RESUMEN

The feasibility of using urine samples for the identification of patients with Gaucher disease and carriers has been investigated. It was found that the pH of a urine sample should be pH 6.0 or lower to ensure stability of lysosomal hydrolases. Two parameters of glucocerebrosidase, which is deficient in Gaucher disease, were studied using urine samples from control subjects, obligate carriers and patients. Firstly, the relative level of glucocerebrosidase activity was measured by relating the activity of the enzyme to that of another lysosomal hydrolase. Secondly, the enzymic activity of glucocerebrosidase per unit of protein was measured using an immunological method. The first method allowed discrimination of nearly all obligate carriers of type 1 Gaucher disease from normal individuals. The second method allowed clear discrimination of the majority of carriers from normal individuals, but some obligate carriers were not distinguishable from normal subjects on the basis of this parameter. However, the combination of both methods allowed discrimination between all obligate carriers examined so far (n = 34) and controls (n = 86). There was variability between healthy individuals in the relative amount of glucocerebrosidase in urine samples. A small proportion of healthy individuals have a relatively high activity of glucocerebrosidase in urine samples, reminiscent of observations made in white blood cells by other investigators. In urine samples from two unrelated parents of Gaucher disease patients a level of glucocerebrosidase activity was present that could not be distinguished from that in samples of patients. These individuals represent cases with subclinical manifestation of Gaucher disease, illustrating once more the remarkable heterogeneity in clinical expression of this disorder.

Asunto(s)

Asunto(s)

RESUMEN

Acid alpha-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297-304). After fusion of spleen cells with myeloma cells, about 10% of the hybrid clones obtained produced antibodies against acid alpha-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid alpha-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major protein bands (mol. wt. 76000 and 70000,) a minor band of mol. wt. 9600 and several minor bands with a mol. wt. of 67000 or lower are seen. Since all these components react with the monoclonal antibodies, they must have at least one antigenic determinant in common.

Asunto(s)

RESUMEN

In the same family, the generalized or infantile form of acid maltase deficiency (glycogenosis type II, Pompe disease) and the muscular or adult-onset form affected different individuals. Autosomal-recessive inheritance for the two clinical forms was demonstrated in this family by assay of acid alpha-glucosidase in muscle, lymphocytes, cultured fibroblasts, and urine of asymptomatic relatives. Current biochemical techniques do not discriminate between persons heterozygous for the generalized form and those heterozygous for the muscular form. To explain the coexistence of both forms in the same family, the infant with the generalized form or her grandfather with the muscular form must have been a genetic compound of different mutant alleles for acid alpha-glucosidase.

Asunto(s)

RESUMEN

In 21 obligate and 9 possible heterozygotes for acid maltase deficiency (AMD) (glycogenosis 2, Pompe's disease), different methods of identifying heterozygotes have been studied. Heterozygosity could not be demonstrated by physical examination, serum CPK assays, morphological examination of muscle biopsy (including light-microscopy, histochemistry and electron-microscopy), or by ultrastructural examination of a skin biopsy. Heterozygotes could be identified to a large, but still limited extent, by measuring the acid alpha-glucosidase activity in urine, cultivated fibroblasts, leucocytes, or skeletal muscle. Heterozygotes for the generalized from of AMD could not be distinguished from those for the muscular form. The limitations of heterozygote identification by means of enzyme assays are discussed, and some practical aspects for genetic counselling are mentioned.

Asunto(s)

RESUMEN

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.

Asunto(s)

Asunto(s)

RESUMEN

1. A method is described for the rapid isolation of alpha-galactosidases A and B (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from normal human liver. 2. When the same method is applied to Fabry liver, most of the alpha-galactosidase activity is recovered in the fraction corresponding to normal alpha-galactosidase B. In agreement with Romeo, G., D'Urso, M., Pisacane, A., Blum, E., De Falco, A. and Ruffilli, A. (1975) Biochem. Genet. 13, 615-628) [18], a small amount of alpha-galactosidase activity is found in the fraction corresponding to normal alpha-galactosidase A. 3. The kinetic properties of the B-like activity from Fabry liver are similar to those of normal alpha-galactosidase B. In agreement with Romeo et al. [18], it was found that the kinetic properties of the A-like activity from Fabry liver are similar to those of normal alpha-galactosidase A. 4. Using antisera raised against normal alpha-galactosidase A and normal alpha-galactosidase B, it is shown that the normal alpha-galactosidase isoenzymes are immunologically distinct and that the B-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B. Furthermore, the A-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B and not to normal alpha-galactosidase A. 5. Normal alpha-galactosidase B is converted into an A-like form during storage. 6. It is concluded that the B-like alpha-galactosidase in Fabry tissues is identical to normal alpha-galactosidase B, and that the small amount of A-like activity found in Fabry material is due to a modified form of alpha-galactosidase B.

Asunto(s)