Proc. Natl. Acad. Sci. USAVol. 75, No. 8, pp. 3979-3983, August 1978Medical Sciences

AB variant of infantile GM2 gangliosidosis: Deficiency of a factornecessary for stimulation of hexosaminidase A-catalyzeddegradation of ganglioside GM2 and glycolipid GA2

(lipid storage disease/activator deficiency)

E. CONZELMANN AND K. SANDHOFFMax-Planck-Institut fur Psychiatrie, Neurochemische Abteilung, Kraepelinstrasse 2, D-8000 Munchen 40, Federal Republic of Germany

Communicated by Saul Roseman, May 24, 1978

ABSTRACT Human kidney extracts heated to 600 and de-void of hexosaminidase activity (2-acetamido-2-deoxy-13-Dglu-coside acetamidodeoxyglucohydrolase EC 3.2.1.30) stimulatemore than 20-fold the hexosaminidase A-catalyzed degradationof ganglioside GM2 and of glycolipid GA2, the neuronal storagecompounds of GM2 gangliosidosis. The stimulating factor of thisextract, which is labile at temperatures above 60°, is also presentin kidney extracts from patients with infantile GM2 gangliosi-dosis having a deficiency of hexosaminidase A (Tay-Sachsdisease, variant B) and a deficiency of hexosaminidases A andB (variant 0). Evidence is presented that this factor is defectivein the AB-variant of infantile GM2 gangliosidosis which ischaracterized by an accumulation of glycolipids GM2 and GAdespite the fact that the degrading enzymes, hexosaminidasesA and B, retain normal activity levels. Thus, variant AB is anexample of a fatal lipid storage disease that is caused not by adefect of a degrading enzyme but rather by a defective factornecessary for the interaction of lipid substrates and the water-soluble hydrolase.Infantile GM2 gangliosidosis is a fatal inherited storage diseasecharacterized by the accumulation of ganglioside GM2 and itsasialo derivative, GA2, in nervous tissue (1). Biochemically, threevariants can be distinguished, two of which could hitherto beexplained in terms of enzyme defects: patients with variant B("Tay-Sachs disease") lack hexosaminidase A (2-acetamido-2-deoxy-f-D-glucoside acetamidodeoxyglucohydrolase, EC3.2.1.30) (2, 3), the enzyme responsible for the further degra-dation of the ganglioside GM2 (4), whereas in patients withvariant 0, major hexosaminidase isoenzymes A and B both aremissing (5, 6), presumably due to the defect of a commonsubunit (7). For the variant AB, however, no such enzyme de-ficiency could be demonstrated. Patients having this variantretain normal or show elevated levels of both hexosaminidasesin all organs examined (3, 6), yet the ganglioside GM2 never-theless accumulates in the brain tissue at the same rate as in theB and 0 variants, and the clinical symptoms and course of thedisease are the same as in these variants. Biochemical and im-munological comparison of an AB patient's hexosaminidase Awith the enzyme from normal subjects revealed no difference(8). In particular, the patient's storage compounds, gangliosideGM2 and glycolipid GA2, were split by the patient's hexosami-nidase A in the presence of detergent at a normal rate, thuspractically ruling out the possibility of a mutant hexosaminidaseA.

Recent publications have demonstrated the necessity for thepresence of a nonenzymic protein to bring about the interactionof some water-soluble hydrolases (9-11) with their sphingolipidsubstrates. Such activator proteins have also been reported for

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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the degradation of gangliosides (10, 12). This paper presentsevidence that a component of an activating factor necessary forthe degradation of ganglioside GM2 and glycolipid GA2 cata-lyzed by hexosaminidase A is defective in variant AB of in-fantile GM2 gangliosidosis.

MATERIALS AND METHODSMaterials. 4-Methylumbelliferyl-2-acetamido-2-deoxy-

,B-D-glucopyranoside (MUFGlcNAc) and N-acetyl-D-galac-tosamine (GalNAc) were from Koch-Light (England); sodiumtaurodeoxycholate, trypsin, and trypsin inhibitor, from SigmaChemical Company (St. Louis, MO); Pronase P (from Strep-tomyces griseus), from Serva (Heidelberg). DEAE-cellulose(DE-52) was obtained from Whatman (Springfield Mill, GreatBritain), Bio-Gel P-200 from Bio-Rad Laboratories (Munich,Federal Republic of Germany). Ganglioside GM2, tritiated inits GalNAc portion (2 ,uCi/,umol) (13), and glycolipid GA2 (190,uCi/,umol), labeled by reduction of the double bond in thesphingosine moiety, were prepared as described (6). All otherreagents were analytically pure or of the best grade available.Organs from patients with variant 0, B, or AB of infantile GM2gangliosidosis and from normals were deep frozen within 6 hrafter death and kept for 2-3 yr at -700 until used.

Preparation of Tissue Extracts. Frozen human tissues (0.5-1g) were thawed, homogenized in 4 vol of distilled water withan Ultra-Turrax homogenizer (Janke & Kunkel, Staufen,Federal Republic of Germany), and centrifuged at 13,000 Xg for 10 min. To obtain a preparation of the activating factorvirtually free of hexosaminidases, the extract was heated at 600for 1 hr, centrifuged, and the precipitated protein was dis-carded. Protein was determined according to Lowry et al. (14)with crystalline bovine serum albumin as standard.

Ion Exchange Chromatography. Heated tissue extract (1.2g of protein in 230 ml) prepared as described above was di-alyzed against 10 mM phosphate buffer (pH 6.5) and loadedonto a DEAE-cellulose column (Whatman DE-52; volume, 300ml) that had been equilibrated with the same buffer. After awashing with 2 vol of buffer, the column was eluted with alinear NaCl gradient, 0-0.5 M in 1 liter of 10 mM phosphatebuffer (pH 6.5). Fractions (25 ml) were collected and assayedfor protein and stimulating factor as described below.

Proteolytic Digestion. Components a and f (50,l each)obtained after gel filtration of the stimulating factor were in-

Abbreviations: MUFGIcNAc, 4-methylumbelliferyl-2-acetamido-2-deoxy-fl-D-glucopyranoside; GaINAc, N-acetyl-D-galactosamine;NeuAc, N-acetylneuraminic acid; Cer, ceramide; glycosphingolipidGA2, gangliotriaosylceramide (GgOse3Cer; GalNAc31-4Gal-f1-4GlI31-1Cer); ganglioside GM2, II3NeuAc-GgOse3Cer (GalN-Acf11-4Gal(3. -2aNeuAc)31-4Glfl-BCer); ganglioside GM3,NeuAca2-3Gal31-4Glcll-lCer.

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3980 Medical Sciences: Conzelmann and Sandhoff

cubated with Pronase P (0.5 mg) at 370 (pH 6.5, 10 hr). Underthese conditions, both components were inactivated by morethan 90%. Controls were run under the same conditions exceptthat the protease was added only after incubation to show thatthe subsequent assay for the stimulating components is notdisturbed by the protease which is inactive at acidic pH.

Preparation of Purified Hexosaminidase A. Hexosamin-idase A was isolated from normal human liver as described (4).Bovine serum albumin (0.1 mg/ml) was added and the enzymesolution was stored at -70°.Enzyme Assays. Hexosaminidase activity was tested as de-

scribed (4), with MUFGlcNAc as substrate; 1 unit of hexo-saminidase activity releases 1 1mol of 4-methylumbelliferoneper min at 37°.

Ganglioside Gm2 N-acetyl-ft-D-galactosaminidase was es-sentially determined according to O'Brien et al. (13). Gangli-oside GM2 (10 nmol), labeled in its N-acetyl-D-galactosamineportion (2 ACi/timol) (12), in chloroform/methanol, 2:1 (vol/vol), was pipetted into the reaction vessel and dried under astream of nitrogen. Citrate buffer (pH 4.2; 4 Mmol) and waterwere added and the mixture was sonicated for 5 sec. As indi-cated in the legends of figures, extract, supernatant of heat-inactivated extract, or detergent and purified hexosaminidasewere added to give a final volume of 40 Al. The mixture-wasvigorously shaken and incubated at 370 for 2-4 hr. The reactionwas stopped by the addition of 750 ll of CHC13/CH30H/2.5M aqueous NH&, 80:201 (vol/vol), followed by 0.5 ml of 1 mMN-acetyl-D-galactosamine in water. After thorough shaking,the phases were separated by centrifugation. The upper phasewas collected and loaded onto a small column (1 ml) ofDEAE-cellulose that had previously been washed with distilledwater. The column was rinsed twice with 850-,ul portions of 1mM N-acetyl-D-galactosamine to elute the enzymically liber-ated GalNAc, the effluents were combined, and their radio-activity was assayed in a liquid scintillation counter.

Degradation of glycolipid GA2 (190 ;&Ci/,gmol) was essen-tially performed as described (4). The 200-,ul mixtures con-tained 0.2 unit of hexosaminidase A and were incubated for 2hr.

Product Identification. [3H]Ganglioside GM2 labeled in itsGalNAc moiety was incubated with purified hexosaminidaseA in the presence of heat-inactivated extracts (1 hr, 600) ofnormal kidneys or of those from variant 0 or B for 5 hr. In thiscase, all quantities were 10 times those described above. Ali-quots were analyzed in two different thin layer systems. (i)Adsorbent was cellulose plates (Merck, Darmstadt, FederalRepublic of Germany); development was by two runs with thenonaqueous phase of the mixture ethyl acetate/pyridine/water,2:1:2 (vol/vol); sugars were visualized with silver nitrate. (i)Adsorbent was silica gel plates (Merck, Darmstadt, FederalRepublic of Germany); the solvent system was n-butanol/eth-anol/water, 2:1:2 (vol/vol); spots were visualized with aceticacid/sulfuric acid/anisaldehyde, 50:1:0.2 (vol/vol), 15 min at1400. Distribution of radioactivity on the thin layer plates wasdetermined with a,8-camera LB 290B (Berthold, Wildbad,Federal Republic of Germany). Besides unreacted [3Hlsubstratethe only tritiated product found comigrated with authenticN-acetyl-D-galactosamine in both systems.

For the identification of lipophilic reaction products [3H]-ganglioside GM2 (4) (16 uCi/,umol) and glycosphingolipid GA2(6) (1901ACi/Amol) labeled in their sphingosine portions wereused as substrates (with 10-fold greater quantities than as de-scribed above). Besides unreacted 3H-labeled substrates, theonly tritiated products found comigrated with authentic gan-glioside GM3 andGalf14Glc1, lCer, respectively, in threedifferent thin-layer systems. The adsorbent was silica gel plates

in all three. The solvent systems were: (i) chloroform/metha-nol/water, 14:6:1 (vol/vol); (if) chloroform/methanol/con-centrated NH4OH, 65:35:4 (vol/vol); and (hi) chloroform/methanol/0.25% NaCl in water, 62.38:8 (vol/vol). Lipids werevisualized with acetic acid/sulfuric acid/anisaldehyde, 50:1:0.2(vol/vol) 15 min at 1400. The distribution of radioactivity onthe plates was measured with a , camera LB 290B (Berthold,Wildbad, Federal Republic of Germany).

RESULTSStimulation of Enzymic Hydrolysis of Glycolipids by

Heated Extracts of Organs. Tissue extracts whose hexosami-nidase activity had been destroyed by heating to 600 for 1 hrstimulated the degradation of ganglioside Gm catalyzed bypurified hexosaminidase A (Fig. 1). Kidney extracts activatedthe enzymic hydrolysis of ganghoside more than 18-fold lin-early with the amount of extract protein added, whereas ex-tracts fromh brain and liver showed only a slight activation atlow protein concentrations (3- to 4-fold) and this diminishedwith higher concentrations of protein. Corresponding obser-vations were obtained for the enzymic degradation of glycolipidGsA. Because kidney samples were available from variant ABas well as from other variants of infantile GM2 gangliosidosis,further investigations on this activating factor were carried outwith these different kidney extracts. The activating factor wasstable up to 600 but labile at higher temperatures (Figs. 2 and3). Because of this stability at 600, it is possible to prepare kidneyextracts rich in activating factor but essentially free of hexo-saminidase activity, so that these extracts degrade neitherganglioside Gm (Fig. 1) nor glycolipid GA2 without the additionof hexosaminidase A.

Unheated extracts from normal kidneys hydrolyzed gan-

S-

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50 100Supernatant added, jug protein/assay

FIG. 1. Stimulation of the hexosaminidase A-catalyzed degra-dation of ganglioside GM2 by extracts from normal human tissues.Tissues were extracted, heated for 60 min at 600, and centrifuged;aliquots of each supernatant were assayed. The incubation mixtures,containing increasing amounts of the supernatants, 30 milliunits ofpurified hexosaminidase A, and 10 nmol of [3Hjganglioside GM2, were

analyzed for [3H]GalNAc formed. Controls lacking hexosaminidaseA were run for each value and subtracted. A, Kidney; 0, brain; 0,liver; X, control incubations with heated kidney extract without ad-ditional hexosaminidase A.

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4-._

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60 70Temperature, 0C

FIG. 2. Thermal inactivation of human kidney hexosaminidaseand of the factor activating the hexosaminidase A-catalyzed degra-dation of ganglioside GM2. Normal human kidney was extracted,heated for 60 min at the indicated temperature, and centrifuged; 20-AIaliquots of each supernatant were assayed. For hexosaminidase (A),the substrate was 4-MUFGIcNAc (4). The supernatant activity (un-heated) was 0.18 units/mg of protein; this value = 100%. For activatingfactor (0), the stimulation of [3H]ganglioside GM2 hydrolysis by 65milliunits of purified hexosaminidase A was measured. Controlslacking hexosaminidase A were subtracted from each value. The 100%value represents the stimulating activity ofunheated supernatant (224pmol of [3H]GalNAc formed/hr per mg of supernatant protein).

glioside GM2 at rates of 0.1-0.2 nmol/hr per mg of protein,whereas unheated extracts of kidney tissues from patients withvariant 0 or B yielded only much lower values (Fig. 4 left).However, in the case of unheated kidney extracts from thepatient having variant AB no significant degradation of gan-glioside Gmg (Fig. 4 left) or glycolipid GA2 (not shown) wasobserved, despite the normal levels of hexosaminidase A andB (0.09 unit/mg of protein compared to 0. 1-0. 15 unit/mg in

1001

20 60 120Inactivation time, min

FIG. 3. Kinetics of thermal inactivation (60°) of normal humankidney hexosaminidase and of the factor activating the hexosamin-idase A-catalyzed degradation of ganglioside GM2. Human kidney wasextracted, heated for the times indicated at 600, and centrifuged; 20-,ulaliquots of each supernatant were assayed. For hexosaminidase (A),the substrate was 4-MUFGlcNAc (4). The supernatant activity (un-heated) was 0.18 unit/mg protein; this value = 100%. For activatingfactor (0), the stimulation of [3H]ganglioside GM2 hydrolysis by 65milliunits of purified hexosaminidase A was measured. The 100%value represents the stimulating activity ofunheated supernatant (224pmol of [3H]GalNAc formed/hr per mg of supernatant protein).

~70 070-

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0 5 10 15 20 0 5 10 15 20Kidney, extract, Al Kidney, extract,Jaid

FIG. 4. Ganglioside Gm2-N-acety1-(l-D-galaCtosaminidase activityof normal and pathological human kidney extracts. Frozen, thawedhuman kidneys were extracted as described in the text, centrifuged,and aliquots of each supernatant assayed for hydrolysis of [3H]gan-glioside GM2 Incubation mixtures containing increasRingamounts ofkidney extract (13 mg of protein per ml) and 10 nmol of [3H]gangli-oside GM2 as substrate were incubated for 2 hr and analyzed for[3HJGalNAc liberated. 0, Kidney extract, normal case 1; +, kidneyextract, normal case 2; X, kidney extract, variant 0; A, kidney extract,variant B (Tay-Sachs disease); o3, kidney extract, variant AB;@0,mixture (1:1, vol/vol) of extracts from normal kidney (case 1) andvariant AB kidney. (Left) Without sodium taurocholate. (Right) With1.85 nmol of sodium taurodeoxycholate per Ag of protein.

control cases 1 and 2) found in these extracts. Mixing experi-ments excluded the presence of an inhibitor for the gangliosideGM2 degradation in the kidney extracts of variant AB. Fur-thermore, the addition to the incubation mixtures of sodiumtaurodeoxycholate, which is known to stimulate the enzymicdegradation of glycolipids (4), resulted in an almost normaldegradation rate of ganglioside Gm2 (Fig. 4 right) and gly-colipid GA2 (not shown), indicating that the hexosaminidasesof extracts from AB variant are able to attack the lipids storedin this disease. The minor acitivity of the extracts from variantsB and 0 in the presence of detergent is probably due to thepresence of hexosaminidase B and S, respectively, in these ex-tracts (4, 15).

Defective Activating Factor in Kidney Extract from Var-iant AB. The findings presented in Figs. 1-4 raised the possi-bility that an activating factor that is present in normal tissueand that can be replaced in vitro by sodium taurodeoxycholatemay be defective in the variant AB tissue. Therefore, heat-inactivated (600, 1 hr) extracts of normal and pathologicalkidneys were compared for their ability to stimulate the en-zymic hydrolysis of glycolipids. Fig. 5 shows the activation(corrected for the "endogeneous" hexosaminidase activity ofthe extracts) of the hexosaminidase A-catalyzed hydrolysis ofganglioside GM2 and glycolipid GA2.The activating capacity exhibited by the extracts from two

variants of Gm2 gangliosidosis was equal (variant B) or evensuperior (variant 0) to that from normal controls, whereas theextract from variant AB kidney showed no activating effect.A mixture (1:1, vol/vol) of variant AB extract with normal ex-tract had about half the activating capacity of the pure normalextract, indicating that deficiency of an activating factor ratherthan presence of an inhibitor was responsible for the lack ofactivation. Furthermore, as shown in Fig. 6 this activating factoralso was absent from unheated kidney extracts of variant AB,ruling out the existence of a more heat labile factor in thisvariant.

Characterization of the Activating Factor. Te activating

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"I .~~~~~.

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FIG. 5. Activation of hexosaminidase A-catalyzed degradationof glycolipids GM2 (Left) and GA2 (Right) by normal and pathologicalhuman kidney extracts. Human kidneys of normal or pathologicalorigin were extracted, heated for 1 hr at 600, and centrifuged. Aliquotsof the supernatants containing increasing amounts of protein were

assayed. (Left) Activation of hexosaminidase A-catalyzed degradationof ganglioside GM2. Standard incubation mixtures containing 65milliunits of purified hexosaminidase A and 10 nmol of [3Hjganglio-side GM2 were analyzed for [3H]GaINAc formed. Blanks were run foreach value without added hexosaminidase A and subtracted. (Right)Activation of hexosaminidase A-catalyzed degradation of glycos-phingolipid GA2. Incubation mixtures containing 200 milliunits pu-

rified hexosaminidase A and 10 nmol glycosphingolipid Gs2 as sub-strate were analyzed for product (Gal(31-4G1c31-1[3HjCer) formed(4). For symbols, see legend of Fig. 4.

factor from normal kidney adsorbed completely to DEAE-cellulose at pH 6.5 and was eluted by a NaCi gradient at a

concentration of 0.15 M.This activating factor was resolved byfiltration on Bio-Gel P-200 into two components, a and ,, eachof which produced only negligible stimulation when assayedalone (Fig. 7). For sigiant stimulation of the hexosaminidaseA-catalyzed degradation of ganglioside Gm, both componentswere required. As estimated from gel filtration experiments,

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FIG. 6. Absence of activating factor from unheated kidney extractof variant AB. Stimulation of the hexosaminidase A-catalyzed hy-drolysis of [3Hjganglioside GM2 was measured in the presence of ex-tracts (13 mg of protein per ml) prepared from human kidneys. In-cubation mixtures containing increasing amounts of kidney extracts,10 nmol of [3H]ganglioside GM2, and 65 milliunits of purified hexo-saminidase A were analyzed for [5H]GaINAc formed. Blanks were runfor each value without additional hexosaminidase A and subtracted.0, Kidney extract, normal case 1; +, kidney extract, variant 0; A,

kidney extract, variant B (Tay-Sachs disease); 0, kidney extract,variant AB; *, mixture (1:1, vol/vol) of extracts from normal kidney(case 1) and variant AB kidney.

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FIG. 7. Resolution of the stimulating factor into two components,a and (3, by gel filtration. Fractions containing the stimulating factorfrom the DEAE-cellulose column were combined, dialyzed againstdistilled water, lyophilizedandtaken up in 20mM phosphate buffer,pH 6.5/25mM NaCl to give a concentration of about 20 mg ofproteinper ml. A 6-ml sample was applied to a 150-ml column of Bio-GelP-200 equilibrated with 20 mM phosphate buffer, pH 6.5/25 mMNaCl. The column was eluted with the same buffer at a flow rate of12 ml/hr. Fractions (5 ml) were collected and assayed for protein (. );10 MI of each fraction was assayed for ability to stimulate the enzymicbreakdown of ganglioside GM2 by hexosaminidase A at 0.1 unit perassay (A). The same ability was also tested in the presence of an ad-ditional 10 Ml of fraction 10, conting component a (-), or fraction15, containing component ft (0). Arrows indicate void volume (V0)and salt volume (Vt) as determined with blue dextran 2000 and[3H]leucine, respectively. a, component a; (3, component (3.

the molecular weights are approximately 25,000-for component(8 and 60,000-80,000 for component a. Both components weredigested by Pronase P. indicating their protein nature. Substi-tution experiments with components a and jB, respectively,showed that extracts prepared from kidney tissue with variantAB contained component a but were deficient in componentft (Table 1).

DISCUSSIONThe interaction of water-soluble lysosomal hydrolases withtheir amphiphilic sphingolipid substrates is in vitro stronglydependent on anionic detergents (e.g., bile salts). In the absenceof detergentsrthese enzymes show only very low activity towardtheir sphingolipid substrates. Their activity in vivo was there-fore puzzling until the discovery of the "activators" (9-11)-water-suble proteins that facilitate the interaction betweenthe lipid substrates and their degrading enzymes. The mostextensively studied example is probably the activator for thedegradation of cerebroside sulfate (9); others have been re-

Table 1. Stimulation of hexosaminidase A-catalyzed degradationof ganglioside GM2 by mixtures of various kidney extracts and

activator components.

Enzyme activity* with addition ofkidney extract fromVariant Variant

H20 AB B Normal

Enzyme 0.06 0.07 0.38 0.34Enzyme + a (5 gi) 0.09 0.09 0.39 0.37Enzyme + * (10 Ml) 0.11 0.44 0.74 0.52

Kidney extracts (6 mg of protein per ml) were heated to 600 for 1hr; activator components a and ft were obtained by gel filtration (seeFig. 7).* nmol of ganglioside GM2 cleaved per hr per unit of hexosaminidaseA.

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ported for the degradation of glucocerebroside (11) and gan-gliosides (10, 12).

This paper demonstrates the presence of an activating factor,composed of two components (a and 0), for the hexosaminidaseA-catalyzed hydrolysis of glycolipids Gm and GA in extractsof various organs. Among those tissues tested, the factor appears

to be most active in kidney extracts. Both components togetherare necessary for the stimulation of the enzymic degradationof lipid substrates by hexosaminidase A but do not affect thehexosaminidase A-catalyzed hydrolysis of synthetic, water-soluble MUFGlcNAc (data not shown). The extent of stimula-tion (up to >2 nmol of ganglioside GM cleaved per hr per en-zyme unit) obtained even with crude extracts, and far fromsaturation conditions, clearly differentiates components a andi from the activators described by Li and Li (10) and Fischerand Jatzkewitz (9). Despite being purified more than 1000-fold,those activators stimulate only up to 0.2 and 1.8 nmol of gan-glioside GM cleaved per hr per unit of hexosaminidase A, re-spectively (4). Furthermore, they are not resolved into twocomponents by gel filtration. On the other hand, the activatorpurified by Hechtman (12, 16) about 100-fold stimulates thehexosaminidase A-catalyzed GM2 degradation to a much lesserextent [V., = 0.1 nmol of ganglioside GM cleaved per hr perunit of hexosaminidase A (16)] but is equally thermolabile andmay be similar or identical to one of the two components de-scribed here (probably the lower molecular weight component,

It was demonstrated that extracts heated to 600 as well asunheated extracts from the kidney of a patient who died fromthe very rare variant AB of infantile Gm gangliosidosis com-pletely fail to enhance the enzymic breakdown of the glycoli-pids GM2 and GA2. This is certainly not due to the presence ofsome stored lipids that might act as inhibitors because othervariants of this disease exhibit a normal or even increased ac-

tivating effect, nor can it be attributed to the presence of a

specific inhibitor (this is ruled out by the results obtained withmixtures of normal and AB variant extracts).From these data and from substitution experiments with

components a and # it may be concluded that there is a defi-ciency of the (B component of the natural activator for the hy-drolysis of glycolipids GM2 and GA by hexosaminidase A. Thisexplains satisfactorily why patients having variant AB of in-fantile GM2 gangliosidosis exhibit and die from the storage ofgangliosides Gm and GA in their nervous tissues, the same waypatients with deficient hexosaminidase A do, although theirhexosaminidases appear to be normal (6, 8). It also explains why

tissue extracts from such patients are able to hydrolyze thestorage compounds in the presence of sodium taurodeoxycho-late. Furthermore, this variant emphasizes the importance ofsuch activators in the catabolism of lipids by demonstrating thatthe defect of this activating factor has the same clinical conse-quences as a deficiency of the enzyme.The nature of the glycolipids stored also allows for some

important conclusions concerning the often-raised question ofthe specificity of the lipid hydrolase activators with respect tosubstrate as well as enzyme. The deficiency of component # ofthe factor described here leads to the storage of only two, closelyrelated, glycolipids (GM2 and GA2) and the severity of the dis-ease clearly indicates that none of the other activators can takeover the task of the defective protein to any significant ex-tent.We are indebted to Dr. A. D. Patrick and Dr. R. Ellis, London, for

providing deep-frozen organs of a patient with variant AB of infantileGM gangliodos and to Mr H. Nehrkom for her excellent asiancein performing the experiments with glycolipid GA2. We thank Dr. Anzilfor his help with the manuscript. This work was supported in part bythe Deutsche Forschungsgemeinschaft (Grant Sa-257/4).

1. Sandhoff, K. (1977) Angew. Chem. Int. Ed. Engl. 16, 273-285.

2. Okada, S. & O'Brien, J. S. (1969) Science 165,698-700.3. Sandhoff, K. (1969) FEBS Lett. 4,351454.4. Sandhoff, K., Conzelmann, E. & Nehrkorn, H. (1977) Hoppe-

Seyler's Z. Physiol. Chem. 358,779-787.5. Sandhoff, K., Andreae, U. & Jatzkewitz, H. (1968) Pathol. Eur.

3,278-285.6. Sandhoff, K., Harzer, K., Wissle, W. & Jatzkewitz, H. (1971) 1.

Neurochem. 18, 2469-2489.7. Geiger, B. & Arnon, R. (1976) Biochemistry 15,3484-3493.8. Conzelmann, E., Sandhoff, K., Nehrkorn, H., Geiger, B. & Arnon,

R. (1978) Eur. J. Biochem. 84,27-33.9. Fischer, G. & Jatzkewitz, H. (1975) Hoppe-Seyler's Z. Physiol.

Chem. 356,605-613.10. Li. S. C. & Li, Y. T. (1976) J. Biol. Chem. 254, 1159-1163.11. Ho, M. W. & O'Brien, J. S. (1971) Proc. Nati. Acad. Sci. USA 68,

2810-2813.12. Hechtman, P. (1977) Can. J. Biochem. 55,315-324.13. O'Brien, J. S., Norden, A. G. W., Miller, A. L., Forst, R. G. &

Kelly, T. (1977) ClIn. Genet. 11, 171-183.14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol. Chem. 193,265-275.15. Geiger, B., Arnon, R. & Sandhoff, K. (1977) Am. J. Hum. Genet.,

29,508-522.16. Hechtman, P. & LeBlanc, D. (1977) Biochem. J. 167, 693-

701.

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