Febs Letters Volume 71, Number 1-2./Volume 72, Number 1-2. [antikvár]

Volume 1976, number 1 IT-BS LETTERS January 1977 CHARACTERIZATION OF (S-D-N-ACETYLHEXOSAMINIDASES C AND S IN FIBROPLASTS FROM CONTROL INDIVIDUALS AND PATIENTS WITH TAY-SACHS DISEASE A. J. J. REUSER and H. GAUAARD Department of Cell Biology and Genetics, Erasmus University. P.O. Box 1738. Rotterdam. The Netherlands Received 25 September 1976 1. Introduction Among the different isoenzymes of (3-D-7V-acetyl-hexoaminidase that occur in human tissues most attention has been paid to the major forms called Hex A and Hex B. A deficiency of Hex A was shown to be responsible for the metabolic defect in GMj-gangliosidosis type B (Tay-Sachs disease) [1 ]. In Sandhoff-Jatzkewitz disease, another form of this lysosomal storage disease, both Hex A and Hex B are deficient [2]. It has been suggested that the simultaneous absence of Hex A (a:(3)„ and Hex B (fiji)^ activity is caused by a defect in the common /3-subunit [3]. A third isoenzyme called Hex C was first described by Hooghwinkel et al [4]. In contrast to Hex A and Hex B it failed to hydrolyse 4-methylum-belliferyl-/3-D-galactosaminide. Subsequent studies revealed other differences between Hex C and the lysosomal isoenzymes Hex A and Hex B such as a neutral pH-optimum, extralysosomal localization and different immunological properties [5-9]. Recently another isoenzyme of /3-hexosaminidase has been characterized and was called Hex S [10-12]. It is the major component of /3-hexosaminidase activity in Sandhoff-Jatzkewitz disease and is absent in tissues from patients with Tay-Sachs disease. At acid pH Hex S exhibits activity towards 4-methylbelliferyl-(3-D-glucosaminide and 4-methylumbelliferyl-/3-D-galacto-saminide. From immunological and biochemical studies it was concluded that Hex S shares the a-subunit with Hex A but not the /3-subunit [11-13]. It has long been a question whether there is a structural relationship between Hex C and Hex A. Ropers et al. [14] suggested a common subunit structure for Hex A and Hex C. This was based on studies that claimed a deficiency of Hex C in tissues from patients with Tay-Sachs disease [4,14]. The isoenzyme pattern of hexosaminidase in man—rodent hybrids seemed to support this hypothesis [15,16]. Other reports however described the presence of Hex C in Tay-Sachs disease [5-9]. To find a reason for this discrepancy we have investigated the electrophoretic pattern of /3-hexosaminidase in fibroblasts from control subjects and patients with Tay-Sachs disease using different staining conditions. With reference to the recent characterization of Hex S special attention was given to the distinction of Hex Cand Hex S. Our findings suggest that there is no evidence for a structural relationship between Hex C and Hex A, and the results rather favor a different interpretation of the studies with man-rodent hybrids. 2. Materials and methods Skin fibroblasts from three different control individuals and six unrelated patients with Tay-Sachs disease were cultured in Falcon Flasks in Ham's FIG medium supplemented with 15% fetal calf serum and antibiotics as described before [17]. Homogenates of 3X10^ cells per 100 fi\ were prepared by sonication in distilled water. Electrophoresis was performed on cellulose acetate gels (Cellogel, Chemetron, Italy) in 50 mM potassium phosphate, pH 6.8, at 4°C, for 90 min at 200 V. 10 |Ul Cell homogenate was used per North-Holland Publishing Company - A msterdam 1