Nicholas G. Beratis

Isozymes of human α-l-fucosidase detectable by starch gel electrophoresis

Abstract Methods are described for the electrophoresis in starch-gel of human α- l -fucosidase and for the detection of the enzyme using the fluorogenic substrate 4-methylumbelliferyl-α- l -fucopyranoside. The electrophoretic pattern of the enzyme was examined in leucocytes, serum, cultured fibroblasts and long-term lymphoid cell lines. The enzyme from cultured lymphoid cell lines was found to consist of up to 6 clearly resolved electrophoretic isozymes plus a diffuse, more anodal region of lower staining intensity. The enzyme from leucocytes and cultured skin fibroblasts was less clearly resolved, but these cell types appeared to have components corresponding in mobility to the isozymes of lymphoid lines. In contrast, the enzyme from serum (or plasma) showed only a diffuse region of activity, with an anodal mobility slightly greater than that of the most anodal lymphoid line isozymes. Evidence is presented which indicates that the electrophoretic heterogeneity of α- l -fucosidase is due in part to the binding of sialic acid to the primary gene product. None of the isozymes was detectable in lymphoid cell lines, serum or cultured fibroblasts from a patient with fucosidosis, an inborn error of metabolism.

Cystathionine synthase deficiency: Heterozygote detection using cultured skin fibroblasta

Summary An assay for cystathionine synthase in cultured fibroblasts, of high sensitivity and not rate-limited for substrate, is described. Enzymatic activity was found to be highest in controls (mean ± SEM = 20.97 ± 1.81 nmole cystathionine/mg protein/hour), intermediate in obligate heterozygotes for synthase deficiency (4.40 ± 0.92), and lowest in patients (0.77 ± 0.42), with no overlap between controls and heterozygotes. One clinically and biochemically atypical patient had a synthase activity at the low end of the heterozygote range. Thus, this method is effective for the detection of heterozygotes for cystathionine synthase deficiency.

Cell-specific differences in membrane β-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 Gauchers 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.

Familial de Lange syndrome. Report of three cases in a sibship.

Familial de Lange syndrome with 3 affected siblings in a sibship of 4 is described. The parents were phenotypically normal. No parental consanguinity was demonstrated. The karyotypes of the affected siblings were normal. Although the etiology of the syndrome is obscure, it is likely that it consists of a heterogeneous group of clinical entities. Morphologic criteria may be useful in distinguishing the sporadic cases from the familial ones.

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.

Properties of placental alkaline phosphatase. II. Interactions of fast- and slow-migrating components.

The relationship between the rapidly (component I) and slowly (components II) migrating components of placental alkaline phosphatase has been studied. Storage of components II resulted in conversion into component I with a parallel increase of activity. The conversion rate increased with temperature. The fastest of the slow-moving components (component IIα) was less stable than the very slow-migrating ones (components IIβ). Component IIα was not identifiable after 25 days at 37 C, while some of the IIβ components were still present after a period of 1 year. No conversion of component I into II was observed. Data from this study suggest that the apparent thermostability of placental alkaline phosphatase results partly from the increase in activity after the conversion of components II into component I.

Homocystinuria: investigations of cystathionine synthase in cultured fetal cells and the prenatal determination of genetic status.

CYSTATHIONINE SYNTHASE DEFICIENCY (Fig. 1), which is inherited in an autosomal recessive manner , results in the clinical syndrome of homocystinuria. The full clinical picture is distinct 1 and may include dislocation of the optic lenses, progressive skeletal changes, severe t h r o m b o e m b o l i c disease , and, of ten, var iable degrees of mental retardation. Synthase activity, and its absence in affected individuals, has been demonstrated in liver and brain2,3,7; more recently it has been found in cultured skin fibroblasts 4, 6 and phytohemagglutininstimulated lymphocytes, s Heterozygotes can now be distinguished from patients and from normal individuals by determinat ion of synthase activity in cultured skin fibroblasts. 6 The utilization of cultured skin fibroblasts in the diagnosis of numerous enzymatic disorders often has been followed by attempts a t spec i f ic prenatal diagnosis by assay of cultured amniotic fluid cells. However, in order to establish proper control data, it is essential to determine optimal assay conditions for cultured amniotic

Properties of placental alkaline phosphatase. III. Thermostability and urea inhibition of isolated components of the three common phenotypes

Different heat inactivation rates were found among the three common homozygous human placental alkaline phosphatase phenotypes with respect to component I (the rapidly migrating component). Phenotype I1 was less stable than F1 and S1, while types F1 and S1 exhibited very similar thermostabilities, F1 being slightly more stable than S1. Components I, IIα (the fastest of the slow-moving components), and IIβ (the group of the very slowly migrating components) had different heat stabilities, IIβ being the most stable and I the least stable. Magnesium greatly increased the heat stability of all tested phenotypes. Placental alkaline phosphatase was found to be less heat resistant than reported previously. All phenotypes were equally inhibited over urea concentrations ranging from 0.5 to 8 m. No difference in the inhibition rate was found among components I, IIα, IIβ, and the crude placental butanol extract. The altered electrophoretic pattern of the crude placental extract obtained by urea starch gel electrophoresis was considered as being most likely due to reversible changes in the folding of the molecules.

Adenosine deaminase. Alterations in activity and isozymes during growth of normal and genetically deficient fibroblasts.

Abstract Adenosine deaminase (ADA) activity was examined in cultured human fibroblasts derived from normal subjects, from a child with ADA deficiency and from two obligate heterozygotes. In all three types of fibroblasts, ADA activity was found to be lowest shortly after passage, a time characterized by rapid increase in cell number and low cell density. In contrast, ADA activity increased in the three cell types approx. 3-fold at late confluency, a period characterized by slow increase in cell number and high cell density. Concomitant with these alterations, the enzyme activity shifted from the low molecular weight isozyme, which exhibits greater anodal mobility on electrophoresis, to the high molecular weight, more slowly migrating isozyme. Reflecting these variations in enzyme activity during cultures, fibroblasts from obligate heterozygotes under these conditions of culture could not be reliably distinguished from those derived from normal subjects.

Ganglioside accumulation in cultured skin fibroblasts from gangliosidosis patients

Abstract At late confluency (21 days after passage), cultured skin fibroblasts from G M1 gangliosidosis, type 1 patients showed approximately a 15-fold increase in G M1 ganglioside, and fibroblasts from Tay-Sachs and Sandhoff disease patients showed a 50- and 30-fold increase in G M2 ganglioside, respectively, when compared to normal fibroblasts. Since demonstration of storage material is important for accurate diagnosis of the lysosomal storage disorders, analysis of the accumulating lipids in late confluency fibroblasts can provide an additional tool for the diagnosis of the gangliosidoses and possibly other lysosomal disorders.