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Biomedical subjects

J Butterworth

Publications and source records attributed to J Butterworth.

At least 73 records · Page 4Linked to original sources
PubMed1981

Susceptibility to neuraminidase of alpha-L-fucosidase and N-acetyl-beta-D-glucosaminidase of cystic fibrosis, I-cell and neuraminidase-deficient fibroblasts.

Intracellular alpha-L-fucosidase and hexosaminidase showed similar isoelectro-focusing patterns in control, cystic fibrosis and neuraminidase-deficient fibroblasts and were unaffected by neuraminidase treatment. An I-cell strain excreted these two enzymes at 3-4 times the rate of the three other cell types. I-cell and neuraminidase-deficient cells excreted more of the electronegative forms of these enzymes than control and cystic fibrosis cells. Extracellular hexosaminidase A and B were both sensitive to neuraminidase for the four cell types. Extracellular alpha-L-fucosidase consisted of a pH 6.1 form insensitive to neuraminidase and other forms that were sensitive and changed to a pI 7.0-7.1 form. Cystic fibrosis extracellular alpha-L-fucosidase and hexosaminidase behaved as for control fibroblasts.

Acetylglucosaminidase↗
PubMed1980

Intracellular and extracellular alpha-D-mannosidase activity of cultured skin fibroblasts: relationship to cystic fibrosis.

Fibroblast intracellular alpha-D-mannosidase was inactivated by 30--50% after 120 min at 50 degrees C. Separation of alpha-D-mannosidase by isoelectrofocusing and wheat germ lectin-Sepharose into neutral and acid components showed the former to be heat labile and the cause of the observed loss. Fetal calf serum acid alpha-D-mannosidase could be inactivated at 60 degrees C if kept at pH 9.0. Fibroblast extracellular alpha-D-mannosidase was inactivated by 50--70% after 120 min at 50 degrees C, reflecting the greater level of the neutral component. Control and cystic fibrosis alpha-D-mannosidase showed no difference.

Body Fluids↗
PubMed1980

Comparison of the substrate 4-methylumbelliferyl-alpha-l-iduronide with phenyl-alpha-L-iduronide for the diagnosis of Hurler's disease in cultured cells.

4-Methylumbelliferyl-alpha-l-iduronide provided a more sensitive method than phenyl-alpha-l-iduronide for the estimation of alpha-l-iduronidase in cultured cells and could be used to diagnose Hurler's disease. The 4-methylumbelliferyl derivative was no more useful than the phenyl derivative for the detection of heterozygotes. All ten lysosomal enzymes tested could be used as reference enzymes when cell extracts were prepared by freeze/thawing in formate buffer pH 3.5 containing 150 mmol/l sodium chloride.

Amniotic Fluid↗
PubMed1979

Carboxypeptidase B activity of cultured skin fibroblasts and relationship to cystic fibrosis.

Enzyme activity against hippuryl-L-arginine was studied in cultured skin fibroblasts from controls and cystic fibrosis patients. The enzyme had a lysosomal distribution and an acid optimum of pH 4.5 with little or no activity present above pH 7.0. Dithiothreitol was required for full activity and the kinetics of thiol activation were different for the control and cystic fibrosis enzyme. The properties and lysosomal distribution of the enzyme indicated that it was a carboxypeptidase B. Substrate affinity, thermolability, pH stability, the fall and rise in activity with subculture, the cyclical pattern of activity through serial passage and the level of activity were similar for the control and cystic fibrosis enzyme.

Arginine↗
PubMed1979

Primary amniotic fluid cell, skin fibroblast and liver alpha-L-fucosidase and its relation to cystic fibrosis.

Cultured skin fibroblast and primary amniotic fluid cell alpha-L-fucosidase had a double optimum of pH 5.0 and 6.0. Alpha-L-fucosidase was largely bound as a single peak to DEAE-cellulose at pH 6.6. Sucrose density isoelectric focusing revealed up to seven components with pI values of 4.9, 5.2, 5.4, 5.8, 6.1, 6.5 and 7.1 with their apparent KM values (77--500 mumol/l) being higher than that (57 mumol/l) of the unfocused enzyme. Liver, skin fibroblast and amniotic fluid cell alpha-L-fucosidase was separated into two peaks by gel filtration. Peak one was more active and stable at low pH and more thermostable at 50 degrees C than peak two, while both peaks had an apparent KM of 52 mumol/l. Apart from the different proportions of the peaks separated by gel filtration, the results for the three tissues were similar. The properties of alpha-L-fucosidase studied were similar for control and cystic fibrosis liver or skin fibroblasts.

Amniotic Fluid↗
PubMed1978

Acid beta-glucosidase and the diagnosis of Gaucher's disease in liver and spleen.

Variable amounts of residual 4-methylumbelliferyl-beta-glucosidase activity gave rise to difficulties in the diagnosis of Gaucher's disease using spleen and particularly liver. Soluble and particulate components of beta-glucosidase, which interfere with the diagnosis, may be eliminated by preincubation of homogenates at pH 3.0 or with 100 mM sodium chloride at pH 4.0. After either of these treatments the optimum of acid beta-glucosidase was pH 4.5 and diagnosis of Gaucher's disease could be more easily made using homogenates of spleen and liver. There was residual acid beta-glucosidase activity in one of the Gaucher livers.

Gaucher Disease↗
PubMed1978

Carboxypeptidase A activity of cultured skin fibroblasts and relationship to cystic fibrosis.

Properties of carboxypeptidase A of cultured skin fibroblasts from control and cystic fibrosis patients were studied using alpha-N-carbobenzoxy-L-glutamyl-L-tyrosine as substrate. Carboxypeptidase A was inhibited by thiomersal, cyanide, iodoacetate and N-ethylmaleimide in a similar manner for control and cystic fibrosis fibroblasts. Both trypsin and dithiothreitol treatment activated the enzyme, but 1,10-phenanthroline inhibited only in the presence of dithiothreitol. Both Zn2+ and Co2+ reversed this inhibition. Trypsin treatment of carboxypeptidase A produced a form of the enzyme having a higher KM value for both control and cystic fibrosis fibroblasts. Dithiothreitol treatment of control fibroblasts resulted in a form with similar properties to the trypsin activated form, but cystic fibrosis fibroblasts yielded a variant form with even higher KM and Vmax values. Since other properties were similar, it seems likely that this difference reflected binding of a molecule to the enzyme rather than of a defect in the enzyme.

Carboxypeptidases↗
PubMed1978

Acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate.

The properties and levels of acid esterase in cultured skin fibroblasts and amniotic fluid cells were investigated using 4-methylumbelliferyl palmitate as substrate. Determinations of acid esterase activity could be made using as little as 1 microgram cell protein. Cardiolipin increased the activity 2--3 fold at the pH optimum 4.0. The apparent KM for both cell types studied was 196 micrometer without and 96 micrometer with cardiolipin. Acid esterase activity was inhibited by cyanide and thiomersal, but not by iodoacetate and N-ethylmaleimide. However activation by cardiolipin was prevented by iodoacetate, N-ethylmaleimide and also sodium chloride. Skin fibroblasts and primary amniotic fluid cells had similar levels with or without cardiolipin. A cyclic activity was found with subculture but no consistent pattern with passage. The acid esterase deficiency in Wolman's and cholesterol ester storage diseases was demonstrated with this substrate.

Amniotic Fluid↗
PubMed1978

alpha-Glucosidase in Pompe's disease.

Isoelectric precipitation at pH 5.0 and the use of the inhibitors, turanose, maltose and citrate, enabled the diagnosis of Pompe's disease to be made in dextran-isolated leucocytes using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate. These techniques were unnecessary with lymphocytes as the deficiency of acid alpha-glucosidase could be shown directly.

Clinical Enzyme Tests↗
PubMed1978

Diagnosis of Gaucher's disease in cultured skin fibroblasts and leucocytes.

Washing skin fibroblasts or leucocytes in 0.25 mol/l sucrose increases the activity of beta-glucosidase at acid pH. This effect is primarily due to removal of low levels of sodium chloride, which inhibit acid beta-glucosidase. A secondary factor for skin fibroblasts in the removal of residual phosphate buffer pH 7.3 used to wash the cells following trypsinization. As the beta-glucosidase activity of water-lysed leucocytes is higher at acid pH than that of a saline suspension of leucocytes, the former are better for the diagnosis of Gaucher's disease. However, more reliable results still may be obtained by assay of this enzyme in cultured skin fibroblasts.

Cells, Cultured↗
PubMed1978

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells.

Assay of alpha-L-iduronidase, heparin sulphamidase, N-acetyl-alpha-D-glucosaminidase, arylsulphatase B, alpha-L-fucosidase, beta-glucuronidase, beta-galactosidase and alpha-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made.

Acetylglucosaminidase↗
PubMed1978

Pompe's disease: diagnosis in kidney and leucocytes using 4-methylumbelliferyl-alpha-D-glucopyranoside.

The diagnosis of Pompe's disease by the assay of acid alpha-glucosidase in kidney and leucocytes was not previously possible because of the presence of another component which had activity at pH 4.0, but was not deficient in the disease. This problem was resolved either by the use of the inhibitors, turanose, maltose and citrate, or by isoelectric precipitation at pH 5.0, which enabled the estimation of acid alpha glucosidase in kidney and leucocytes.

Glucosides↗
PubMed1978

Low arylsulphatase A activity in a family without metachromatic leukodystrophy.

A low arylsulphatase A activity was noted in the leukocytes and cultured skin fibroblasts of a child without any other symptoms of metachromatic leukodystrophy. Although the mother had a level of arylsulphatase commensurate with heterozygosity for the classical metachromatic leukodystrophy gene, the father had a variant gene giving an unusually low in vitro level of this enzyme. In combination (the proband), these two genes gave rise to a very low in vitro activity without any apparent disease. In screening for metachromatic leukodystrophy, a low arylsulphatase A level is not necessarily indicative of this disease, if a clinically normal parent shows an unusually low level of this enzyme.

Arylsulfatases↗
PubMed1977

Diagnosis of Pompe's disease in cultured skin fibroblasts and primary amniotic fluid cells using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate.

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.

Amniotic Fluid↗
PubMed1977

The diagnosis of Gaucher's disease in liver using 4-methylumbelliferyl-beta-D-glucopyranoside.

1. Cases of Gaucher's disease could not be distinguished from controls by the assay of beta-glucosidase activity in water homogenates of liver using 4-methylumbelliferyl-beta-D glucopyranoside. 2. Two peaks of beta-glucosidase activity were separated by Sephadex G-150 gel filtration in control and Gaucher livers. In the presence of the elution buffer pH profiles of peak I showed a deficiency at pH 3.5-4.5 in Gaucher's disease. Gaucher and control peak II had similar pH profiles with little or no activity at pH 3.0-4.0. 3. A clear distinction between homogenates of Gaucher and control liver was obtained by assay at pH 4.0 in the presence of elution buffer, or of sodium chloride, a component of the elution buffer.

Gaucher Disease↗