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Biomedical subjects

B M Turner

Publications and source records attributed to B M Turner.

108 records · Page 6Linked to original sources
PubMed1978

Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease.

Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease.

Adolescent↗
PubMed1977

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.

Cell Membrane↗
PubMed1977

alpha-L-fucosidase in cultured skin fibroblasts from normal subjects and fucosidosis patients.

alpha-L-Fucosidase (EC 3.2.1.51) activity was studied in cultured skin fibroblasts obtained from 23 members of a family in which two cases of fucosidosis type 2 had occurred and in the fibroblasts of a patient with fucosidosis type 1. Both the 4-methylumbelliferyl glycoside and the p-nitrophenyl derivative were used as substrates. pH activity profiles showed two major peaks of activity. With the fluorogenic substrate pH optimum was at 4.5, whereas with the colorigenic substrate maximum activity was at pH 5.7. No activity was found in the fibroblasts of the three patients with the colorimetric assay. With the fluorometric assay the mean activity in the two patients with fucosidosis type 2 was 4.1 and 2.8 (range 1.3-9.9); activity in the patient with fucosidosis type 1 was 4.6 nmol 4-methylumbelliferone/mg protein/hr (range 1.0-8.6). The specific activity in the lysates of the patients' fibroblasts decreased as the amount of cellular protein used per assay increased. Fucosidosis fibroblasts cultured for 3 and 5 days in medium without fetal calf serum showed almost the same levels of apparent residual activity as fibroblasts cultured in medium containing fetal calf serum. Maximum activity in the deficient fibroblasts was at pH 4.5-4.75. Mixing experiments between lysates of both of fucosidosis and a normal fibroblast strain showed the expected enzymatic activities. The mean alpha-fucosidase activity in four heterozygotes for fucosidosis was 37.6 (range 24.1-48.7) and 30.3 (range 19.0-44.1) nmol final product split/mg protein/hr with the fluorogenic and the colorigenic substrate, respectively. In 12 normal fibroblast strains the mean activity +/- SD was 85.3 +/- 24.4 (range 50.8-129.3) and 67.6 +/- 21.1 (range 31.1-118.3). However, in four family members in which the alpha-L-fucosidase phenotype (by isoelectric focusing) was type 2-1, and who should therefore be carrying two normal alleles, the activity was within the heterozygote range. This indicates that occasional overlap between normal subjects and carriers may be present in cultured skin fibroblasts. Increased specific activity of alpha-L-fucosidase at pH 3.2-4.0 was observed after incubation of cell lysates with neuraminidase. The alpha-L-fucosidase activity did not show any increase after fusion between fucosidosis fibroblasts type 1 and 2.

Biopsy↗
PubMed1976

alpha-L-Fucosidase in normal and deficient individuals.

Deficiency of the enzyme alpha-L-fucosidase has been demonstrated in peripheral leukocytes, cultured skin fibroblasts and long-term lymphoid lines from two sibs with fucosidosis 2. Reliable identification of heterozygotes for this disease was accomplished by enzyme assay of mononuclear cells isolated from peripheral blood. Isoelectric focusing of alpha-L-fucosidase isozymes in leukocyte extracts from normal individuals revealed a common polymorphism which was shown to result from two autosomal alleles at a single, autosomal locus. Evidence was obtained for a third, "silent" allele segregating in the family of the two affected children. It is probable that the disease fucosidosis type 2 results from homozygosity for this "silent" allele. Preliminary experiments have shown that cultured skin fibroblasts from deficient individuals can incorporate and retain purified alpha-L-fucosidase added to the culture medium.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1975

An investigation of some factors affecting levels of erythrocyte inorganic pyrophosphatase activity.

A reproducible method has been devised for assaying erythrocyte inorganic pyrophosphatase. The activity level in healthy individuals appears to be a constant characteristic. Population surveys have been carried out in 100 Europeans and 34 Indians. In each population an otherwise normal individual was found with activity approximately three standard deviations from the mean. Investigation of the family of one such individual indicated that this was an inherited characteristic. A few individuals with clinical abnormalities were examined and some were found to have activity levels above the normal range. High activity levels appeared to be associated with those diseases in which raised circulating reticulocyte levels are found. The rate of in vivo decay of erythrocyte inorganice pyrophosphatase has been measured in healthy individuals. The decay pattern appears to be of the constant logarithmic type. An estimate has been made of the activity in circulating reticulocytes, and the half life of the enzymes has been calculated.

Adolescent↗
PubMed1975

Fucosidosis: detection of the carrier state in peripheral blood leukocytes.

We have utilized the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside to measure the activity of alpha-L-fucosidase in white blood cells and serum. We have compared the findings with those using the P-nitrophenyl derivative. pH activity curves showed two major peaks of activity in leukocyte lysates, with different specificities to these substrates. alpha-L-Fucosidase activity was determined in peripheral leukocytes. Isolated mononuclear cells (mainly lymphocytes), and granulocytes in 21 members of a family in which fucosidosis had occurred and in normal control subjects. The activity in the leukocytes, lymphocytes, and granulocytes of the normal subjects was 300.7 +/- 79.8, 190.1 +/- 43.9, and 281.9 +/- 73.1 nmoles 4-methylumbelliferone/mg protein/hour with the fluorogenic substrate, and 150.0 +/- 31.8, 154.8 +/- 21.0, and 148.3 +/- 48.3 nmoles p-nitrophenol/mg protein/hour with the colorigenic substrate, respectively. No activity was detected in the patients' cells with the colorigenic substrate, whereas with the fluorogenic substrate the apparent activity varied from 0.5 to 1.1. In the lymphocytes of both of the patients' parents, two grandparents, and six other potential carriers, the activity fell between the normal and patients' values. Great variation in alpha-L-fucosidase activity, with broad overlap between normal subjects and heterozygotes, was observed in serum and plasma. Our findings indicate that detection of carriers for fucosidosis is possible by direct fucosidase determinations in isolated mononuclear cells.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1975

Arylsulfatase B deficiency in Maroteaux-Lamy syndrome: Cellular studies and carrier identification.

Arylsulfatase B deficiency was demonstrated in peripheral leukocytes, cultured skin fibroblasts, and a lymphoid line derived from a patient with MLS. The patient's parents demonstrated levels of arylsulfatase B that were intermediate between those found in patient and those in control subjects. The activity (mean plus or minus SD) in leukocytes from normal subjects, the patient's parents, and the patient was 113.7 plus or minus 36.2, 31.0, and 5.2 nmol 4-nitrocatechol/mg protein/hr, respectively. In skin fibroblasts of the same subjects the activity was 145.2 plus or minus 41.6, 58.5, and 7.0, respectively. Nine other lysosomal enzymes were normal in skin fibroblasts of the patient. No arylsulfatase B activity was detected in a lymphoid line established from the patient with MLS. The arylsulfatase B activity in cultured amniotic fluid cells from 10 normal pregnancies was 203.2 plus or minus 49.9.

Amniotic Fluid↗
PubMed1975

Polymorphism of human alpha fucosidase.

A common polymorphism of human alpha fucosidase has been detected by the technique of isoelectric focusing on thin layer acrylamide gel. Family studies have shown that the three common phenotypes Fu 1, Fu 2, and Fu 2-1 represent homozygosity or heterozygosity for two autosomal codominat alleles, Fu1 and Fu2. Frequencies of the Fu1 and Fu2 alleles in the New York populations studied were .753 and .247 among whites and .926 and .074 among blacks, respectively.

Alleles↗