The cerebroside sulfate activator from pig kidney: Derivitization, cerebroside sulfate binding, and metabolic correction

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

47, 86-96 (1992)

The Cerebroside Sulfate Activator from Pig Kidney: Derivitization, Cerebroside Sulfate Binding, and Metabolic Correction ARVAN

University

L. FLUHARTY,’

WILLIAM E. MEEK, ZOLTAN AND KATHERINE K. TSAY

of California at Los Angeles School Group, Lanterman Developmental

KATONA,

of Medicine, Mental Retardation Research Center, Pomona, California 91769

Center

Received October 31, 1991 Highly purified cerebroside sulfate activator from pig kidneys was characterized by a number of chemical and biological procedures. Methods for chemical modifications were evaluated in an attempt to obtain biologically active derivatives. Iodination, dabsylation, and to a lesser degree reductive methylation provided useful products with good retention of cerebroside sulfate activator activity. Other procedures resulted in largely inactive derivatives or losses in both protein and biological activities. Attempts at renaturation of cerebroside sulfate activator subjected to various denaturing conditions appeared to be successful in many instances, but it was uncertain if the protein structure had actually been disrupted. The binding of cerebroside sulfate by activator was estimated by gel filtration under conditions similar to those of its assay. The formation of a relatively stable 1: 1 complex was observed, collaborating results with the human protein. The complex was stable enough to be isolated and shown to be an efficient substrate for arylsulfatase A. The effectiveness of the pig kidney cerebroside sulfate activator for correcting the metabolic defect in activator-deficient human fibroblasts was compared with human materials. The pig kidney protein was taken up more efficiently by the cells and resulted in a better metabolic correction than material from human liver, but was somewhat less effective than a preparation from human urine. 0 1992 Academic Press, ILK.

Cerebroside sulfate activator (CS-Act) is a heat-stable protein which binds a number of sphingolipids making their hydrophilic head groups available to hydrolytic enzymes (1). The specificity of lipid binding and the number of reactions for which this protein can act as a facilitator is somewhat unclear. It was initially thought that the protein was specific for the cerebroside sulfate (CS) hydrolysis by arylsulfatase A (ARSA). However, when CS-Act was found to be identical to the GM1 ganglioside and globotriaosylceramide activators (2) and that it could also potentiate the degradation of a variety of other sphingolipids by a number of enzymes, the protein was renamed sphingolipid activator protein 1 (SAP-l) by ’ To whom correspondence should be addressed at UCLA-MRRC Research Group, Lanterman Developmental Center, P.O. Box 100-R, Pomona, CA 91769. Fax: (714)-5944709. 86 0885-4505192 $3.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

CHARACTERIZATION

OF PIG KIDNEY

CS-ACT

87

Inui and Wenger (3) and nonspecific activator protein by Li et al. (4). More recently CS-Act was shown to be derived from a precursor protein containing four structurally similar regions, each of which gives rise to a distinct sphingolipidactivator protein (5). This precursor was called prosaposin and the individual subcomponents referred to as saposins, CS-Act being saposin B. Prosaposin has been found to be closely related to the major glycoprotein excreted by rat Sertoli cells (6) and distantly related to the precursor to one class of lung surfactant proteins (7). It appears that CS-Act is one of a widely distributed family of proteins involved in lipid binding and metabolism (vide infra). The companion paper reports the purification of CS-Act from pig kidney in high yield and details certain chemical and physical characterizations of the molecule (8). This communication describes attempts at preparing derivatives of the protein for use in monitoring chromatographic and cell interaction studies. The effects of denaturation-renaturation protocols on the activity of the protein are presented. The binding of CS by the activator and the protein’s ability to correct the metabolic defect in CS-Act-deficient human fibroblasts are also documented. METHODS Materials. The preparation of CS-Act from pig kidney and its usual assay are presented in the accompanying paper (8). The fibroblast culture from a patient with the activator deficient form of metachromatic leukodystrophy and the cerebroside sulfate loading test have been described previously (9,lO). Most radioisotopically labeled materials ([3H]NaBH,, [‘4C]iodoacetic acid and acetic anhydride, [1251]iodide) were obtained from New England Nuclear while [35S]CS was biosynthesized (11). Isotopes were counted in a Searle Isocap 300 liquid scintillation spectrophotometer using the manufacturer’s preset channels for each of the isotopes employed, with 35S being counted in the r4C channel. In most instances Ready Safe liquid scintillation cocktail from Beckman has replaced a dioxanebased cocktail used in early portions of the study. Dabsyl chloride, dansyl chloride, and IODO-GEN were obtained from Pierce; iodoacetic acid and guanidine-HCl were from Sigma; urea was from Mallinckrodt AR; octyl glucoside was from either Pfanstiehl or Calbiochem; Sephadex G-25, octyl Sepharose, and Sephacryl S-300 were from Pharmacia; Ultragel 34 was from LKB; and sodium borohydride was from Alfa Inorganics. Other chemicals were of analytical or better grade and were obtained from various chemical suppliers. CS-Act assay with radioactively labeled protein. The presence of radioactive label in CS-Act interfered with the [35S]sulfate-based activator assay. This was circumvented by introducing an octyl Sepharose-binding step in the assay protocol. The 0.5-ml of the washed upper phase normally used for counting of the released inorganic sulfate was passed through a O.l- to 0.2-ml bed of buffer-equilibrated octyl Sepharose in a l-ml plastic syringe, and the resin rinsed through with 0.5 ml 25 mM Tris-Cl buffer, pH 7.5. The combined eluate and wash fraction were counted together. Little breakthrough of protein label was encountered if the CSAct had been purified through octyl Sepharose before use. Protein modification studies. A variety of protein modification procedures were tested and only those which provided useful derivatives are described in detail.

88

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ET AL.

Zodination. Preliminary iodination studies were carried out by the procedure of Thorell and Johansson (12). To 1 ml of pig kidney CS-Act (6.2 mg) was added 10 ~1 of 250 mM K13 and 10 ,ul of 30% Hz02 and mixed. Additional lo-cl.1 aliquots of the K13 and peroxide solutions were added and mixed twice. The mixture was then allowed to incubate at room temperature for 10 min before reactants and products were separated by passing the sample through a 20-ml Sephadex G-25 desalting column. lz51 labeling was carried out by the Pierce IODO-GEN procedure (13). Activator solution (1 ml) was added to 320 pg of IODO-GEN which had been evaporated on the surface of a glass tube. This was followed by 10 ~1 of [‘251]iodide solution. The mixture was allowed to react for 0.5 to 1 h with shaking approximately every 5 min. The mixture was then fractionated through a small BioRad 10DG desalting column equilibrated with 50 mM Tris-Cl, pH 7.5. Protein containing fractions (-3.5 ml) were stored in the cold after the addition of 100 ~1 of 5 mg/ml NaI, 5 mg/ml dithiothreitol in 50 mM Tris-Cl, pH 7.5. Just before use -300 pg of labeled activator stock was added to the top of a 0.5-ml octyl Sepharose column in a disposable tuberculin syringe and washed through with 4 ml of buffer. The resin retained about 80 % of the radioactivity in a fresh preparation which decreased to about 50% after 4 months storage. Bound material was eluted with 2 ml of 25.5 mg/ml octyl glucoside in buffer, which displaced 80-100% of the bound radioactivity. The eluate was dialyzed (Spectrapor 3 membranes) overnight against buffer to remove detergent. Rechromatography of an aliquot through a second octyl Sepharose column resulted in 94% binding and 80% recovery on detergent elution. An overall protein recovery of 64% was achieved with a CS-Act sp act of 1700 U/mg and a specific radioactivity of approximately 25 &i/mg. Dabsylution. Dabsylation was carried out by the procedure of Parkinson and Redshaw (14). About 10 mg of CS-Act (sp act 2500 U/mg) in 1 ml was made 0.1 M in sodium bicarbonate, pH 9.0. Then 100 ~1 of 3.3 mg/ml dabsyl chloride (Pierce) was added while vortexing. After incubation at 37°C for 2 h the reaction was stopped by adding 10 mg lysine. After an additional hour the mixture was applied to a 1 x 26 cm column of Sephadex G-25 and eluted with 50 mM TrisCl buffer at a flow rate of 0.5 ml/min. The initial peak, eluting between 16 and 28 min, was pooled and concentrated to the starting volume. Recovery of the visibly orange derivative was 56% on a protein basis with a CS-Act sp act of 1300-1400 U/mg. Figure 1 shows the results of an SDS gel comparison of the dabsylated CS-Act with the unmodified starting material. Both the starting material and the colored derivative migrate just behind the dye front in the SDS micelle band. The bright orange band visible in the unstained gel corresponds with the protein band visualized by Coomassie blue in both the starting material and derivative. Reductive methylution. The procedure presented by Tack and Wilder (15) was used as a guide for attempts at reductive methylation. A 2-mg sample of CS-Act was made 0.2 M in pH 9.0 borate buffer and 0.4 ml of 37% formaldehyde was added (0.15 mg). After 5 min, 70 pg of NaBH, containing 100 Z.&i of 3H was added in a well-vented hood and allowed to react for 10 min at 0°C. Activator was recovered by octyl Sepharose affinity, the mixture being slowly passed through

CHARACTERIZATION

OF PIG KIDNEY

CS-ACT

89

a l-ml column of resin and washed with 3 ml of 50 mM Tris-Cl buffer, pH 7.5. The column was eluted with 3 ml of 20 mg/ml octyl glucoside in buffer and OSml aliquots were collected and monitored. About 0.2 &i of 3H-labeled protein was recovered which retained greater than 50% of the initial CS-Act specific activity. Modifications resulting in substantial loss of activity. Dansylation by the procedure of Hsieh and Matthews (16) gave good protein recovery but more than a 90% loss in activity. Esterification of carboxyl residues with methanol-HCl by the technique of Williams and Gratzer (17) on a sample of lyophilized activator resulted in a complete loss of activity and only a 14% protein recovery. Alkylation (18) with [‘“Cl’ IOd oacetate resulted in a 75% loss of activity and little apparent incorporation of radioactivity (4-6 x 10’ cpm from 20 PCi). Dye coupling to Drimarene brilliant blud K BL (Pierce) by the method of Bosshard and Datyner (19) resulted in a nearly complete loss of activity, although dye did appear to have been bound to the protein. Carrying out the reaction in the presence of urea did not improve the results. Acetylation with acetic anhydride in the presence of dioxane led to a complete loss of activity. Sulfonation using the approach of Chan (20) inactivated the CS-activator and only a small amount of activity (-25%) could be recovered when refolding in the presence of urea and mercaptoethanol was attempted. Periodate oxidation followed by borohydride reduction appeared to be more successful with 25-50% activity being recovered and only a moderate decrease in specific activity. However, only a small amount of radioactivity was incorporated when tritiated reductant was employed. Denaturation-renaturation studies. A 2.5-mg aliquot of pig kidney activator (sp act 1600 U/mg) in 0.5 ml 50 mM Tris-Cl buffer, pH 7.5, was added to 2 ml of SDS-mercaptoethanol solution (SDS-PAGE sample buffer) and heated for 2 min in boiling water. After dialysis in Spectrapor 3 membranes against 250 ml of 5 M urea in 50 mM Tris-Cl, pH 7.5, for 1.5 h, the tubing was transferred to a like volume of 4 M urea for 1 to 2 h. The sample was then progressively dialyzed against 3, 2, 1, and 0.5 M urea solutions in a similar manner. The material was finally dialyzed overnight against 2 liters of urea-free buffer and put through a Sephadex G-25 desalting column to remove a small amount of residual dye which had not completely dialyzed away. The breakthrough fractions were concentrated and subsequently chromatographed through Sephacryl S-300. Another CS-Act sample was made 6 M in urea (without prior SDS or heat treatments) and dialyzed to urea-free buffer through a similar series of decreasing urea concentrations. For neutral guanidine-HCI treatment, CS-Act was dialyzed overnight (Spectrapor 3 membranes) into 6 M guanidine-HCl in 50 mM Tris-Cl, pH 7.5. The sample was then subjected to gel filtration in the presence of denaturant. Peak fractions were collected and dialyzed back into the Tris-Cl buffer overnight. A more severe treatment involved making the activator solution 6 M in guanidineHCl in 50 mM sodium acetate buffer, pH 4.5, and heating the sample in a 100°C bath for 2 min. This material was then chromatographed through Sephacryl S300 in the presence of the pH 4.5 acetate buffer. Cerebroside sulfate binding. Radioactive CS was allowed to interact with CSAct in 0.16 M sodium acetate buffer, pH 4.0. The mixture in a total volume of

90

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ET AL.

275 ~1 was incubated at 37°C for 1 to 2 h and then placed on a 1 x 18 cm column of Ultragel 34 preequilibrated with 50 mM Tris-Cl, pH 7.5. The column was developed with 20 ml of buffer at room temperature and l-ml factions were collected. Column breakthrough occurred in fraction 5 or 6 and CS-Act peaked in fraction 10. The majority of the radioactive lipid was retained on the column requiring that a fresh column be used for each sample. Sephadex G-75 Superfine was also explored for this purpose (21) but did not provide as effective a separation of reactant and product. Metabolic correction of activator-deficient cells. CS-Act was added to serumcontaining medium (Medium 199) and passed through a 0.45-pm filter unit. Medium-containing [35S]CS was prepared as previously described (22). The test solution applied to the cells was obtained by mixing appropriate proportions of these supplemented media with unsupplemented media. For studies involving ‘=Ilabeled activator, uptake was measured in a parallel set of culture flasks to which radioactive CS was not added. RESULTS Chemical modiJications. Attempts at acetylation, dansylation, sulfonation, and reductive glycosylation all inactivated the protein to a large extent. Reductive methylation procedures gave a reasonable retention of activity, but only a small amount of radioactivity was incorporated when tritiated sodium borohydride was employed as reductant. While this reductive methylation product was useful for monitoring CS-Act on electrophoretic and chromatographic procedures, its radioactivity was not high enough to evaluate cell uptake. An attempt to attach a pentamannose monophosphate residue by reductive glycosylation (23) was not successful. Dabsylation was more productive with a derivatized activator protein recovered in 56% yield. This material still retained 60% of its initial CS-Act specific activity. The orange-colored protein derivative has been extremely useful in monitoring the electrophoretic properties of CS-Act. An example is shown in Fig. 1 where the behavior of dabsyl CS-Act is compared with the unmodified protein. The dabsylated protein which could be observed throughout the run accurately marked the position of the unmodified protein. Similar results were obtained with gel isoelectric focusing, but staining of residual ampholites in glutaraldehyde-fixed gels has made them difficult to reproduce for publication. CS-Act retained its activity on iodination and a useful ‘251-labeled derivative was obtained. Octyl Sepharose affinity chromatography provided a simple way to purify the iodinated derivative. The process eliminated any protein inactivated during the iodination reaction and removed deiodination products which accumulated on storage. Octyl Sepharose binding also provided a way to separate labeled protein from [35S]sulfate in the determination of the CS-Act activity of the 12’1-iodinated protein. Autoradiographs of the crude and purified ‘251-labeled protein are shown in Fig. 2. Denatzuation-renaturation. SDS-mercaptoethanol denaturation followed by a urea gradient renaturation resulted in a protein recovery of 27% (0.68 mg) and a specific activity increase of about 40% (41% recovery on a unit basis). The

CHARACTERIZATION

OF PIG KIDNEY

CS-ACT

91

-DF

I-

(+) ’ 2 3 4 5 6 7 6 FIG. 1. SDS gel electrophoresis of dabsylated CS-Act. Samples were heated in SDS-mercaptoethanol sample buffer and applied to a gel with a 10% acrylamide running gel and a 5% stacking gel. Electrophoresis, glutaraldehyde fixation, and Coomassie blue staining were as described in the previous paper (8). Lanes 1 and 2 contained 4 pg each of carbonic anhydrase (CA), myoglobin (M), ribonuclease (R), and insulin (I) whose migration positions are indicated at the edge of the gel. Lanes 4 and S contained 33 pg of dabsylated CS-Act with the position of the orange band in the unstained gel marked by the upper set of pinholes in these lanes. Lanes 7 and 8 contained 36 pg of unmodified es-Act. The positions of the top of the separating gel (0) and the dye front (DF) are indicated on the right side of the gel.

molecular weight of the denatured-renatured protein was unchanged as judged by its migration relative to the myoglobin standard on Sephacryl S-300 chromatography. The use of 6 M urea as the initial denaturant gave a similar result, except for a moderate decrease in specific activity.

; :", -CA

, ‘” ” ;

=y -I

sfFc

(+)

’ 2

FIG. 2. Gel electrophoresis of ‘ZSI-labeled CS-Act. (A) Silver-stained 10% acrylamide SDS gel with 3 pg CS-Act in lane 1 and 0.5 pg each of the protein standards in lane 2. (B) Autoradiograph of 5 x 105 cpm [‘?]CS-Act lane on the dried gel exposed for 6 h. (C) Autoradiograph of the crude ‘Z51-labeled CS-Act preparation before purification with octyl Sepharose. In this case the exposure was extended to 4 days to reveal labeled minor components. A substantial contaminant moving ahead of the dye front and a minor contaminant at the level of the myoglobin standard could be seen. Neither of these remained after the octyl Sepharose purification.

92

FLUHARTY

2

6

ET AL.

10

14

18

FRACTION NUMBER FIG. 3. Evaluation of CS binding by Ultragel 34 gel filtration. Conditions for sample preparation and fractionation are described in the text. The samples contained 60 nmol CS with 4400 cpm/nmol. The dashed line is the control sample with no CS-Act, while the solid line is a sample containing 5 nmol CS-Act.

Treatment with 6 M guanidine-HCI at room temperature and neutral pH had previously been shown to cause the apparent dissociation of CS-Act into its subunits with the molecular mass reduced to 7000-8000 Da (8). When the putative subunits generated in this fashion were collected from the gel filtration column and dialyzed back into denaturant-free buffer there was a 50% recovery of protein, a slight increase in specific activity, and a restoration of the usual S-300 chromatographic properties. However, when the activator had been heated in acidic 6 M guanidine-HCl, the renatured protein proved to be somewhat smaller than the starting material (-13,000 Da) and there was a substantial loss (-80%) in specific activity. Cerebroside sulfate binding. In the absence of activator, two peaks of radioactivity were eluted from the Ultragel 34 columns, each representing about 5% of the applied [35S]cerebroside sulfate. The remainder apparently was entrapped within the column. The first peak occurred at the breakthrough volume, the other just ahead of the column volume in fractions 16 and 17. Both proved to be cerebroside sulfate when examined by TLC. In the presence of CS-Act an additional peak of 35S radioactivity occurred at about fraction 10, intermediate between the other two (Fig. 3). This corresponded to the elution position of 3Hlabeled pig kidney CS-Act. The amount of radioactivity in the intermediate peak depended on the concentration of CS-Act in the reaction mixture and presumably consisted of activator-bound CS. When recovered and rechromatographed, this material gave a single peak at fraction 10 without substantial loss of radioactivity on the column. Incubation of this fraction with ARSA in the absence of detergent or additional CS-Act resulted in the rapid release of radioactive sulfate. The occurrence of activator-bound material was pH dependent with maximum binding at pH 4 or below, half maximum at pH 4.5, and only lo-15% of maximum above pH 5.0.

CHARACTERIZATION

OF PIG KIDNEY

nmole Activator I

260

CS-ACT

93

( mw 2x10 4 ) I

4&l

I

660

units Activator FIG. 4. CS binding by pig kidney CS-Act as a function of activator concentration. In the presence of 60 nmol of CS (open circles) each molecule of activator bound 0.95 mol lipid, but at 10 nmol CS (open triangles) binding was less efficient. The nanomoles of activator was based on a molecular weight of 20,000.

Figure 4 shows the CS binding as a function of activator concentration in the presence of excess (60 nmol) sulfolipid. There is a roughly linear dependence with a 0.95 : 1 mol ratio based on a CS-Act molecular weight of 20,000. This represents 1 nmol CS per 34 units of CS-Act based on the specific activity of 1800 U/mg for this preparation. Partially purified activator fractions also fell on this line if plotted on the basis of CS-Act units employed. In the presence of lower cerebroside sulfate (10 nmol) binding was not as efficient and less than half the available sulfolipid was bound even in the presence of a twofold molar excess of CS-Act. CS-Act correction of deficient fibroblasts. The pig kidney CS-Act could ameliorate the defect in CS catabolism in activator-deficient human fibroblasts. After a period of 8 days, only 0.4% of the added ‘251-labeled CS-Act was found within the cells. However, CS hydrolysis was enhanced nearly fourfold, to about onehalf of that observed in the normal control. The pig kidney protein proved to be several-fold better at metabolic correction than a sample of purified human liver CS-Act tested simultaneously, but was less effective than a CS-Act fraction prepared from human urine. Comparisons were made on basis of equivalent CS-Act activities (see Table 1). It should be noted that the cell line employed in this investigation was more metabolically impaired and less responsive to CS-Act correction than when it had been examined in an earlier study (10). DISCUSSION Variable success was achieved in generating derivatives of the pig kidney CSAct which could be used to monitor chemical and biological properties of the molecule. There appeared to be available amino and tyrosyl residues which could

94

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TABLE 1 Comparison of CS Activator Correction of Activator-Deficient Activator sample Pig kidney Human liver Human urine None Normal cell (control)

Units per flask 21 25 21 0 -

% [‘?]CS-Act incorporated 0.41 0.16 0.49 -

Fibroblasts

% CS assimilated 8 days

% Assimilated CS hydrolyzed

21.3 14.7 25.4 17.5 35.4

45.2 16.8 56.9 12.1 96.0

be modified without substantial loss of activity. Both iodinated and dabsylated derivatives were prepared which retained much of their CS-Act activity. These derivatives were used to monitor electrophoretic and cell uptake properties of the molecule. Only limited success was achieved with reductive methylation. While there was retention of activity, only small amounts of radioactive label were incorporated. A periodate oxidation-reduction protocol also failed to give useful incorporation of radioactivity. An attempt to attach a pentamannose phosphate substituent by reductive glycosylation and thereby enhance cell uptake was also not successful. Attempts at acetylation, acylation, sulfonation, carboxylate esterification, and Drimarene Blue coupling led to severe loss of activity. Fair success was achieved in recovering CS-activator activity after cycles of denaturation-renaturation. Upon treatment with 6 M urea, 6 M guanidine-HCl, or hot SDS-mercaptoethanol active CS-Act could be recovered through gradual renaturation protocols. Renatured material showed normal chromatographic properties on Sephacryl S-300 gel filtration columns and even had an enhanced specific activity in some instances. However, there was always some uncertainty if true denaturation had in fact been achieved. Only a small proportion (lo-15%) of the cysteine residues could be substituted with [‘4C]iodoacetate in the SDS-mercaptoethanol-treated material even after the heating period at 100°C had been extended to 30 min. There was evidence for subunit dissociation in 6 M guanidineHCl, but complete unfolding could not be documented. Heating the protein in acidic guanidine-HCl did appear to cause more profound structural alterations, but only a small proportion of the original CS-Act activity could be recovered after renaturation. The pig kidney CS-Act bound 1 mol of CS per mol of the -20,000 molecular weight dimer under conditions approximating those of the activator assay. This complex was sufficiently stable to be recovered and rechromatographed without loss of the bound lipid. The CS in this isolated CS-CS-Act complex was rapidly hydrolyzed by ARSA without any requirement for additional activator or detergent. It is therefore likely that the complex is the true substrate for the cerebroside sulfatase activity of the enzyme. The activator isolated from pig kidney was able to correct the CS hydrolytic defect in CS-Act-deficient human fibroblasts. It was nearly as good as CS-Act from human urine in its correction activity when compared on the basis of equal CS-Act activity. The pig kidney protein was substantially better than a sample of

CHARACTERIZATION

OF PIG KIDNEY

CS-ACT

95

human liver CS-Act evaluated simultaneously. The difference appeared to be due to a less efficient uptake of the liver protein. While it is unclear what mechanism is involved in CS-Act uptake it does not appear to utilize the phosphomannose receptor system characteristic of many other lysosomal proteins including arylsulfatase A (unpublished data). The availability of substantial quantities of highly purified pig kidney CS-Act should facilitate more detailed studies on its uptake mechanism(s). REFERENCES 1. Fisher G, Jatzkewitz H. The activator of cerebroside sulphatase: Binding studies with enzyme and substrate demonstrating the detergent function on the activator protein. Biochim Siophys Acta 481:561-572, 1977. 2. Li S-C, Kihara H, Serizawa S, Li Y-T, Fluharty AL, Mayes JS, Shapiro LJ. Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. J Biol Chem 260:1867-1871, 1985. 3. Inui K, Wenger DA. Biochemical, immunological and structural studies on a sphingolipid activator protein (SAP-l). Arch Biochem Biophys 233:556-564, 1984. 4. Li S-C, Sonnino S, Tettamanti G, Li Y-T. Characterization of a nonspecific activator protein for the enzymatic hydrolysis of glycolipids. J Biol Chem 263:6588-6591, 1988. 5. O’Brien JS, Kishimoto Y. Saposin proteins: Structure, function, and role in human lysosomal storage disorders. FASEB J 5~301-308, 1991. 6. Collard MW, Sylvester SR, Tsuruta JK, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: Sequence similarity with the 70-kilodalton precursor to sulfatide/GMl activator. Biochemistry 27~4557-4564, 1988. 7. Patthy L. Homology of the precursor of pulmonary surfactant-associated protein SP-B with prosaposin and sulfated glycoprotein 1. J Biol Chem 266~6035-6-037, 1991. 8. Fluharty AL, Katona Z, Meek WE, Frei K, Fowler AV. The cerebroside sulfate activator from pig kidney: Purification and molecular structure. Biochem Med Metab Biol47:66-85, 1992. 9. Shapiro LJ, Alec KA, Kaback MM, Itabashi H, Desnick RJ, Brand N, Stevens RL, Fluharty AL, Kihara H. Metachromatic leukodystrophy without arylsulfatase A deficiency. Pediatr Res 13:1179-1181, 1979. 10. Stevens RL, Fluharty AL, Kihara H, Kaback MM, Shapiro LJ, Marsh B, Sandhoff K, Fischer G. Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy. Am J Hum Genet 33~900-906, 1981. 11. Fluharty AL, Davis ML, Kihara H, Kritchevsky G. Simplified procedure for preparation of %labeled brain sulfatide. Lipids 9~865-869, 1974. 12. ThorelI JI, Johansson BG. Enzymatic iodination of polypeptides with “‘1 to high specific activity. Biochim Biophys Acta 251:363-369, 1971. 13. Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun 80~849-857, 1978. 14. Parkinson D, Redshaw JD. Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride. Anal Biochem 141:121-126, 1984. 15. Tack BF, Wilder RL. Titration of proteins to high specific activity: Application to radio immuno assay. In Methods in Enzymology (Langone JJ, Van Vunakis H, Eds.), Vol. 73. San Diego: Academic Press, 1981, pp. 138-147. 16. Hsieh WT, Matthews KS. Lactose repressor protein modified with dansyl chloride: Activity effects and fluorescence properties. Biochemistry X3043-3049, 1985. 17. Williams JG, Gratzer WB. Limitations of the detergent-polyacrylamide gel electrophoresis method for molecular weight determination of proteins. J Chromatogr 57:121-125, 1971. 18. Crestfield AM, Moore S, Stein WH. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem 238~622-627, 1963. 19. Bosshard HF, Datyner A. The use of a new reactive dye for quantitation of prestained proteins on polyacrylamide gels. Anal Biochem 82~327-333, 1977.

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20. Chan WW-C. A method for the complete S sulfonation of cysteine residues in proteins. Biochembtry 7:4247-4254, 1968. 21. Mitsuyama T, Gasa S., Nojima T, Taniguchi N, Makita A. Purification and properties of galactosylcemmide sulfatase activator from human liver. J Biochem 98:605-613, 1985. 22. Kihara H, Ho C-K, Fluharty AL, Tsay KK, Hartlage PL. Prenatal diagnosis of metachromatic leukodystrophy in a family with pseudo arylsulfatase A deficiency by the cerebroside sulfate loading test. Pediat Res 14:224-227, 1980. 23. Ko KW, Storrie B. Targeting of lacto-peroxidase to phosphomannosyl-specific receptors on fibroblasts. Cell Biol Int Rep 6~1019-1023, 1982.