Molecular Forms of β‐N‐Acetylhexosaminidase in Epstein‐Barr Virus‐Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Tay‐Sachs Disease

Abstract

In whole leukocytes and in lymphocytes from normal subjects, the percentage activity of heat‐stable β‐N‐acetylhexosaminidase (30 ± 5% and 45 ± 5%, respectively) was higher than in the transformed lymphoid cell line (19 ± 3%). In Tay‐Sachs transformed cells as well as non‐transformed lymphocytes, β‐N‐acetylhexosaminidase was almost completely heat‐stable (95–98%). In the transformed cells from normal subjects, the β‐N‐acetylhexosaminidase B (Hex B) activity (5% of total) was significantly lower than in blood lymphocytes (average 25–30% of total activity), whereas Hex A and Hex I were similar in the either cell type. Blood lymphocytes and lymphoid cell lines established from a Tay‐Sachs patient lacked heat‐labile Hex A and expressed high heat‐stable Hex I and Hex B activities (3–6‐fold). After neuraminidase treatment, Hex A peak sharpened while Hex I peaks switched to higher pI than normal Hex I, in the region of Hex B. PreHex A/S pI was not affected. Hydrolytic properties using the both substrates (4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside and 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranoside) of each molecular form were similar in transformed and non‐transformed cells. Data derived from the use of a mixture of substrates were consistent with the model which proposes a common active site for either substrate in the case of preHex A, Hex B and Hex I, but not for Hex A.Thus Epstein‐Barr virus‐transformed lymphoid cell lines represent an accurate model system for studies on Tay‐Sachs disease.

Molecular forms of sphingomyelinase and non‐specific phosphodiesterases in Epstein‐Barr virus‐transformed lymphoid cell lines from Niemann‐Pick disease types A and B

“…Also, p-Nacetylglucosaminidase activity focussed in the two classical peaks (Fig. 1, left, inset) one of which (form B) is reduced in LCL as previously reported [25]. The data show that sphingomyelinase activity which in normal cells focussed as a single peak (PI = 5.60 & 0.1) was absent from those derived from NPD types A and B patients (Fig.…”

“…Activities of BMP and BMPP phosphodiesterases were assayed according to Besley [6] and Fensom et al [5], respectively. p-N-Acetylglucosaminidase was tested as previously described [25].…”

ß-Glucosidase Isoenzymes in Epstein-Barr Virus-Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Type 1 Gaucher Disease

“…Our group has demonstrated the validity of such a cellular system for the study of Fabry disease [Salvayre et al, , 1985, Tay-Sachs disease [Salvayre et al, 1983], Sandhoff disease [Maret et al, 1985], Niemann-Pick disease [Levade et al, 1984] and Wolman disease [Nègre et al, 1984], and we report here our results about Gaucher LCL.…”

New Tools for the Study of Niemann-Pick Disease: Analogues of Natural Substrate and Epstein-Barr Virus-transformed Lymphoid Cell Lines

“…RPMI 1640 and fetal calf serum were from Gibco (Grand Island, NY) and other reagents were from Merck (Darmstadt, F. R. G. Determination of molecular forms. Analysis of sphingomyelinase was carried out using preparative electrofocusing as previously reported for hexosaminidase (30).…”