in 500 ,ul of buffer C and dialysed for 16 h at 4°C against 200 ml of buffer D. The suspension was centrifuged at 4000 g for 10 min and the supernatant was applied to a 3 ml PBE 94 column equilibrated in buffer D. The column was washed with 15 ml of buffer D before the application of 30 ml of a 1:8 dilution of Polybuffer 74/HCI, pH 4.0, followed by 6 ml of the same buffer containing 500 mM-NaCl. Skin fibroblast 6S activity was detected by activity towards GIcNAc6S-IdoA2S-anM6S and GlcNAc6S-IdOA.…”

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties

“…Human livers, lungs and kidneys, obtained from autopsies of normal adults with intervals post mortem ranging from 24 to 72 h, were stored at -20 'C. Cultured skin fibroblasts from normal individuals, were grown and harvested, and the homogenates for enzyme assay were prepared as described previously [15,16]. Pharmacia Fine Chemicals (Uppsala, Sweden) supplied concanavalin A-Sepharose, DEAE-Sephacel, octyl-Sepharose, CM-Sepharose, the chromatofocusing media PBE 94 and Polybuffer 74 and the molecular-mass standard kit for SDS/polyacrylamide-gel electrophoresis.…”