Neurochemical Research (1) 275-298 (1976)
BRAIN LYSOSOMAL HYDROLASES: I. S O L U B I L I Z A T I O N A N D E L E C T R O P H O R E T I C B E H A V I O R OF A C I D H Y D R O L A S E S IN N E R V E - E N D I N G A N D MITOC H O N DRI A L - L Y S OS O M A L F R A C T I O N S FROM R A T B R A I N . E F F E C T S OF A U T O L Y S I S , N E U R A M I N I D A S E , A N D STORAGE ABDUSSAMAD PATEL AND HAROLD KOENIG Neurology Service, Veterans Administration Lakeside Hospital, and Department of Neurology Northwestern University Medical School Chicago, Illinois 60611
Accepted F e b r u a r y 23, 1976
In solubility studies of 7 acid hydrolases, the extent of solubilization by sonic disruption varied with the e n z y m e species and increased with increasing p H and Triton X-100 concentration of the s u s p e n s i o n m e d i u m . H y d r o l a s e s in the nerveending (NE) fraction were more resistant to solubilization than t h o s e in the m i t o c h o n d r i a l - l y s o s o m a l (M-L) fraction, but nearly quantitative solubilization was attained by sonication in an alkaline t~uffer containing 0.5% Triton X-100. Polyacrylamide gel electrophoresis of extracts revealed multiple c o m p o n e n t s of acid p h o s p h a t a s e , acid esterase, arylsulfatase, /3-glucuronidase, and/3-N-acetylh e x o s a m i n i d a s e . T h e e n z y m e patterns varied with the subcellular fraction and the c o m p o s i t i o n of the m e d i u m . In general, the acidic (anodic) f o r m s of t h e s e hydrolases were more readily solubilized by sonication in acidic buffer, alkaline p H and Triton X-100 being required to solubilize the basic (cationic) c o m p o nents. The acidic forms of t h e s e e n z y m e s were c o n v e r t e d to less anodic or cathodic forms, or both, during autolysis at p H 6 at 0 and 37~ and during storage at - 2 0 ~
275 9 1976Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 1001I. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher,
276
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INTRODUCTION The various acid hydrolases in brain, as in other tissues, are sequestered in a latent state within lysosomes. Brain lysosomes vary widely in size, fine structure, physical properties, and enzymatic composition (1,2). Some of the lysosomal hydrolases in brain--for example,/3-N-acetylhexosaminidase (3-5), acid phosphatase (6), and arylsulfatase (7,8)--occur in two or more molecular forms or isoenzymes. Previous investigations in this laboratory have indicated that many of the acid hydrolases in rat kidney and liver lysosomes are glycoprotein enzymes that occur in two or more forms differing in solubility, electrophoretic mobility (9-11), and isoelectric points (12-14), and that the electronegative charge of these glycoenzymes is related to their N-acetylneuraminic acid content (1114). Further, we have shown that the solubility, electrophoretic mobility (10), and isoelectric points (12,15) of 5 acid hydrolases in rat kidney vary markedly according to their subcellular locations. The synaptosomal or nerve-ending fraction from rat brain contains substantial acid hydrolase activities (1,2) that have been localized to intrasynaptosomal lysosomes (16,17), synaptosomes (18); extrasynaptosomal lysosomes (18,19), and Golgi elements (20). Verity and coworkers (18) have reported differences in latency properties of the acid phosphatase associated with two subsynaptosomal fractions. As part of a series of studies of the molecular heterogeneity of lysosomal enzymes in brain, we have investigated a number of acid hydrolases in the nerve-ending and mitochondrial-lysosomal fractions with respect to their solubilities and electrophoretic behavior in polyacrylamide gels. We also report the effects of autoincubation, bacterial neuraminidase, and storage on the electrophoretic mobility of these enzymes. Some of these findings have appeared in abstract form (20-24).
EXPERIMENTAL PROCEDURE
Chemicals Phenolphthalein glucuronic acid, p-nitrophenylphosphate (disodium salt), a-naphthyl phosphate, a-naphthyl acetate, naphthol AS-BI-/3-D-glucuronide, and neuraminidase (CI. perfringens, Type VI, specific activity 1.1 U/Ing) were purchased from Sigma Chemical Co., St. Louis, Mo. p-Nitrophenyl-N-acetyl-/3-D-glucosaminide,p-nitrophenyl-/3-D-galactopyranoside, 4-methylumbelliferyl-N-acetyl-/~-D-glucosaminide,and 4-methylumbelliferyl/3-D-galactopyranoside were purchased from Pierce Chemical Co., Rockford, Ill. Triton
BRAIN LYSOSOMAL HYDROLASES
277
X-100 was obtained from Rohm & Haas, Philadelphia, Pa. All other chemicals were of reagent grade.
Preparation of Nerve-Ending-and Lysosorne-Rich Fractions Female Sprague-Dawley rats (Holtzman Co., Madison, Wisconsin) weighing 140-180 g were killed by decapitation, and the brains were quickly removed and placed in chilled 0.32 M sucrose. All subsequent operations were carried out at 0-4 ~ C. Tile cerebral cortex was dissected free of most of the white matter, suspended in 9 vol 0.32 M sucrose, and homogenized in a Potter-Elvehjem type glass homogenizer by 3 rapid passes with a motordriven Teflon pestle. The homogenate was filtered through surgical cotton gauze and centrifuged at 1,000g for 10 rain to remove nuclei, cell debris, and red cells. The supernatant was centrifuged at 16,000g for 20 min to obtain a crude mitochondrial pellet, and the supernatant was discarded. The crude mitochondrial fraction was fractionated by isopycnic centrifugation on a discontinuous sucrose density gradient. The mitochondrial pellets were suspended in 6 ml 0.32 M sucrose, layered on top of a gradient consisting of 0.8 M and 1.2 M sucrose, and centrifuged in the Spinco swinging-bucket type 25.1 rotor at 63,500g for 2 h. The nerve-ending (NE) fraction was collected at the 0.8-1.2 M sucrose interface and the mitochondrial-lysosomal (M-L) fraction as a pellet from 1.2 M sucrose. The NE fraction was diluted with 0.32 M sucrose to a final concentration of 0.6 M sucrose and centrifuged for 30 rain at 96,500g to pellet the fraction. The NE and M-L pellets were suspended in a small volume of 0.32 M sucrose and used fresh or stored at - 7 0 ~ C. In several experiments, an M-L fraction was prepared from cat cerebral cortex.
Solubilization of Acid Hydrolases The acid hydrolases in freshly prepared or deep-frozen ( - 7 0 ~ C) fractions were solubilized by a single direct extraction or by a stepwise extraction procedure using a variety of media. The following extraction media were used: pH 5 buffer (0.15 M sodium acetate), pH 7 buffer (0.15 M Tris-HC1), pH 9 buffer (0.15 M glycine-NaOH), 2.0% (vol/ vol) Triton X-100 in H~O, and various concentrations of Triton X-100 in pH 9 buffer. Direct Extraction. The NE and M-L fractions were suspended in these media to give a protein concentration of 3-5 mg/ml. Suspensions were sonicated for 2 min at 0~ (in 4 30sec bursts separated by 15-sec rest periods to minimize heating) with a Branson Sonifier Model W185D (Heat Systems-Ultrasonics, Inc., Plainview, N.Y.) at the output setting of 40 W. For gel electrophoresis experiments, fractions frozen at -70~ in plastic tubes were covered with 0.5% (vol/vol) Triton X-100, 0.15 M glycine-NaOH buffer, pH 9, and left undisturbed for 20-30 min at -6-7~ Fractions were then subjected to 3 or 4 mild freezethaw cycles, performed by alternately immersing and swirling in a dry ice-propanol mixture and in an ice-water mixture. This was followed by a 2-rain period of sonication as described above, except that the suspension was immersed in chilled water kept at - 6 7~ The disruptates were clarified by centrifuging at 96,500g for 30 rain at 0~ Stepwise Extraction. The fraction was suspended in the pH 5 buffer (with or without Triton X-100) and sonicated for 2 rain at 0~ as already described, and the sonicate was clarified by high-speed centrifugation. The pellet was resuspended in pH 7 buffer (with or without Triton X-100), and the process of sonication and centrifugation was repeated. The pH 7 residue was resuspended in the pH 9 buffer (with or without Triton X-100), sonicated, and centrifuged. Finally, the pH 9 residue was resuspended in the pH 5 buffer containing 0.2% Triton X-100 (vol/vol).
278
PATEL AND KOENIG
Enzyme Assays Acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) was assayed with p-nitrophenyl phosphate as substrate (25). Arylsulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was assayed with p-nitrocatechol sulfate as substrate (26). /3Glucuronidase (/3-D-glucuronide glucuronohydrolase, EC 3.2.1.31) was measured with phenolphthalein/3-glucuronide as substrate (27). The activity of/3-N-acetylhexosaminidase (/3-2-acetamido-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) and /3galactosidase (/3-D-galactoside galactohydrolase, EC 3.2.1.23) was assayed spectrophotometrically using p-nitrophenyl (PNP)/3-D-glycosides and fluorometrically using 4-methylumbelliferyl (MU)-/3-D-glycosides as fluorogenic substrates (28,29). The assay conditions for measuring the enzyme activity with the PNP-substrates were: 3 mM (PNP)-N-acetyl/3-D-glucosaminide, 80 mM sodium citrate-phosphate buffer, pH 4.5, for fl-N-acetylhexosaminidase, and 3 mM (PNP)-/3-D-galactopyranoside, 80 mM sodium citrate-phosphate buffer, p H 3.6, for/3-galactoside. The reaction was terminated by adding NaOH, and the absorbance of the liberated p-nitrophenol was read at 410 rim. For the fluorometric determination of enzyme activity, the incubation mixture was the same as used in the spectrophotometric method, except that 0.056 mM MU-derivative was substituted for the PNP-derivative. The reaction was terminated by adding 3 ml 0.133 M glycine-NaOH buffer, pH 10.45. The fuorescence was measured in the Aminco Bowman spectrophotofluorimeter at an excitation wavelength of 370 nm and an emission wavelength of 475 nm. Cathepsin D (EC 3.4.4.23) was determined with denatured hemoglobin as substrate (30). The reaction was stopped by adding 0.5 ml 30% trichloroacetic acid (wt/vol), and released tyrosine was measured colorimetrically (31). Acid esterase was assayed by an azo dyecoupling technique with a-naphthyl acetate as substrate and fast blue B as the diazonium salt (32). The red color was extracted in 3 ml ethyl acetate, and the absorbance measured at 500 nm. Protein was determined by the method of Lowry et al. (31).
Polyacrylamide Gel Electrophoresis Disc electrophoresis was carried out in 5% polyacrylamide gels in 0.2 M glycine-Tris buffer, pH 8.8, by the procedure of Davis (33). The enzyme extracts were layered directly on top of the polymerized gels in 0.2 ml 20% sucrose. Electrophoresis was conducted for 90-120 min at 2.5 mA/gel at 4~ The gels were fixed for 10 min in 4% buffered formaldehyde, pH 6.0, at 4~ transferred to beakers containing ice-cold distilled water to remove excess formaldehyde, and stored overnight at 0~
Demonstration of Enzyme Activities in Gels Acid phosphatase,/3-glucuronidase, and acid esterase activities were demonstrated by a simultaneous coupling azo dye method with hexazonium pararosalinine as a capture agent. For acid phosphatase, the substrate was a-naphthyl phosphate (4 mM in 0.05 M sodium acetate buffer, pH 5.4) (34). For/3-glucuronidase, the substrate was naphthol AS-BI-fl-Dglucuronide (0.25 mM in 0.1 M sodium acetate buffer, pH 5.2) (35). For acid esterase, the substrate was a-naphthyl acetate (7.5 mM in 0.1 M sodium citrate-phosphate buffer, pH 5.4, with 10-n M diisopropylfluorophosphate) (34). Incubation was for 60 rain at 37~ Arylsulfatase was detected by incubating gels in l0 mM p-nitrocatechol sulfate in 0.2 M sodium acetate buffer, pH 5.5, for 30 rain at 37~ (26). The red color of the enzyme bands
BRAIN LYSOSOMAL HYDROLASES
2,79
was developed by adding 0.5 N NaOH. (PNP)-fl-N-Acetylhexosaminidasewas stained by incubating gels in 0.05 M sodium citrate, pH 5.5, containing 5 mM (PNP)-N-acetyl-fl-oglucosaminadide (36). The yellow bands of enzyme activity were stained with 2,3,5triphenyltetrazolium chloride in a postcoupling procedure (37). (MU)-fl-N-Acetylhexosaminidase was demonstrated by incubating the gels in 0.056 mM (MU)-N-acetyl-fl-Dglucosaminide in 0.1 M sodium citrate-phosphate buffer, pH 4.5, for 15 rain at 37~ (38). The fluorescent bands of liberated 4-methylumbelliferone were developed by adding 0.133 M glycine-NaOH buffer, pH 10.45, and visualized or photographed in near UV light.
Neuraminidase Treatment, Autoincubation, and Storage of Enzyme Preparations For these studies, the M-L fraction was extracted by sonicating in 2% (vol/vol) Triton X-100 in H20 as already described. Portions of the extract were incubated in 0.1 M sodium acetate buffer, pH 6.0, at 37~ for 3 h in the presence or absence of 0.5 mg neuraminidase/ml, or kept at 0~ for 18 h without added neuraminidase. In another set of experiments, the Triton X-100 extracts were stored without buffer at -20~ for 1-5 weeks. Experimental and untreated control extracts were subjected to polyacrylamide gel electrophoresis and incubated for enzyme activities as described.
RESULTS
Solubilization of Acid Hydrolases in NE and M-L Fractions The extent to which the particulate acid hydrolases were released into solution by sonication varied according to the subcellular fraction, the specific enzyme, the pH of the buffer, and the presence or absence of Triton X-100. The results with a single direct extraction are given in Table I. In general, the various hydrolases and protein in the M-L fraction were much more readily solubilized than those in the NE fraction, and the solubility of the hydrolases increased with increasing pH of the buffer. Arylsulfatase, fl-glucuronidase, (PNP)- and (MU)-flN-acetylhexosaminidase, (MU)-fl-galactosidase, and cathepsin D were readily solubilized in buffered media, whereas (PNP)-fl-galactosidase, acid phosphatase, and acid esterase were more resistant to solubilization. Thus, when the M-L fraction was sonicated in pH 9 buffer without detergent, the enzymes in the former group were nearly completely (8698%) solubilized, while only 60, 52, and 26% of the (PNP)-fl-galactosidase, acid phosphatase, and acid esterase were solubilized. Similar differences between these two groups of hydrolases were observed in the NE fraction. Furthermore, the latter 3 enzymes in the M-L and the NE fractions showed a smaller increment in solubility with increasing pH than the more soluble hydrolases.
280
PATEL AND KOENIG
Triton X-100 (2%, vol/vol) in water, by contrast, solubilized much of the acid phosphatase, (MU)-/3-galactosidase, acid esterase, and arylsulfatase (58-68% and 61-79% of the total in the NE and the M-L fraction, respectively), but was less effective in solubilizing the other hydrolases (18--38% and 24-59% of the total in the NE and the M-L fraction, respectively). The combination of Triton X-100 and alkaline buffer proved to be the most efficient medium for extracting the various hydrolases. It is noteworthy that the solubility of/3-N-acetylhexosaminidase and /3-galactosidase differed substantially, depending on the substrate used for assay. The proportion of these enzyme activities that remained sedimentable under various conditions of extraction was generally greater when the assays were performed with the (PNP)-/3-Dglycoside than with the (MU)-/3-D-glycoside substrate. In a second set of experiments, we studied the effect on the solubilization of acid hydrolases and protein of sequential extraction of the NE and M-L fractions in buffers of increasing p H values with and without Triton X-100. The results are given in Table II. The addition of Triton X-100 to the buffers increased the solubilization of the particulate acid hydrolases and protein in proportion to the concentration of the detergent. Under these conditions, the sequential treatment with buffers containing 2% (vol/vol) Triton X-100 completely solubilized all the hydrolases in the M-L fraction. The enzymes and proteins in the NE fraction were less easily solubilized by sequential extraction with buffers than those in the M-L fraction. Nevertheless, 88--89% of the acid phosphatase and (PNP)-/3-galactosidase and 95-100% of the other hydrolases in the NE fraction were solubilized by the complete buffer sequence containing 2% (vol/vol) Triton X-100.
Electrophoretic Patterns of Acid Hydrolases in Direct Extracts of M-L and NE Fractions For polyacrylamide gel electrophoresis, 0.5% (vol/vol) Triton X-100, 0.15 M glycine-NaOH buffer, pH 9, was generally used for the extraction of the NE and M-L fractions, as this medium solubilized between 75 and 99% of the acid hydrolases in these fractions. The electrophoretic patterns of the hydrolases in extracts of the NE and M-L fractions were sharply divergent. In general, the enzyme patterns obtained from the NE fraction showed a major cathodic form and a minor anodic form, moving faster than the corresponding anodic form from the M-L fraction. The M-L fraction contained at least 7 acid
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BRAIN LYSOSOMAL HYDROLASES: I. S O L U B I L I Z A T I O N A N D E L E C T R O P H O R E T I C B E H A V I O R OF A C I D H Y D R O L A S E S IN N E R V E - E N D I N G A N D MITOC H O N DRI A L - L Y S OS O M A L F R A C T I O N S FROM R A T B R A I N . E F F E C T S OF A U T O L Y S I S , N E U R A M I N I D A S E , A N D STORAGE ABDUSSAMAD PATEL AND HAROLD KOENIG Neurology Service, Veterans Administration Lakeside Hospital, and Department of Neurology Northwestern University Medical School Chicago, Illinois 60611
Accepted F e b r u a r y 23, 1976
In solubility studies of 7 acid hydrolases, the extent of solubilization by sonic disruption varied with the e n z y m e species and increased with increasing p H and Triton X-100 concentration of the s u s p e n s i o n m e d i u m . H y d r o l a s e s in the nerveending (NE) fraction were more resistant to solubilization than t h o s e in the m i t o c h o n d r i a l - l y s o s o m a l (M-L) fraction, but nearly quantitative solubilization was attained by sonication in an alkaline t~uffer containing 0.5% Triton X-100. Polyacrylamide gel electrophoresis of extracts revealed multiple c o m p o n e n t s of acid p h o s p h a t a s e , acid esterase, arylsulfatase, /3-glucuronidase, and/3-N-acetylh e x o s a m i n i d a s e . T h e e n z y m e patterns varied with the subcellular fraction and the c o m p o s i t i o n of the m e d i u m . In general, the acidic (anodic) f o r m s of t h e s e hydrolases were more readily solubilized by sonication in acidic buffer, alkaline p H and Triton X-100 being required to solubilize the basic (cationic) c o m p o nents. The acidic forms of t h e s e e n z y m e s were c o n v e r t e d to less anodic or cathodic forms, or both, during autolysis at p H 6 at 0 and 37~ and during storage at - 2 0 ~
275 9 1976Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 1001I. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher,
276
PATEL AND KOENIG
INTRODUCTION The various acid hydrolases in brain, as in other tissues, are sequestered in a latent state within lysosomes. Brain lysosomes vary widely in size, fine structure, physical properties, and enzymatic composition (1,2). Some of the lysosomal hydrolases in brain--for example,/3-N-acetylhexosaminidase (3-5), acid phosphatase (6), and arylsulfatase (7,8)--occur in two or more molecular forms or isoenzymes. Previous investigations in this laboratory have indicated that many of the acid hydrolases in rat kidney and liver lysosomes are glycoprotein enzymes that occur in two or more forms differing in solubility, electrophoretic mobility (9-11), and isoelectric points (12-14), and that the electronegative charge of these glycoenzymes is related to their N-acetylneuraminic acid content (1114). Further, we have shown that the solubility, electrophoretic mobility (10), and isoelectric points (12,15) of 5 acid hydrolases in rat kidney vary markedly according to their subcellular locations. The synaptosomal or nerve-ending fraction from rat brain contains substantial acid hydrolase activities (1,2) that have been localized to intrasynaptosomal lysosomes (16,17), synaptosomes (18); extrasynaptosomal lysosomes (18,19), and Golgi elements (20). Verity and coworkers (18) have reported differences in latency properties of the acid phosphatase associated with two subsynaptosomal fractions. As part of a series of studies of the molecular heterogeneity of lysosomal enzymes in brain, we have investigated a number of acid hydrolases in the nerve-ending and mitochondrial-lysosomal fractions with respect to their solubilities and electrophoretic behavior in polyacrylamide gels. We also report the effects of autoincubation, bacterial neuraminidase, and storage on the electrophoretic mobility of these enzymes. Some of these findings have appeared in abstract form (20-24).
EXPERIMENTAL PROCEDURE
Chemicals Phenolphthalein glucuronic acid, p-nitrophenylphosphate (disodium salt), a-naphthyl phosphate, a-naphthyl acetate, naphthol AS-BI-/3-D-glucuronide, and neuraminidase (CI. perfringens, Type VI, specific activity 1.1 U/Ing) were purchased from Sigma Chemical Co., St. Louis, Mo. p-Nitrophenyl-N-acetyl-/3-D-glucosaminide,p-nitrophenyl-/3-D-galactopyranoside, 4-methylumbelliferyl-N-acetyl-/~-D-glucosaminide,and 4-methylumbelliferyl/3-D-galactopyranoside were purchased from Pierce Chemical Co., Rockford, Ill. Triton
BRAIN LYSOSOMAL HYDROLASES
277
X-100 was obtained from Rohm & Haas, Philadelphia, Pa. All other chemicals were of reagent grade.
Preparation of Nerve-Ending-and Lysosorne-Rich Fractions Female Sprague-Dawley rats (Holtzman Co., Madison, Wisconsin) weighing 140-180 g were killed by decapitation, and the brains were quickly removed and placed in chilled 0.32 M sucrose. All subsequent operations were carried out at 0-4 ~ C. Tile cerebral cortex was dissected free of most of the white matter, suspended in 9 vol 0.32 M sucrose, and homogenized in a Potter-Elvehjem type glass homogenizer by 3 rapid passes with a motordriven Teflon pestle. The homogenate was filtered through surgical cotton gauze and centrifuged at 1,000g for 10 rain to remove nuclei, cell debris, and red cells. The supernatant was centrifuged at 16,000g for 20 min to obtain a crude mitochondrial pellet, and the supernatant was discarded. The crude mitochondrial fraction was fractionated by isopycnic centrifugation on a discontinuous sucrose density gradient. The mitochondrial pellets were suspended in 6 ml 0.32 M sucrose, layered on top of a gradient consisting of 0.8 M and 1.2 M sucrose, and centrifuged in the Spinco swinging-bucket type 25.1 rotor at 63,500g for 2 h. The nerve-ending (NE) fraction was collected at the 0.8-1.2 M sucrose interface and the mitochondrial-lysosomal (M-L) fraction as a pellet from 1.2 M sucrose. The NE fraction was diluted with 0.32 M sucrose to a final concentration of 0.6 M sucrose and centrifuged for 30 rain at 96,500g to pellet the fraction. The NE and M-L pellets were suspended in a small volume of 0.32 M sucrose and used fresh or stored at - 7 0 ~ C. In several experiments, an M-L fraction was prepared from cat cerebral cortex.
Solubilization of Acid Hydrolases The acid hydrolases in freshly prepared or deep-frozen ( - 7 0 ~ C) fractions were solubilized by a single direct extraction or by a stepwise extraction procedure using a variety of media. The following extraction media were used: pH 5 buffer (0.15 M sodium acetate), pH 7 buffer (0.15 M Tris-HC1), pH 9 buffer (0.15 M glycine-NaOH), 2.0% (vol/ vol) Triton X-100 in H~O, and various concentrations of Triton X-100 in pH 9 buffer. Direct Extraction. The NE and M-L fractions were suspended in these media to give a protein concentration of 3-5 mg/ml. Suspensions were sonicated for 2 min at 0~ (in 4 30sec bursts separated by 15-sec rest periods to minimize heating) with a Branson Sonifier Model W185D (Heat Systems-Ultrasonics, Inc., Plainview, N.Y.) at the output setting of 40 W. For gel electrophoresis experiments, fractions frozen at -70~ in plastic tubes were covered with 0.5% (vol/vol) Triton X-100, 0.15 M glycine-NaOH buffer, pH 9, and left undisturbed for 20-30 min at -6-7~ Fractions were then subjected to 3 or 4 mild freezethaw cycles, performed by alternately immersing and swirling in a dry ice-propanol mixture and in an ice-water mixture. This was followed by a 2-rain period of sonication as described above, except that the suspension was immersed in chilled water kept at - 6 7~ The disruptates were clarified by centrifuging at 96,500g for 30 rain at 0~ Stepwise Extraction. The fraction was suspended in the pH 5 buffer (with or without Triton X-100) and sonicated for 2 rain at 0~ as already described, and the sonicate was clarified by high-speed centrifugation. The pellet was resuspended in pH 7 buffer (with or without Triton X-100), and the process of sonication and centrifugation was repeated. The pH 7 residue was resuspended in the pH 9 buffer (with or without Triton X-100), sonicated, and centrifuged. Finally, the pH 9 residue was resuspended in the pH 5 buffer containing 0.2% Triton X-100 (vol/vol).
278
PATEL AND KOENIG
Enzyme Assays Acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) was assayed with p-nitrophenyl phosphate as substrate (25). Arylsulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was assayed with p-nitrocatechol sulfate as substrate (26). /3Glucuronidase (/3-D-glucuronide glucuronohydrolase, EC 3.2.1.31) was measured with phenolphthalein/3-glucuronide as substrate (27). The activity of/3-N-acetylhexosaminidase (/3-2-acetamido-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) and /3galactosidase (/3-D-galactoside galactohydrolase, EC 3.2.1.23) was assayed spectrophotometrically using p-nitrophenyl (PNP)/3-D-glycosides and fluorometrically using 4-methylumbelliferyl (MU)-/3-D-glycosides as fluorogenic substrates (28,29). The assay conditions for measuring the enzyme activity with the PNP-substrates were: 3 mM (PNP)-N-acetyl/3-D-glucosaminide, 80 mM sodium citrate-phosphate buffer, pH 4.5, for fl-N-acetylhexosaminidase, and 3 mM (PNP)-/3-D-galactopyranoside, 80 mM sodium citrate-phosphate buffer, p H 3.6, for/3-galactoside. The reaction was terminated by adding NaOH, and the absorbance of the liberated p-nitrophenol was read at 410 rim. For the fluorometric determination of enzyme activity, the incubation mixture was the same as used in the spectrophotometric method, except that 0.056 mM MU-derivative was substituted for the PNP-derivative. The reaction was terminated by adding 3 ml 0.133 M glycine-NaOH buffer, pH 10.45. The fuorescence was measured in the Aminco Bowman spectrophotofluorimeter at an excitation wavelength of 370 nm and an emission wavelength of 475 nm. Cathepsin D (EC 3.4.4.23) was determined with denatured hemoglobin as substrate (30). The reaction was stopped by adding 0.5 ml 30% trichloroacetic acid (wt/vol), and released tyrosine was measured colorimetrically (31). Acid esterase was assayed by an azo dyecoupling technique with a-naphthyl acetate as substrate and fast blue B as the diazonium salt (32). The red color was extracted in 3 ml ethyl acetate, and the absorbance measured at 500 nm. Protein was determined by the method of Lowry et al. (31).
Polyacrylamide Gel Electrophoresis Disc electrophoresis was carried out in 5% polyacrylamide gels in 0.2 M glycine-Tris buffer, pH 8.8, by the procedure of Davis (33). The enzyme extracts were layered directly on top of the polymerized gels in 0.2 ml 20% sucrose. Electrophoresis was conducted for 90-120 min at 2.5 mA/gel at 4~ The gels were fixed for 10 min in 4% buffered formaldehyde, pH 6.0, at 4~ transferred to beakers containing ice-cold distilled water to remove excess formaldehyde, and stored overnight at 0~
Demonstration of Enzyme Activities in Gels Acid phosphatase,/3-glucuronidase, and acid esterase activities were demonstrated by a simultaneous coupling azo dye method with hexazonium pararosalinine as a capture agent. For acid phosphatase, the substrate was a-naphthyl phosphate (4 mM in 0.05 M sodium acetate buffer, pH 5.4) (34). For/3-glucuronidase, the substrate was naphthol AS-BI-fl-Dglucuronide (0.25 mM in 0.1 M sodium acetate buffer, pH 5.2) (35). For acid esterase, the substrate was a-naphthyl acetate (7.5 mM in 0.1 M sodium citrate-phosphate buffer, pH 5.4, with 10-n M diisopropylfluorophosphate) (34). Incubation was for 60 rain at 37~ Arylsulfatase was detected by incubating gels in l0 mM p-nitrocatechol sulfate in 0.2 M sodium acetate buffer, pH 5.5, for 30 rain at 37~ (26). The red color of the enzyme bands
BRAIN LYSOSOMAL HYDROLASES
2,79
was developed by adding 0.5 N NaOH. (PNP)-fl-N-Acetylhexosaminidasewas stained by incubating gels in 0.05 M sodium citrate, pH 5.5, containing 5 mM (PNP)-N-acetyl-fl-oglucosaminadide (36). The yellow bands of enzyme activity were stained with 2,3,5triphenyltetrazolium chloride in a postcoupling procedure (37). (MU)-fl-N-Acetylhexosaminidase was demonstrated by incubating the gels in 0.056 mM (MU)-N-acetyl-fl-Dglucosaminide in 0.1 M sodium citrate-phosphate buffer, pH 4.5, for 15 rain at 37~ (38). The fluorescent bands of liberated 4-methylumbelliferone were developed by adding 0.133 M glycine-NaOH buffer, pH 10.45, and visualized or photographed in near UV light.
Neuraminidase Treatment, Autoincubation, and Storage of Enzyme Preparations For these studies, the M-L fraction was extracted by sonicating in 2% (vol/vol) Triton X-100 in H20 as already described. Portions of the extract were incubated in 0.1 M sodium acetate buffer, pH 6.0, at 37~ for 3 h in the presence or absence of 0.5 mg neuraminidase/ml, or kept at 0~ for 18 h without added neuraminidase. In another set of experiments, the Triton X-100 extracts were stored without buffer at -20~ for 1-5 weeks. Experimental and untreated control extracts were subjected to polyacrylamide gel electrophoresis and incubated for enzyme activities as described.
RESULTS
Solubilization of Acid Hydrolases in NE and M-L Fractions The extent to which the particulate acid hydrolases were released into solution by sonication varied according to the subcellular fraction, the specific enzyme, the pH of the buffer, and the presence or absence of Triton X-100. The results with a single direct extraction are given in Table I. In general, the various hydrolases and protein in the M-L fraction were much more readily solubilized than those in the NE fraction, and the solubility of the hydrolases increased with increasing pH of the buffer. Arylsulfatase, fl-glucuronidase, (PNP)- and (MU)-flN-acetylhexosaminidase, (MU)-fl-galactosidase, and cathepsin D were readily solubilized in buffered media, whereas (PNP)-fl-galactosidase, acid phosphatase, and acid esterase were more resistant to solubilization. Thus, when the M-L fraction was sonicated in pH 9 buffer without detergent, the enzymes in the former group were nearly completely (8698%) solubilized, while only 60, 52, and 26% of the (PNP)-fl-galactosidase, acid phosphatase, and acid esterase were solubilized. Similar differences between these two groups of hydrolases were observed in the NE fraction. Furthermore, the latter 3 enzymes in the M-L and the NE fractions showed a smaller increment in solubility with increasing pH than the more soluble hydrolases.
280
PATEL AND KOENIG
Triton X-100 (2%, vol/vol) in water, by contrast, solubilized much of the acid phosphatase, (MU)-/3-galactosidase, acid esterase, and arylsulfatase (58-68% and 61-79% of the total in the NE and the M-L fraction, respectively), but was less effective in solubilizing the other hydrolases (18--38% and 24-59% of the total in the NE and the M-L fraction, respectively). The combination of Triton X-100 and alkaline buffer proved to be the most efficient medium for extracting the various hydrolases. It is noteworthy that the solubility of/3-N-acetylhexosaminidase and /3-galactosidase differed substantially, depending on the substrate used for assay. The proportion of these enzyme activities that remained sedimentable under various conditions of extraction was generally greater when the assays were performed with the (PNP)-/3-Dglycoside than with the (MU)-/3-D-glycoside substrate. In a second set of experiments, we studied the effect on the solubilization of acid hydrolases and protein of sequential extraction of the NE and M-L fractions in buffers of increasing p H values with and without Triton X-100. The results are given in Table II. The addition of Triton X-100 to the buffers increased the solubilization of the particulate acid hydrolases and protein in proportion to the concentration of the detergent. Under these conditions, the sequential treatment with buffers containing 2% (vol/vol) Triton X-100 completely solubilized all the hydrolases in the M-L fraction. The enzymes and proteins in the NE fraction were less easily solubilized by sequential extraction with buffers than those in the M-L fraction. Nevertheless, 88--89% of the acid phosphatase and (PNP)-/3-galactosidase and 95-100% of the other hydrolases in the NE fraction were solubilized by the complete buffer sequence containing 2% (vol/vol) Triton X-100.
Electrophoretic Patterns of Acid Hydrolases in Direct Extracts of M-L and NE Fractions For polyacrylamide gel electrophoresis, 0.5% (vol/vol) Triton X-100, 0.15 M glycine-NaOH buffer, pH 9, was generally used for the extraction of the NE and M-L fractions, as this medium solubilized between 75 and 99% of the acid hydrolases in these fractions. The electrophoretic patterns of the hydrolases in extracts of the NE and M-L fractions were sharply divergent. In general, the enzyme patterns obtained from the NE fraction showed a major cathodic form and a minor anodic form, moving faster than the corresponding anodic form from the M-L fraction. The M-L fraction contained at least 7 acid
BRAIN LYSOSOMAL
281
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