John F. Tallman

Replacement Therapy for Inherited Enzyme Deficiency: Use of Purified Ceramidetrihexosidase in Fabry's Disease

Abstract The effect of intravenous administration of glucocerebrosidase isolated from human placenta was investigated in two patients with Gauchers disease who are deficient in this enzyme. The first received one injection of 1.5 x 106 units of glucocerebrosidase, and the second an injection of 1.65 x 106 units on two successive days. Liver biopsies were obtained before and 24 hours after injection of enzyme. Glucocerebroside in the liver of the first patient decreased from 702 to 519 μg per gram wet weight and from 1634 to 1214 μg per gram in the second after infusion of glucocerebrosidase. The quantity of glucocerebroside in erythrocytes of the two patients before infusion was 7.4 and 6.2 μg per milliliter of cells respectively and 2.9 and 2.6 μg per milliliter of cells 72 hours afterward. These findings indicate that exogenous glucocerebrosidase causes definite decreases in the quantity of accumulated lipid in patients with Gauchers disease. (N Engl J Med 291:989–993, 1974)

The metabolism of Tay-Sachs ganglioside: catabolic studies with lysosomal enzymes from normal and Tay-Sachs brain tissue

The catabolism of Tay-Sachs ganglioside, N-acetylgalactosaminyl- (N-acetylneuraminosyl) -galactosylglucosylceramide, has been studied in lysosomal preparations from normal human brain and brain obtained at biopsy from Tay-Sachs patients. Utilizing Tay-Sachs ganglioside labeled with (14)C in the N-acetylgalactosaminyl portion or (3)H in the N-acetylneuraminosyl portion, the catabolism of Tay-Sachs ganglioside may be initiated by either the removal of the molecule of N-acetylgalactosamine or N-acetylneuraminic acid. The activity of the N-acetylgalactosamine-cleaving enzyme (hexosaminidase) is drastically diminished in such preparations from Tay-Sachs brain whereas the activity of the N-acetylneuraminic acid-cleaving enzyme (neuraminidase) is at a normal level. Total hexosaminidase activity as measured with an artificial fluorogenic substrate is increased in tissues obtained from patients with the B variant form of Tay-Sachs disease and it is virtually absent in the O-variant patients. The addition of purified neuraminidase and various purified hexosaminidases exerted only a minimal synergistic effect on the hydrolysis of Tay-Sachs ganglioside in the lysosomal preparations from the control or patient with the O variant of Tay-Sachs disease.

The purification and properties of a mammalian neuraminidase (sialidase)

Abstract A method for the purification of neuraminidase (sialidase) from mammalian tissue is described. The resulting enzyme was approximately 3500-fold purified when G d 1a was utilized as substrate and possesses activity towards both mono and polysialogangliosides. The enzyme also was active when fetuin was utilized as substrate but did not possess catalytic activity towards neuraminlactose. Studies of the kinetic parameters of the enzyme were performed and a total scheme for the hydrolysis of gangliosides starting with G + 1 is presented.

An Investigation of the Metabolism of Tay-Sachs Ganglioside Specifically Labeled in Critical Portions of the Molecule

Investigations which provided for a demonstration of an enzymatic deficiency in Tay-Sachs disease varied considerably in design from the basic pattern of experimentation which proved so useful for unravelling the metabolic abnormalities in related disorders of lipid metabolism. For example, in order to elucidate the etiology of Gaucher’s disease, Niemann-Pick disease, and Fabry’s disease, the principal sphingolipid which accumulates in the tissues of patients with these disorders was labeled with radiocarbon-14C or radiohydrogen-3H in a critical portion of the molecule. The metabolism of these labeled compounds was then examined in the following fashion. Enzymes which catalyzed the initial step in the catabolism of the accumulating substances were identified and partially purified from appropriate mammalian tissues. The conditions which influenced the activity of these enzymes were ascertained. The optimal pH for the reactions was determined, the Michaelis constant for each enzyme was defined, and the products of the reactions were identified. When these parameters were clearly established in control human tissue preparations, the activity of the enzyme suspected of being involved in the pathogenesis of the disease was determined in similar specimens obtained from patients with these disorders of lipid metabolism. This technique was used with success for identifying the deficiency of glucocerebrosidase in Gaucher’s disease, sphingomyelinase in Niemann-Pick disease, and ceramidetrihexosidase in Fabry’s disease(1).

[51] The preparation of Tay-Sachs ganglioside specifically labeled in either the N-acetylneuraminosyl or N-acetylgalactosaminyl portion of the molecule

Publisher Summary Knowledge of the pathways of metabolism of Tay-Sachs ganglioside, N - acetylgalactosaminyl- ( N -acetylneuraminosyl) – galactosylglucosylceramide (G M2 ) is essential for an understanding of the pathogenesis of Tay-Sachs disease. The enzymatic hydrolysis of G M2 in vitro has been difficult to demonstrate in the past because of the lack of an assay with sufficient sensitivity to detect the small amounts of reaction products which result. However, through specific labeling of G M2 with radioactivity as described in this method the minimum detectable level of enzymatic activity is greatly reduced.